Receptor tyrosine kinase which mediates the pleiotropic actions of insulin. Binding of insulin leads to phosphorylation of several intracellular substrates, including, insulin receptor substrates (IRS1, 2, 3, 4), SHC, GAB1, CBL and other signaling intermediates. Each of these phosphorylated proteins serve as docking proteins for other signaling proteins that contain Src-homology-2 domains (SH2 domain) that specifically recognize different phosphotyrosines residues, including the p85 regulatory subunit of PI3K and SHP2. Phosphorylation of IRSs proteins lead to the activation of two main signaling pathways: the PI3K-AKT/PKB pathway, which is responsible for most of the metabolic actions of insulin, and the Ras-MAPK pathway, which regulates expression of some genes and cooperates with the PI3K pathway to control cell growth and differentiation. Binding of the SH2 domains of PI3K to phosphotyrosines on IRS1 leads to the activation of PI3K and the generation of phosphatidylinositol-(3, 4, 5)-triphosphate (PIP3), a lipid second messenger, which activates several PIP3-dependent serine/threonine kinases, such as PDPK1 and subsequently AKT/PKB. The net effect of this pathway is to produce a translocation of the glucose transporter SLC2A4/GLUT4 from cytoplasmic vesicles to the cell membrane to facilitate glucose transport. Moreover, upon insulin stimulation, activated AKT/PKB is responsible for: anti-apoptotic effect of insulin by inducing phosphorylation of BAD; regulates the expression of gluconeogenic and lipogenic enzymes by controlling the activity of the winged helix or forkhead (FOX) class of transcription factors. Another pathway regulated by PI3K-AKT/PKB activation is mTORC1 signaling pathway which regulates cell growth and metabolism and integrates signals from insulin. AKT mediates insulin-stimulated protein synthesis by phosphorylating TSC2 thereby activating mTORC1 pathway. The Ras/RAF/MAP2K/MAPK pathway is mainly involved in mediating cell growth, survival and cellular differentiation of insulin. Phosphorylated IRS1 recruits GRB2/SOS complex, which triggers the activation of the Ras/RAF/MAP2K/MAPK pathway. In addition to binding insulin, the insulin receptor can bind insulin-like growth factors (IGFI and IGFII).
In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IR(Delta43)) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IR(Delta43) (L6(IRDelta43)) also showed no insulin effect on glucose uptake and glycogen synthase, accompanied by a >80% decrease in insulin induction of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) activity and tyrosine phosphorylation and of protein kinase B (PKB) phosphorylation at Thr(308). Insulin induced the phosphatidylinositol 3 kinase-dependent coprecipitation of PDK-1 with wild-type IR (IR(WT)), but not IR(Delta43). Based on overlay blotting, PDK-1 directly bound IR(WT), but not IR(Delta43). Insulin-activated IR(WT), and not IR(Delta43), phosphorylated PDK-1 at tyrosines 9, 373, and 376. The IR C-terminal 43-amino-acid peptide (C-terminal peptide) inhibited in vitro PDK-1 tyrosine phosphorylation by the IR. Tyr-->Phe substitution prevented this inhibitory action. In the L6(hIR) cells, the C-terminal peptide coprecipitated with PDK-1 in an insulin-stimulated fashion. This peptide simultaneously impaired the insulin effect on PDK-1 coprecipitation with IR(WT), on PDK-1 tyrosine phosphorylation, on PKB phosphorylation at Thr(308), and on glucose uptake. Upon insulin exposure, PDK-1 membrane persistence was significantly reduced in L6(IRDelta43) compared to control cells. In L6 cells expressing IR(WT), the C-terminal peptide also impaired insulin-dependent PDK-1 membrane persistence. Thus, PDK-1 directly binds to the insulin receptor, followed by PDK-1 activation and insulin metabolic effects.
Protein microsequencing of human placental IGF-I receptors purified by immunoaffinity chromatography using IGF-I receptor specific monoclonal antibody revealed amino acid sequences of both IGF-I and insulin receptors. Since this finding indicated the presence of IGF-I/insulin receptor hybrids, hybrid receptors were further purified by immunoaffinity chromatography using insulin receptor specific monoclonal antibody. The molecular size of the nonreduced hybrid receptor was approximately 350K, indicating that the IGF-I and insulin receptor alpha beta halves were disulfide-linked. The ratio of IGF/insulin binding activity of purified hybrid receptors was approximately 25 when measured using tracer amounts of radioactive ligands. 125I-IGF binding to these receptors was inhibited by IGF-I and insulin with IC50s of approximately 2 and approximately 1000 nM, respectively. 125I-Insulin binding to these receptors was similarly inhibited by IGF-I and insulin with IC50 of approximately 3 nM. Autophosphorylation and kinase activities of the hybrid receptor were stimulated by IGF-I more effectively than insulin in a dose-dependent manner. Thus, the present studies indicate that hybrid receptors purified from human placenta have the functional properties of an IGF-I receptor.
The insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) have a highly homologous structure, but different biological effects. Insulin and IGF-I half-receptors can heterodimerize, leading to the formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) that bind IGF-I with high affinity. As the IR exists in two isoforms (IR-A and IR-B), we evaluated whether the assembly of the IGF-IR with either IR-A or IR-B moieties may differently affect Hybrid-R signaling and biological role. Three different models were studied: (a) 3T3-like mouse fibroblasts with a disrupted IGF-IR gene (R(-) cells) cotransfected with the human IGF-IR and with either the IR-A or IR-B cDNA; (b) a panel of human cell lines variably expressing the two IR isoforms; and (c) HepG2 human hepatoblastoma cells predominantly expressing either IR-A or IR-B, depending on their differentiation state. We found that Hybrid-Rs containing IR-A (Hybrid-Rs(A)) bound to and were activated by IGF-I, IGF-II, and insulin. By binding to Hybrid-Rs(A), insulin activated the IGF-I half-receptor beta-subunit and the IGF-IR-specific substrate CrkII. In contrast, Hybrid-Rs(B) bound to and were activated with high affinity by IGF-I, with low affinity by IGF-II, and insignificantly by insulin. As a consequence, cell proliferation and migration in response to both insulin and IGFs were more effectively stimulated in Hybrid-R(A)-containing cells than in Hybrid-R(B)-containing cells. The relative abundance of IR isoforms therefore affects IGF system activation through Hybrid-Rs, with important consequences for tissue-specific responses to both insulin and IGFs.
We have previously shown that phosphatidylinositol (PtdIns) 3'-kinase is activated by the binding of proteins or peptides containing the phosphorylated motif Y(P)XXM. In the present study, we examine interactions between PtdIns 3'-kinase and the human insulin receptor, which contains a C-terminal phosphorylation site in the sequence Y1322THM. Partially purified insulin receptors bound tightly to bacterial fusion proteins containing the N- or C-terminal SH2 domains from PtdIns 3'-kinase regulatory subunit (p85). In contrast, a mutant insulin receptor, truncated by 43 amino acids at the C terminus (IR delta CT), bound poorly to the SH2 domains; these mutant receptors have normal kinase activity but lack the Y1322THM motif. Similarly, incubation with wild-type receptors increased the activity of immunopurified PtdIns 3'-kinase, whereas incubation with IR delta CT receptors did not affect PtdIns 3'-kinase activity. Activation of PtdIns 3'-kinase by the wild-type receptor was mimicked by a tyrosyl phosphopeptide derived from the insulin receptor C terminus and containing the Y1322THM motif; non-phosphorylated peptide did not affect activity. Thus, the insulin receptor C terminus activates PtdIns 3'-kinase in vitro by binding to the SH2 domains of the 85-kDa regulatory subunit. These data support the hypothesis that binding of tyrosyl-phosphorylated receptors to p85 SH2 domains is a general mechanism for PtdIns 3'-kinase activation, and they suggest that direct interactions between the insulin receptor and PtdIns 3'-kinase may provide an alternative pathway for the activation of this enzyme by insulin.
Insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) are both from the same subgroup of receptor tyrosine kinases that exist as covalently bound receptor dimers at the cell surface. For both IR and IGF-IR, the most described forms are homodimer receptors. However, hybrid receptors consisting of one-half IR and one-half IGF-IR are also present at the cell surface. Two splice variants of IR are expressed that enable formation of two isoforms of the IGF-IR/IR hybrid receptor. In this study, these two splice variants of hybrid receptors were studied with respect to binding affinities of insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II). Unlike previously published data, in which semipurified receptors have been studied, we found that the two hybrid receptor splice variants had similar binding characteristics with respect to insulin, IGF-I, and IGF-II binding. We studied both semipurified and purified hybrid receptors. In all cases we found that IGF-I had at least 50-fold higher affinity than insulin, irrespective of the splice variant. The binding characteristics of insulin and IGF-I to both splice variants of the hybrid receptors were similar to classical homodimer IGF-IR.
Insulin-like growth factor II (IGF-II) is a peptide growth factor that is homologous to both insulin-like growth factor I (IGF-I) and insulin and plays an important role in embryonic development and carcinogenesis. IGF-II is believed to mediate its cellular signaling via the transmembrane tyrosine kinase type 1 insulin-like growth factor receptor (IGF-I-R), which is also the receptor for IGF-I. Earlier studies with both cultured cells and transgenic mice, however, have suggested that in the embryo the insulin receptor (IR) may also be a receptor for IGF-II. In most cells and tissues, IR binds IGF-II with relatively low affinity. The IR is expressed in two isoforms (IR-A and IR-B) differing by 12 amino acids due to the alternative splicing of exon 11. In the present study we found that IR-A but not IR-B bound IGF-II with an affinity close to that of insulin. Moreover, IGF-II bound to IR-A with an affinity equal to that of IGF-II binding to the IGF-I-R. Activation of IR-A by insulin led primarily to metabolic effects, whereas activation of IR-A by IGF-II led primarily to mitogenic effects. These differences in the biological effects of IR-A when activated by either IGF-II or insulin were associated with differential recruitment and activation of intracellular substrates. IR-A was preferentially expressed in fetal cells such as fetal fibroblasts, muscle, liver and kidney and had a relatively increased proportion of isoform A. IR-A expression was also increased in several tumors including those of the breast and colon. These data indicate, therefore, that there are two receptors for IGF-II, both IGF-I-R and IR-A. Further, they suggest that interaction of IGF-II with IR-A may play a role both in fetal growth and cancer biology.
Eur. J. Biochem. 250, 411-417 (1997)[PubMed:9428692]
We have screened a human placenta library using the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor. Doing so, we trapped a cDNA clone which encodes the Stat 5B region comprising amino acids 469 to 786. We show that interaction between Stat 5B and the receptor requires a functional insulin-receptor kinase, Tyr960 of insulin receptor is implicated in the interaction with Stat 5B, whereas asparagine and proline forming the NPEY960-motif are not, and Stat 5B mutated at Thr684, a potential phosphorylation site of mitogen-activated protein kinase, loses its ability to interact with the insulin receptor. Further, we found that insulin promotes rapid tyrosine phosphorylation of endogenous Stat 5B in 293 EBNA cells overexpressing insulin receptor and in NHIR cells. Taken together, our findings suggest that Stat 5B corresponds to a substrate for the insulin-receptor kinase, and this widens the repertoire of insulin-signaling pathways.
Biochem. J. 290 ( Pt 2), 419-426 (1993)[PubMed:8452530]
Hybrid insulin/insulin-like growth factor-I (IGF-I) receptors have previously been described in human placenta, but it has not been possible to study their properties in the presence of classical insulin receptors and type I IGF receptors. To facilitate the purification of hybrids, we produced an anti-peptide monoclonal antibody IGFR 1-2, directed against the C-terminal peptide of the type I IGF receptor beta-subunit. The antibody bound native human and rat type I IGF receptors, and reacted specifically with the beta-subunit on immunoblots. Solubilized placental microsomal membranes were depleted of classical type I IGF receptors by incubation with an immobilized monoclonal antibody IGFR 24-55, which reacts well with type I receptors but very poorly with hybrid receptors. Residual hybrid receptors were then isolated by incubation with immobilized antibody IGFR 1-2, and recovered by elution with excess of synthetic peptide antigen. Binding properties of hybrids were compared with those of immuno-affinity-purified insulin receptors and type I IGF receptors, by using the radioligands 125I-IGF-I and 125I-insulin. Hybrids bound approx. 20 times as much 125I-IGF-I as 125I-insulin at tracer concentrations (approx. 0.1 nM). The binding of 125I-insulin, but not 125I-IGF-I, to hybrids increased after treatment with dithiothreitol to reduce disulphide bonds between the alpha-subunits. Hybrids behaved very similarly to type I receptors with respect to the inhibition of 125I-IGF-I binding by unlabelled IGF-I and insulin. By contrast, the affinity of hybrids for insulin was approx. 10-fold lower than that of classical insulin receptors, as assessed by inhibition of 125I-insulin binding by unlabelled hormone. It is concluded that the properties of insulin receptors, but not IGF receptors, are markedly affected by assembly as hybrid compared with classical structures, and that hybrids are more likely to be responsive to IGF-I than insulin under physiological conditions.
Isoform
Short
Has a higher affinity for IGFII binding. When present in a hybrid receptor with IGF1R, binds IGF1. PubMed12138094 shows that hybrid receptors composed of IGF1R and INSR isoform Long are activated with a high affinity by IGF1, with low affinity by IGF2 and not significantly activated by insulin, and that hybrid receptors composed of IGF1R and INSR isoform Short are activated by IGF1, IGF2 and insulin. In contrast, PubMed16831875 shows that hybrid receptors composed of IGF1R and INSR isoform Long and hybrid receptors composed of IGF1R and INSR isoform Short have similar binding characteristics, both bind IGF1 and have a low affinity for insulin.
Protein microsequencing of human placental IGF-I receptors purified by immunoaffinity chromatography using IGF-I receptor specific monoclonal antibody revealed amino acid sequences of both IGF-I and insulin receptors. Since this finding indicated the presence of IGF-I/insulin receptor hybrids, hybrid receptors were further purified by immunoaffinity chromatography using insulin receptor specific monoclonal antibody. The molecular size of the nonreduced hybrid receptor was approximately 350K, indicating that the IGF-I and insulin receptor alpha beta halves were disulfide-linked. The ratio of IGF/insulin binding activity of purified hybrid receptors was approximately 25 when measured using tracer amounts of radioactive ligands. 125I-IGF binding to these receptors was inhibited by IGF-I and insulin with IC50s of approximately 2 and approximately 1000 nM, respectively. 125I-Insulin binding to these receptors was similarly inhibited by IGF-I and insulin with IC50 of approximately 3 nM. Autophosphorylation and kinase activities of the hybrid receptor were stimulated by IGF-I more effectively than insulin in a dose-dependent manner. Thus, the present studies indicate that hybrid receptors purified from human placenta have the functional properties of an IGF-I receptor.
Biochem. J. 290 ( Pt 2), 419-426 (1993)[PubMed:8452530]
Hybrid insulin/insulin-like growth factor-I (IGF-I) receptors have previously been described in human placenta, but it has not been possible to study their properties in the presence of classical insulin receptors and type I IGF receptors. To facilitate the purification of hybrids, we produced an anti-peptide monoclonal antibody IGFR 1-2, directed against the C-terminal peptide of the type I IGF receptor beta-subunit. The antibody bound native human and rat type I IGF receptors, and reacted specifically with the beta-subunit on immunoblots. Solubilized placental microsomal membranes were depleted of classical type I IGF receptors by incubation with an immobilized monoclonal antibody IGFR 24-55, which reacts well with type I receptors but very poorly with hybrid receptors. Residual hybrid receptors were then isolated by incubation with immobilized antibody IGFR 1-2, and recovered by elution with excess of synthetic peptide antigen. Binding properties of hybrids were compared with those of immuno-affinity-purified insulin receptors and type I IGF receptors, by using the radioligands 125I-IGF-I and 125I-insulin. Hybrids bound approx. 20 times as much 125I-IGF-I as 125I-insulin at tracer concentrations (approx. 0.1 nM). The binding of 125I-insulin, but not 125I-IGF-I, to hybrids increased after treatment with dithiothreitol to reduce disulphide bonds between the alpha-subunits. Hybrids behaved very similarly to type I receptors with respect to the inhibition of 125I-IGF-I binding by unlabelled IGF-I and insulin. By contrast, the affinity of hybrids for insulin was approx. 10-fold lower than that of classical insulin receptors, as assessed by inhibition of 125I-insulin binding by unlabelled hormone. It is concluded that the properties of insulin receptors, but not IGF receptors, are markedly affected by assembly as hybrid compared with classical structures, and that hybrids are more likely to be responsive to IGF-I than insulin under physiological conditions.
Eur. J. Biochem. 250, 411-417 (1997)[PubMed:9428692]
We have screened a human placenta library using the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor. Doing so, we trapped a cDNA clone which encodes the Stat 5B region comprising amino acids 469 to 786. We show that interaction between Stat 5B and the receptor requires a functional insulin-receptor kinase, Tyr960 of insulin receptor is implicated in the interaction with Stat 5B, whereas asparagine and proline forming the NPEY960-motif are not, and Stat 5B mutated at Thr684, a potential phosphorylation site of mitogen-activated protein kinase, loses its ability to interact with the insulin receptor. Further, we found that insulin promotes rapid tyrosine phosphorylation of endogenous Stat 5B in 293 EBNA cells overexpressing insulin receptor and in NHIR cells. Taken together, our findings suggest that Stat 5B corresponds to a substrate for the insulin-receptor kinase, and this widens the repertoire of insulin-signaling pathways.
Insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) are both from the same subgroup of receptor tyrosine kinases that exist as covalently bound receptor dimers at the cell surface. For both IR and IGF-IR, the most described forms are homodimer receptors. However, hybrid receptors consisting of one-half IR and one-half IGF-IR are also present at the cell surface. Two splice variants of IR are expressed that enable formation of two isoforms of the IGF-IR/IR hybrid receptor. In this study, these two splice variants of hybrid receptors were studied with respect to binding affinities of insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II). Unlike previously published data, in which semipurified receptors have been studied, we found that the two hybrid receptor splice variants had similar binding characteristics with respect to insulin, IGF-I, and IGF-II binding. We studied both semipurified and purified hybrid receptors. In all cases we found that IGF-I had at least 50-fold higher affinity than insulin, irrespective of the splice variant. The binding characteristics of insulin and IGF-I to both splice variants of the hybrid receptors were similar to classical homodimer IGF-IR.
Insulin-like growth factor II (IGF-II) is a peptide growth factor that is homologous to both insulin-like growth factor I (IGF-I) and insulin and plays an important role in embryonic development and carcinogenesis. IGF-II is believed to mediate its cellular signaling via the transmembrane tyrosine kinase type 1 insulin-like growth factor receptor (IGF-I-R), which is also the receptor for IGF-I. Earlier studies with both cultured cells and transgenic mice, however, have suggested that in the embryo the insulin receptor (IR) may also be a receptor for IGF-II. In most cells and tissues, IR binds IGF-II with relatively low affinity. The IR is expressed in two isoforms (IR-A and IR-B) differing by 12 amino acids due to the alternative splicing of exon 11. In the present study we found that IR-A but not IR-B bound IGF-II with an affinity close to that of insulin. Moreover, IGF-II bound to IR-A with an affinity equal to that of IGF-II binding to the IGF-I-R. Activation of IR-A by insulin led primarily to metabolic effects, whereas activation of IR-A by IGF-II led primarily to mitogenic effects. These differences in the biological effects of IR-A when activated by either IGF-II or insulin were associated with differential recruitment and activation of intracellular substrates. IR-A was preferentially expressed in fetal cells such as fetal fibroblasts, muscle, liver and kidney and had a relatively increased proportion of isoform A. IR-A expression was also increased in several tumors including those of the breast and colon. These data indicate, therefore, that there are two receptors for IGF-II, both IGF-I-R and IR-A. Further, they suggest that interaction of IGF-II with IR-A may play a role both in fetal growth and cancer biology.
The insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) have a highly homologous structure, but different biological effects. Insulin and IGF-I half-receptors can heterodimerize, leading to the formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) that bind IGF-I with high affinity. As the IR exists in two isoforms (IR-A and IR-B), we evaluated whether the assembly of the IGF-IR with either IR-A or IR-B moieties may differently affect Hybrid-R signaling and biological role. Three different models were studied: (a) 3T3-like mouse fibroblasts with a disrupted IGF-IR gene (R(-) cells) cotransfected with the human IGF-IR and with either the IR-A or IR-B cDNA; (b) a panel of human cell lines variably expressing the two IR isoforms; and (c) HepG2 human hepatoblastoma cells predominantly expressing either IR-A or IR-B, depending on their differentiation state. We found that Hybrid-Rs containing IR-A (Hybrid-Rs(A)) bound to and were activated by IGF-I, IGF-II, and insulin. By binding to Hybrid-Rs(A), insulin activated the IGF-I half-receptor beta-subunit and the IGF-IR-specific substrate CrkII. In contrast, Hybrid-Rs(B) bound to and were activated with high affinity by IGF-I, with low affinity by IGF-II, and insignificantly by insulin. As a consequence, cell proliferation and migration in response to both insulin and IGFs were more effectively stimulated in Hybrid-R(A)-containing cells than in Hybrid-R(B)-containing cells. The relative abundance of IR isoforms therefore affects IGF system activation through Hybrid-Rs, with important consequences for tissue-specific responses to both insulin and IGFs.
In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IR(Delta43)) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IR(Delta43) (L6(IRDelta43)) also showed no insulin effect on glucose uptake and glycogen synthase, accompanied by a >80% decrease in insulin induction of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) activity and tyrosine phosphorylation and of protein kinase B (PKB) phosphorylation at Thr(308). Insulin induced the phosphatidylinositol 3 kinase-dependent coprecipitation of PDK-1 with wild-type IR (IR(WT)), but not IR(Delta43). Based on overlay blotting, PDK-1 directly bound IR(WT), but not IR(Delta43). Insulin-activated IR(WT), and not IR(Delta43), phosphorylated PDK-1 at tyrosines 9, 373, and 376. The IR C-terminal 43-amino-acid peptide (C-terminal peptide) inhibited in vitro PDK-1 tyrosine phosphorylation by the IR. Tyr-->Phe substitution prevented this inhibitory action. In the L6(hIR) cells, the C-terminal peptide coprecipitated with PDK-1 in an insulin-stimulated fashion. This peptide simultaneously impaired the insulin effect on PDK-1 coprecipitation with IR(WT), on PDK-1 tyrosine phosphorylation, on PKB phosphorylation at Thr(308), and on glucose uptake. Upon insulin exposure, PDK-1 membrane persistence was significantly reduced in L6(IRDelta43) compared to control cells. In L6 cells expressing IR(WT), the C-terminal peptide also impaired insulin-dependent PDK-1 membrane persistence. Thus, PDK-1 directly binds to the insulin receptor, followed by PDK-1 activation and insulin metabolic effects.
We have previously shown that phosphatidylinositol (PtdIns) 3'-kinase is activated by the binding of proteins or peptides containing the phosphorylated motif Y(P)XXM. In the present study, we examine interactions between PtdIns 3'-kinase and the human insulin receptor, which contains a C-terminal phosphorylation site in the sequence Y1322THM. Partially purified insulin receptors bound tightly to bacterial fusion proteins containing the N- or C-terminal SH2 domains from PtdIns 3'-kinase regulatory subunit (p85). In contrast, a mutant insulin receptor, truncated by 43 amino acids at the C terminus (IR delta CT), bound poorly to the SH2 domains; these mutant receptors have normal kinase activity but lack the Y1322THM motif. Similarly, incubation with wild-type receptors increased the activity of immunopurified PtdIns 3'-kinase, whereas incubation with IR delta CT receptors did not affect PtdIns 3'-kinase activity. Activation of PtdIns 3'-kinase by the wild-type receptor was mimicked by a tyrosyl phosphopeptide derived from the insulin receptor C terminus and containing the Y1322THM motif; non-phosphorylated peptide did not affect activity. Thus, the insulin receptor C terminus activates PtdIns 3'-kinase in vitro by binding to the SH2 domains of the 85-kDa regulatory subunit. These data support the hypothesis that binding of tyrosyl-phosphorylated receptors to p85 SH2 domains is a general mechanism for PtdIns 3'-kinase activation, and they suggest that direct interactions between the insulin receptor and PtdIns 3'-kinase may provide an alternative pathway for the activation of this enzyme by insulin.
Highly purified preparations of insulin receptor catalyzed the phosphorylation of the 95,000-dalton subunit of the insulin receptor. This subunit of the insulin receptor was also labeled with [alpha-32P]8-azidoadenosine 5'-triphosphate, a photoaffinity label for adenosine triphosphate binding sites. The identity of the 95,000-dalton band was confirmed in both cases by precipitation with a monoclonal antibody to the insulin receptor. These results suggest that the insulin receptor is itself a protein kinase.
J. Biol. Chem. 272, 10135-10143 (1997)[PubMed:9092559]
Human fat cells possess a multireceptor-linked H2O2-generating system that is activated by insulin. Previous studies revealed that manganese was the sole cofactor required for a hormonal regulation of NADPH-dependent H2O2 generation in vitro. In this report it is shown that the synergistic activation of NADPH-dependent H2O2 generation by Mn2+ and insulin was blocked by GDPbetaS (guanosine 5'-O-(2-thiodiphosphate)), pertussis toxin and COOH-terminal anti-Galphai1-2 or the corresponding peptide. Consistently, manganese could be replaced by micromolar concentrations of GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), which increased NADPH-dependent H2O2 generation by 20-40%. Insulin shifted the dose response curve for GTPgammaS to the left (>10-fold) and increased the maximal response. In the presence of 10 microM GTPgammaS, the hormone was active at picomolar concentrations, indicating that insulin acted via its cognate receptor. The insulin receptor and Gi were co-adsorbed on anti-Galphai and anti-insulin receptor beta-subunit (anti-IRbeta) affinity columns. Partially purified insulin receptor preparations contained Galphas, Galphai2, and Gbetagamma (but no Galphai1 or Galphai3). The functional nature of the insulin receptor-Gi2 complex was made evident by insulin's ability to modulate labeling of Gi by bacterial toxins. Insulin action was mimicked by activated Galphai, but not by Galphao or Gbetagamma, indicating that insulin's signal was transduced via Galphai2. Thus, NADPH oxidase is the first example of an effector system that is coupled to the insulin receptor via a heterotrimeric G protein.
Interacting selectively and non-covalently with insulin, a polypeptide hormone produced by the islets of Langerhans of the pancreas in mammals, and by the homologous organs of other organisms.
Tissue-specific alternative splicing of exon 11 of the insulin receptor gene results in 2 mRNAs that differ by 36 nucleotides within the coding region. The 2 transcripts encode 2 protein isoforms with (Ex11+) or without (Ex11-) 12 additional amino acids at the carboxy-terminus of the receptor alpha-subunit. Previous studies of the 2 isoforms of the human insulin receptor expressed in mammalian cell transfectants have revealed small functional differences at the levels of equilibrium insulin binding affinity and acute ligand-induced receptor internalization. In the present study, we determined the biochemical basis for differential insulin binding affinity. Further functional characterization of the 2 receptor isoforms was also performed. The results obtained were as follows. 1) Studies of ligand association demonstrated a faster (1.8-fold) "on rate" for Ex11- receptors than for Ex11+ receptors, as determined by the kinetics of [125I]insulin binding to transfected cells. In addition, dissociation of prebound [125I]insulin from Ex11- receptors was characterized by an accelerated "off rate" relative to that of Ex11+ receptors. 2) Using both intact Chinese hamster ovary (CHO) cells and partially purified solubilized insulin receptors, the ability of insulin-like growth factor-I to compete for [125I]insulin binding to either isoform differed markedly. The mean IC50 for Ex11- was 40 nM vs. 350 nM for Ex11+. 3) Both Ex11- and Ex11+ receptors were equally capable of hybrid formation with endogenous CHO cell insulin-like growth factor-I receptors. 4) The relative abilities of 2 inhibitory polyclonal antiinsulin receptor antisera to displace [125I]insulin binding did not differ between the two isoforms. 5) Studies of insulin-induced (300 nM) receptor down-regulation in CHO cell transfectants suggested preferential down-regulation of Ex11- receptors; however, no down-regulation difference was observed when Rat 1 cell transfectants expressing the two splice variants were studied. These findings further support the idea that the 2 isoforms of the insulin receptor are functionally distinct in important ways.
Evidence
2:
Inferred from Physical InteractionBHF-UCL
Biochem. J. 290 ( Pt 2), 419-426 (1993)[PubMed:8452530]
Hybrid insulin/insulin-like growth factor-I (IGF-I) receptors have previously been described in human placenta, but it has not been possible to study their properties in the presence of classical insulin receptors and type I IGF receptors. To facilitate the purification of hybrids, we produced an anti-peptide monoclonal antibody IGFR 1-2, directed against the C-terminal peptide of the type I IGF receptor beta-subunit. The antibody bound native human and rat type I IGF receptors, and reacted specifically with the beta-subunit on immunoblots. Solubilized placental microsomal membranes were depleted of classical type I IGF receptors by incubation with an immobilized monoclonal antibody IGFR 24-55, which reacts well with type I receptors but very poorly with hybrid receptors. Residual hybrid receptors were then isolated by incubation with immobilized antibody IGFR 1-2, and recovered by elution with excess of synthetic peptide antigen. Binding properties of hybrids were compared with those of immuno-affinity-purified insulin receptors and type I IGF receptors, by using the radioligands 125I-IGF-I and 125I-insulin. Hybrids bound approx. 20 times as much 125I-IGF-I as 125I-insulin at tracer concentrations (approx. 0.1 nM). The binding of 125I-insulin, but not 125I-IGF-I, to hybrids increased after treatment with dithiothreitol to reduce disulphide bonds between the alpha-subunits. Hybrids behaved very similarly to type I receptors with respect to the inhibition of 125I-IGF-I binding by unlabelled IGF-I and insulin. By contrast, the affinity of hybrids for insulin was approx. 10-fold lower than that of classical insulin receptors, as assessed by inhibition of 125I-insulin binding by unlabelled hormone. It is concluded that the properties of insulin receptors, but not IGF receptors, are markedly affected by assembly as hybrid compared with classical structures, and that hybrids are more likely to be responsive to IGF-I than insulin under physiological conditions.
Interacting selectively and non-covalently with any of the insulin receptor substrate (IRS) proteins, adaptor proteins that bind to the transphosphorylated insulin and insulin-like growth factor receptors, are themselves phosphorylated and in turn recruit SH2 domain-containing signaling molecules to form a productive signaling complex.
Evidence
1:
Inferred from Physical InteractionUniProtKB
J. Biol. Chem. 270, 23258-23262 (1995)[PubMed:7559478]
Insulin receptor substrate 1 (IRS-1) and src homology and collagen protein (SHC) are signaling proteins which are rapidly phosphorylated on tyrosines after insulin receptor (IR) activation. We have recently shown that both SHC and IRS-1 interact with the tyrosine-phosphorylated NPEY motif of the IR and insulin-like growth factor I receptor via non-SH2 domains (Gustafson, T. A., He, W., Craparo, A., Schaub, C. D., and O'Neill, T. J. (1995) Mol. Cell. Biol. 15, 2500-2508; O'Neill, T. J., Craparo, A., and Gustafson, T. A. (1994) Mol. Cell. Biol. 14, 6433-6442; Craparo, A., O'Neill, T. J., and Gustafson, T. A. (1995) J. Biol. Chem. 270, 15639-15643). In this study we characterize these interactions by examining the effects of 18 amino acid substitutions within and around the IR NPEY motif upon interaction with SHC and IRS-1. We confirm that Tyr-960 within the NPEY motif of the IR is essential for both IRS-1 and SHC interaction and that Asn-957 and Pro-958 are essential for IRS-1 interaction and important but not critical for SHC interaction. Additional mutations surrounding the NPEY motif revealed completely distinct patterns of interaction for SHC and IRS-1. Specifically, mutation of Leu-952 or Tyr-953 (at positions -7 and -8 from Tyr-960) markedly reduced IRS-1 interaction but had no effect upon SHC interaction. Likewise, mutation of Ala-963 (+3) reduced IRS-1 but not SHC interaction. Conversely, substitution of Leu-961 (+1) with either Ala or Arg reduced SHC interaction by 70 and 90%, respectively, yet had no effect upon interaction with IRS-1. Our data show that the sequences within and surrounding the NPEY contribute differentially to either SHC or IRS-1 recognition. Our findings suggest mechanisms by which the differential interaction of known receptors with IRS-1 and SHC may be mediated.
Evidence
2:
Inferred from Physical InteractionUniProtKB
The SHC proteins have been implicated in insulin receptor (IR) signaling. In this study, we used the sensitive two-hybrid assay of protein-protein interaction to demonstrate that SHC interacts directly with the IR. The interaction is mediated by SHC amino acids 1 to 238 and is therefore independent of the Src homology 2 domain. The interaction is dependent upon IR autophosphorylation, since the interaction is eliminated by mutation of the IR ATP-binding site. In addition, mutational analysis of the Asn-Pro-Glu-Tyr (NPEY) motif within the juxtamembrane domain of the IR showed the importance of the Asn, Pro, and Tyr residues to both SHC and IR substrate 1 (IRS-1) binding. We conclude that SHC interacts directly with the IR and that phosphorylation of Tyr-960 within the IR juxtamembrane domain is necessary for efficient interaction. This interaction is highly reminiscent of that of IRS-1 with the IR, and we show that the SHC IR-binding domain can substitute for that of IRS-1 in yeast and COS cells. We identify a homologous region within the IR-binding domains of SHC and IRS-1, which we term the SAIN (SHC and IRS-1 NPXY-binding) domain, which may explain the basis of these interactions. The SAIN domain appears to represent a novel motif which is able to interact with autophosphorylated receptors such as the IR.
Evidence
3:
Inferred from Physical InteractionBHF-UCL
J. Biol. Chem. 270, 29189-29193 (1995)[PubMed:7493946]
Insulin signal transduction involves the multisite docking protein insulin receptor substrate-1 (IRS-1) and a number of Src homology-2 (SH2) domain factors, including p85/p110 phosphatidylinositol 3-kinase, p110 GTPase-activating protein, and the phosphotyrosine-specific phosphatase PTP1D. In transfected baby hamster kidney cells, Rat1 fibroblasts, and normal IM9 lymphoblasts, PTP1D directly binds activated insulin receptor. This interaction is mediated by catalytic domain-proximal SH2 determinants of the phosphatase and phosphotyrosine 1146 of the activated insulin receptor. While the receptor and the phosphatase do not serve as substrates for each other, their interaction promotes IRS-1 binding to the receptor, indicating that PTP1D functions as an adapter for insulin receptor and IRS-1. The formation of a multiprotein signaling complex involving the insulin receptor, PTP1D, and IRS-1 enhances cellular glucose uptake, a critical process in the physiological action of insulin.
Highly purified preparations of insulin receptor catalyzed the phosphorylation of the 95,000-dalton subunit of the insulin receptor. This subunit of the insulin receptor was also labeled with [alpha-32P]8-azidoadenosine 5'-triphosphate, a photoaffinity label for adenosine triphosphate binding sites. The identity of the 95,000-dalton band was confirmed in both cases by precipitation with a monoclonal antibody to the insulin receptor. These results suggest that the insulin receptor is itself a protein kinase.
Tissue-specific alternative splicing of exon 11 of the insulin receptor gene results in 2 mRNAs that differ by 36 nucleotides within the coding region. The 2 transcripts encode 2 protein isoforms with (Ex11+) or without (Ex11-) 12 additional amino acids at the carboxy-terminus of the receptor alpha-subunit. Previous studies of the 2 isoforms of the human insulin receptor expressed in mammalian cell transfectants have revealed small functional differences at the levels of equilibrium insulin binding affinity and acute ligand-induced receptor internalization. In the present study, we determined the biochemical basis for differential insulin binding affinity. Further functional characterization of the 2 receptor isoforms was also performed. The results obtained were as follows. 1) Studies of ligand association demonstrated a faster (1.8-fold) "on rate" for Ex11- receptors than for Ex11+ receptors, as determined by the kinetics of [125I]insulin binding to transfected cells. In addition, dissociation of prebound [125I]insulin from Ex11- receptors was characterized by an accelerated "off rate" relative to that of Ex11+ receptors. 2) Using both intact Chinese hamster ovary (CHO) cells and partially purified solubilized insulin receptors, the ability of insulin-like growth factor-I to compete for [125I]insulin binding to either isoform differed markedly. The mean IC50 for Ex11- was 40 nM vs. 350 nM for Ex11+. 3) Both Ex11- and Ex11+ receptors were equally capable of hybrid formation with endogenous CHO cell insulin-like growth factor-I receptors. 4) The relative abilities of 2 inhibitory polyclonal antiinsulin receptor antisera to displace [125I]insulin binding did not differ between the two isoforms. 5) Studies of insulin-induced (300 nM) receptor down-regulation in CHO cell transfectants suggested preferential down-regulation of Ex11- receptors; however, no down-regulation difference was observed when Rat 1 cell transfectants expressing the two splice variants were studied. These findings further support the idea that the 2 isoforms of the insulin receptor are functionally distinct in important ways.
Biochem. J. 290 ( Pt 2), 419-426 (1993)[PubMed:8452530]
Hybrid insulin/insulin-like growth factor-I (IGF-I) receptors have previously been described in human placenta, but it has not been possible to study their properties in the presence of classical insulin receptors and type I IGF receptors. To facilitate the purification of hybrids, we produced an anti-peptide monoclonal antibody IGFR 1-2, directed against the C-terminal peptide of the type I IGF receptor beta-subunit. The antibody bound native human and rat type I IGF receptors, and reacted specifically with the beta-subunit on immunoblots. Solubilized placental microsomal membranes were depleted of classical type I IGF receptors by incubation with an immobilized monoclonal antibody IGFR 24-55, which reacts well with type I receptors but very poorly with hybrid receptors. Residual hybrid receptors were then isolated by incubation with immobilized antibody IGFR 1-2, and recovered by elution with excess of synthetic peptide antigen. Binding properties of hybrids were compared with those of immuno-affinity-purified insulin receptors and type I IGF receptors, by using the radioligands 125I-IGF-I and 125I-insulin. Hybrids bound approx. 20 times as much 125I-IGF-I as 125I-insulin at tracer concentrations (approx. 0.1 nM). The binding of 125I-insulin, but not 125I-IGF-I, to hybrids increased after treatment with dithiothreitol to reduce disulphide bonds between the alpha-subunits. Hybrids behaved very similarly to type I receptors with respect to the inhibition of 125I-IGF-I binding by unlabelled IGF-I and insulin. By contrast, the affinity of hybrids for insulin was approx. 10-fold lower than that of classical insulin receptors, as assessed by inhibition of 125I-insulin binding by unlabelled hormone. It is concluded that the properties of insulin receptors, but not IGF receptors, are markedly affected by assembly as hybrid compared with classical structures, and that hybrids are more likely to be responsive to IGF-I than insulin under physiological conditions.
The insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) have a highly homologous structure, but different biological effects. Insulin and IGF-I half-receptors can heterodimerize, leading to the formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) that bind IGF-I with high affinity. As the IR exists in two isoforms (IR-A and IR-B), we evaluated whether the assembly of the IGF-IR with either IR-A or IR-B moieties may differently affect Hybrid-R signaling and biological role. Three different models were studied: (a) 3T3-like mouse fibroblasts with a disrupted IGF-IR gene (R(-) cells) cotransfected with the human IGF-IR and with either the IR-A or IR-B cDNA; (b) a panel of human cell lines variably expressing the two IR isoforms; and (c) HepG2 human hepatoblastoma cells predominantly expressing either IR-A or IR-B, depending on their differentiation state. We found that Hybrid-Rs containing IR-A (Hybrid-Rs(A)) bound to and were activated by IGF-I, IGF-II, and insulin. By binding to Hybrid-Rs(A), insulin activated the IGF-I half-receptor beta-subunit and the IGF-IR-specific substrate CrkII. In contrast, Hybrid-Rs(B) bound to and were activated with high affinity by IGF-I, with low affinity by IGF-II, and insignificantly by insulin. As a consequence, cell proliferation and migration in response to both insulin and IGFs were more effectively stimulated in Hybrid-R(A)-containing cells than in Hybrid-R(B)-containing cells. The relative abundance of IR isoforms therefore affects IGF system activation through Hybrid-Rs, with important consequences for tissue-specific responses to both insulin and IGFs.
Biochem. J. 290 ( Pt 2), 419-426 (1993)[PubMed:8452530]
Hybrid insulin/insulin-like growth factor-I (IGF-I) receptors have previously been described in human placenta, but it has not been possible to study their properties in the presence of classical insulin receptors and type I IGF receptors. To facilitate the purification of hybrids, we produced an anti-peptide monoclonal antibody IGFR 1-2, directed against the C-terminal peptide of the type I IGF receptor beta-subunit. The antibody bound native human and rat type I IGF receptors, and reacted specifically with the beta-subunit on immunoblots. Solubilized placental microsomal membranes were depleted of classical type I IGF receptors by incubation with an immobilized monoclonal antibody IGFR 24-55, which reacts well with type I receptors but very poorly with hybrid receptors. Residual hybrid receptors were then isolated by incubation with immobilized antibody IGFR 1-2, and recovered by elution with excess of synthetic peptide antigen. Binding properties of hybrids were compared with those of immuno-affinity-purified insulin receptors and type I IGF receptors, by using the radioligands 125I-IGF-I and 125I-insulin. Hybrids bound approx. 20 times as much 125I-IGF-I as 125I-insulin at tracer concentrations (approx. 0.1 nM). The binding of 125I-insulin, but not 125I-IGF-I, to hybrids increased after treatment with dithiothreitol to reduce disulphide bonds between the alpha-subunits. Hybrids behaved very similarly to type I receptors with respect to the inhibition of 125I-IGF-I binding by unlabelled IGF-I and insulin. By contrast, the affinity of hybrids for insulin was approx. 10-fold lower than that of classical insulin receptors, as assessed by inhibition of 125I-insulin binding by unlabelled hormone. It is concluded that the properties of insulin receptors, but not IGF receptors, are markedly affected by assembly as hybrid compared with classical structures, and that hybrids are more likely to be responsive to IGF-I than insulin under physiological conditions.
Interacting selectively and non-covalently with a phosphatidylinositol 3-kinase, any enzyme that catalyzes the addition of a phosphate group to an inositol lipid at the 3' position of the inositol ring.
Evidence
1:
Inferred from Physical InteractionUniProtKB
The SHC proteins have been implicated in insulin receptor (IR) signaling. In this study, we used the sensitive two-hybrid assay of protein-protein interaction to demonstrate that SHC interacts directly with the IR. The interaction is mediated by SHC amino acids 1 to 238 and is therefore independent of the Src homology 2 domain. The interaction is dependent upon IR autophosphorylation, since the interaction is eliminated by mutation of the IR ATP-binding site. In addition, mutational analysis of the Asn-Pro-Glu-Tyr (NPEY) motif within the juxtamembrane domain of the IR showed the importance of the Asn, Pro, and Tyr residues to both SHC and IR substrate 1 (IRS-1) binding. We conclude that SHC interacts directly with the IR and that phosphorylation of Tyr-960 within the IR juxtamembrane domain is necessary for efficient interaction. This interaction is highly reminiscent of that of IRS-1 with the IR, and we show that the SHC IR-binding domain can substitute for that of IRS-1 in yeast and COS cells. We identify a homologous region within the IR-binding domains of SHC and IRS-1, which we term the SAIN (SHC and IRS-1 NPXY-binding) domain, which may explain the basis of these interactions. The SAIN domain appears to represent a novel motif which is able to interact with autophosphorylated receptors such as the IR.
Evidence
2:
Inferred from Physical InteractionUniProtKB
We have previously shown that phosphatidylinositol (PtdIns) 3'-kinase is activated by the binding of proteins or peptides containing the phosphorylated motif Y(P)XXM. In the present study, we examine interactions between PtdIns 3'-kinase and the human insulin receptor, which contains a C-terminal phosphorylation site in the sequence Y1322THM. Partially purified insulin receptors bound tightly to bacterial fusion proteins containing the N- or C-terminal SH2 domains from PtdIns 3'-kinase regulatory subunit (p85). In contrast, a mutant insulin receptor, truncated by 43 amino acids at the C terminus (IR delta CT), bound poorly to the SH2 domains; these mutant receptors have normal kinase activity but lack the Y1322THM motif. Similarly, incubation with wild-type receptors increased the activity of immunopurified PtdIns 3'-kinase, whereas incubation with IR delta CT receptors did not affect PtdIns 3'-kinase activity. Activation of PtdIns 3'-kinase by the wild-type receptor was mimicked by a tyrosyl phosphopeptide derived from the insulin receptor C terminus and containing the Y1322THM motif; non-phosphorylated peptide did not affect activity. Thus, the insulin receptor C terminus activates PtdIns 3'-kinase in vitro by binding to the SH2 domains of the 85-kDa regulatory subunit. These data support the hypothesis that binding of tyrosyl-phosphorylated receptors to p85 SH2 domains is a general mechanism for PtdIns 3'-kinase activation, and they suggest that direct interactions between the insulin receptor and PtdIns 3'-kinase may provide an alternative pathway for the activation of this enzyme by insulin.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionUniProtKB
SH3P12/CAP/ponsin, a gene product with a sorbin homology domain and three consecutive SH3 domains in the carboxy-terminus, has been isolated from murine adipocytes and identified as an important adaptor during insulin signaling. Here we describe the cloning, mapping, and expression of the human homologue, termed SORBS1 (sorbin and SH3 domain containing 1). Multiple transcripts of this gene with different mRNA isoforms were observed among different tissues. Here we report 13 alternatively spliced exons, which were ascertained from the full-length cDNA cloned in adipose, liver, and skeletal muscle tissues. Among the major isoforms, the shortest, 2223-bp, open reading frame (ORF) encodes a protein with a predicted molecular weight of 81.5 kDa, while the longest, 3879-bp, ORF encodes a protein of about 142.2 kDa. This gene was mapped to human chromosome 10q23.3-q24.1, which is a candidate region for insulin resistance found in Pima Indians. In human hepatoma Hep3B cells, SORBS1 was partly dissociated from the insulin receptor complex and bound to c-Abl protein upon insulin stimulation. This interaction with c-Abl was through the third SH3 domain and a possible conformational change of SORBS1 induced by insulin. Our data suggest that c-Abl oncoprotein via SORBS1 might play a role in the insulin signaling pathway.
Evidence
2:
Inferred from Physical InteractionBHF-UCL
When overexpressed, the membrane glycoprotein PC-1 may play a role in human insulin resistance through the inhibition of insulin receptor (IR) autophosphorylation. A PC-1 variant (K121Q, with lysine 121 replaced by glutamine) is also associated with whole-body insulin resistance when not overexpressed. To better understand the effects of the Q allele on IR function and downstream signaling, we transfected cultured cells with cDNAs for either the Q or the K alleles. In human MCF-7 cells, the Q allele was severalfold more effective (P < 0.05-0.01) than the K allele in reducing insulin stimulation of IR autophosphorylation, insulin receptor substrate-1 phosphorylation, phosphatidylinositol 3-kinase activity, glycogen synthesis, and cell proliferation. Similar data on IR autophosphorylation inhibition were also obtained in mouse R-/hIR and human HEK 293 cell lines. In transfected MCF-7 cells, 125I-labeled insulin binding and IR content were unchanged, and PC-1 overexpression did not influence IGF-1 stimulation of IGF-1 receptor autophosphorylation. Both the Q and K alleles directly interacted with the IR, as documented by coimmunoprecipitation assays. This interaction was greater for the Q allele than for the K allele (P < 0.01), suggesting that direct PC-1-IR interactions are important for the PC-1 inhibitory effect on insulin signaling. In conclusion, the Q allele has stronger inhibitory activity on IR function and insulin action than the more common K allele, and this is likely a consequence of the intrinsic characteristics of the molecule, which more strongly interacts with the IR.
Evidence
3:
Inferred from Physical InteractionUniProtKB
The SHC proteins have been implicated in insulin receptor (IR) signaling. In this study, we used the sensitive two-hybrid assay of protein-protein interaction to demonstrate that SHC interacts directly with the IR. The interaction is mediated by SHC amino acids 1 to 238 and is therefore independent of the Src homology 2 domain. The interaction is dependent upon IR autophosphorylation, since the interaction is eliminated by mutation of the IR ATP-binding site. In addition, mutational analysis of the Asn-Pro-Glu-Tyr (NPEY) motif within the juxtamembrane domain of the IR showed the importance of the Asn, Pro, and Tyr residues to both SHC and IR substrate 1 (IRS-1) binding. We conclude that SHC interacts directly with the IR and that phosphorylation of Tyr-960 within the IR juxtamembrane domain is necessary for efficient interaction. This interaction is highly reminiscent of that of IRS-1 with the IR, and we show that the SHC IR-binding domain can substitute for that of IRS-1 in yeast and COS cells. We identify a homologous region within the IR-binding domains of SHC and IRS-1, which we term the SAIN (SHC and IRS-1 NPXY-binding) domain, which may explain the basis of these interactions. The SAIN domain appears to represent a novel motif which is able to interact with autophosphorylated receptors such as the IR.
Evidence
4:
Inferred from Physical InteractionBHF-UCL
Evidence for Short
J. Biol. Chem. 270, 29189-29193 (1995)[PubMed:7493946]
Insulin signal transduction involves the multisite docking protein insulin receptor substrate-1 (IRS-1) and a number of Src homology-2 (SH2) domain factors, including p85/p110 phosphatidylinositol 3-kinase, p110 GTPase-activating protein, and the phosphotyrosine-specific phosphatase PTP1D. In transfected baby hamster kidney cells, Rat1 fibroblasts, and normal IM9 lymphoblasts, PTP1D directly binds activated insulin receptor. This interaction is mediated by catalytic domain-proximal SH2 determinants of the phosphatase and phosphotyrosine 1146 of the activated insulin receptor. While the receptor and the phosphatase do not serve as substrates for each other, their interaction promotes IRS-1 binding to the receptor, indicating that PTP1D functions as an adapter for insulin receptor and IRS-1. The formation of a multiprotein signaling complex involving the insulin receptor, PTP1D, and IRS-1 enhances cellular glucose uptake, a critical process in the physiological action of insulin.
Evidence
5:
Inferred from Physical InteractionUniProtKB
J. Biol. Chem. 270, 23258-23262 (1995)[PubMed:7559478]
Insulin receptor substrate 1 (IRS-1) and src homology and collagen protein (SHC) are signaling proteins which are rapidly phosphorylated on tyrosines after insulin receptor (IR) activation. We have recently shown that both SHC and IRS-1 interact with the tyrosine-phosphorylated NPEY motif of the IR and insulin-like growth factor I receptor via non-SH2 domains (Gustafson, T. A., He, W., Craparo, A., Schaub, C. D., and O'Neill, T. J. (1995) Mol. Cell. Biol. 15, 2500-2508; O'Neill, T. J., Craparo, A., and Gustafson, T. A. (1994) Mol. Cell. Biol. 14, 6433-6442; Craparo, A., O'Neill, T. J., and Gustafson, T. A. (1995) J. Biol. Chem. 270, 15639-15643). In this study we characterize these interactions by examining the effects of 18 amino acid substitutions within and around the IR NPEY motif upon interaction with SHC and IRS-1. We confirm that Tyr-960 within the NPEY motif of the IR is essential for both IRS-1 and SHC interaction and that Asn-957 and Pro-958 are essential for IRS-1 interaction and important but not critical for SHC interaction. Additional mutations surrounding the NPEY motif revealed completely distinct patterns of interaction for SHC and IRS-1. Specifically, mutation of Leu-952 or Tyr-953 (at positions -7 and -8 from Tyr-960) markedly reduced IRS-1 interaction but had no effect upon SHC interaction. Likewise, mutation of Ala-963 (+3) reduced IRS-1 but not SHC interaction. Conversely, substitution of Leu-961 (+1) with either Ala or Arg reduced SHC interaction by 70 and 90%, respectively, yet had no effect upon interaction with IRS-1. Our data show that the sequences within and surrounding the NPEY contribute differentially to either SHC or IRS-1 recognition. Our findings suggest mechanisms by which the differential interaction of known receptors with IRS-1 and SHC may be mediated.
Evidence
6:
Inferred from Physical InteractionHGNC
Sorting nexins (SNX) comprise a family of proteins with homology to several yeast proteins, including Vps5p and Mvp1p, that are required for the sorting of proteins to the yeast vacuole. Human SNX1, -2, and -4 have been proposed to play a role in receptor trafficking and have been shown to bind to several receptor tyrosine kinases, including receptors for epidermal growth factor, platelet-derived growth factor, and insulin as well as the long form of the leptin receptor, a glycoprotein 130-associated receptor. We now describe a novel member of this family, SNX6, which interacts with members of the transforming growth factor-beta family of receptor serine-threonine kinases. These receptors belong to two classes: type II receptors that bind ligand, and type I receptors that are subsequently recruited to transduce the signal. Of the type II receptors, SNX6 was found to interact strongly with ActRIIB and more moderately with wild type and kinase-defective mutants of TbetaRII. Of the type I receptors, SNX6 was found to interact only with inactivated TbetaRI. SNXs 1-4 also interacted with the transforming growth factor-beta receptor family, showing different receptor preferences. Conversely, SNX6 behaved similarly to the other SNX proteins in its interactions with receptor tyrosine kinases. Strong heteromeric interactions were also seen among SNX1, -2, -4, and -6, suggesting the formation in vivo of oligomeric complexes. These findings are the first evidence for the association of the SNX family of molecules with receptor serine-threonine kinases.
J. Biol. Chem. 268, 11256-11264 (1993)[PubMed:8496180]
Insulin-stimulated autophosphorylation of the cytoplasmic juxtamembrane region of the human insulin receptor was examined by Tricine/SDS-PAGE. Various mutant receptor molecules were used to identify two tryptic phosphopeptides associated with the juxtamembrane region which accounts for 15% of the autophosphorylation of partially purified insulin receptor. These phosphopeptides were immunoprecipitated with an antipeptide antibody against the juxtamembrane sequence and were phosphorylated exclusively on tyrosine. Substitution of both Tyr960 and Tyr953 with alanine eliminated insulin-stimulated phosphorylation of the juxtamembrane region without affecting tyrosine autophosphorylation in the C terminus or regulatory regions. Monosubstitution of Tyr960 with phenylalanine or alanine reduced phosphorylation in the juxtamembrane region by more than 50%, and manual Edman degradation indicated that Tyr960 was phosphorylated in wild-type receptor. In vivo, phosphorylation of the juxtamembrane region accounts for one-third of the insulin receptor phosphorylation and contains both phosphoserine and phosphotyrosine. Deletion of Tyr960 and 11 adjacent amino acids eliminated insulin-stimulated phosphorylation of the juxtamembrane region. Substitution of Tyr960 reduced this phosphorylation by more than 50%. The insulin receptor also undergoes serine phosphorylation outside of the juxtamembrane region which depends on the presence of Tyr1151. Together with our previous studies, this report suggests that phosphorylation of Tyr960 may play an important role in signal transduction by the insulin receptor.
Evidence
2:
Inferred from Mutant PhenotypeUniProtKB
The SHC proteins have been implicated in insulin receptor (IR) signaling. In this study, we used the sensitive two-hybrid assay of protein-protein interaction to demonstrate that SHC interacts directly with the IR. The interaction is mediated by SHC amino acids 1 to 238 and is therefore independent of the Src homology 2 domain. The interaction is dependent upon IR autophosphorylation, since the interaction is eliminated by mutation of the IR ATP-binding site. In addition, mutational analysis of the Asn-Pro-Glu-Tyr (NPEY) motif within the juxtamembrane domain of the IR showed the importance of the Asn, Pro, and Tyr residues to both SHC and IR substrate 1 (IRS-1) binding. We conclude that SHC interacts directly with the IR and that phosphorylation of Tyr-960 within the IR juxtamembrane domain is necessary for efficient interaction. This interaction is highly reminiscent of that of IRS-1 with the IR, and we show that the SHC IR-binding domain can substitute for that of IRS-1 in yeast and COS cells. We identify a homologous region within the IR-binding domains of SHC and IRS-1, which we term the SAIN (SHC and IRS-1 NPXY-binding) domain, which may explain the basis of these interactions. The SAIN domain appears to represent a novel motif which is able to interact with autophosphorylated receptors such as the IR.
J. Biol. Chem. 270, 23258-23262 (1995)[PubMed:7559478]
Insulin receptor substrate 1 (IRS-1) and src homology and collagen protein (SHC) are signaling proteins which are rapidly phosphorylated on tyrosines after insulin receptor (IR) activation. We have recently shown that both SHC and IRS-1 interact with the tyrosine-phosphorylated NPEY motif of the IR and insulin-like growth factor I receptor via non-SH2 domains (Gustafson, T. A., He, W., Craparo, A., Schaub, C. D., and O'Neill, T. J. (1995) Mol. Cell. Biol. 15, 2500-2508; O'Neill, T. J., Craparo, A., and Gustafson, T. A. (1994) Mol. Cell. Biol. 14, 6433-6442; Craparo, A., O'Neill, T. J., and Gustafson, T. A. (1995) J. Biol. Chem. 270, 15639-15643). In this study we characterize these interactions by examining the effects of 18 amino acid substitutions within and around the IR NPEY motif upon interaction with SHC and IRS-1. We confirm that Tyr-960 within the NPEY motif of the IR is essential for both IRS-1 and SHC interaction and that Asn-957 and Pro-958 are essential for IRS-1 interaction and important but not critical for SHC interaction. Additional mutations surrounding the NPEY motif revealed completely distinct patterns of interaction for SHC and IRS-1. Specifically, mutation of Leu-952 or Tyr-953 (at positions -7 and -8 from Tyr-960) markedly reduced IRS-1 interaction but had no effect upon SHC interaction. Likewise, mutation of Ala-963 (+3) reduced IRS-1 but not SHC interaction. Conversely, substitution of Leu-961 (+1) with either Ala or Arg reduced SHC interaction by 70 and 90%, respectively, yet had no effect upon interaction with IRS-1. Our data show that the sequences within and surrounding the NPEY contribute differentially to either SHC or IRS-1 recognition. Our findings suggest mechanisms by which the differential interaction of known receptors with IRS-1 and SHC may be mediated.
Conveys a signal from an upstream receptor or intracellular signal transducer by catalysis of the reaction: ATP + a protein-L-tyrosine = ADP + a protein-L-tyrosine phosphate.
Highly purified preparations of insulin receptor catalyzed the phosphorylation of the 95,000-dalton subunit of the insulin receptor. This subunit of the insulin receptor was also labeled with [alpha-32P]8-azidoadenosine 5'-triphosphate, a photoaffinity label for adenosine triphosphate binding sites. The identity of the 95,000-dalton band was confirmed in both cases by precipitation with a monoclonal antibody to the insulin receptor. These results suggest that the insulin receptor is itself a protein kinase.
Interacting selectively and non-covalently with a SH2 domain (Src homology 2) of a protein, a protein domain of about 100 amino-acid residues and belonging to the alpha + beta domain class.
Evidence
1:
Inferred from Physical InteractionUniProtKB
We have previously shown that phosphatidylinositol (PtdIns) 3'-kinase is activated by the binding of proteins or peptides containing the phosphorylated motif Y(P)XXM. In the present study, we examine interactions between PtdIns 3'-kinase and the human insulin receptor, which contains a C-terminal phosphorylation site in the sequence Y1322THM. Partially purified insulin receptors bound tightly to bacterial fusion proteins containing the N- or C-terminal SH2 domains from PtdIns 3'-kinase regulatory subunit (p85). In contrast, a mutant insulin receptor, truncated by 43 amino acids at the C terminus (IR delta CT), bound poorly to the SH2 domains; these mutant receptors have normal kinase activity but lack the Y1322THM motif. Similarly, incubation with wild-type receptors increased the activity of immunopurified PtdIns 3'-kinase, whereas incubation with IR delta CT receptors did not affect PtdIns 3'-kinase activity. Activation of PtdIns 3'-kinase by the wild-type receptor was mimicked by a tyrosyl phosphopeptide derived from the insulin receptor C terminus and containing the Y1322THM motif; non-phosphorylated peptide did not affect activity. Thus, the insulin receptor C terminus activates PtdIns 3'-kinase in vitro by binding to the SH2 domains of the 85-kDa regulatory subunit. These data support the hypothesis that binding of tyrosyl-phosphorylated receptors to p85 SH2 domains is a general mechanism for PtdIns 3'-kinase activation, and they suggest that direct interactions between the insulin receptor and PtdIns 3'-kinase may provide an alternative pathway for the activation of this enzyme by insulin.
Kaposi sarcoma (KS), a multifocal neoplasm of the skin that can spread to visceral organs, is the most prevalent malignant tumor in acquired immuno deficiency syndrome (AIDS) patients. KS-associated herpesvirus (KSHV or HHV8) is considered the primary etiological factor of this malignancy, as well as of primary effusion lymphoma and multicentric Castleman's disease. KS lesions are characterized by proliferating spindle cells of endothelial cell (EC) origin. The action of the insulin-like growth factor (IGF) system has been implicated in many malignancies, and recent data have demonstrated that the IGF-I receptor (IGF-IR) is required for in vitro growth of the KS-derived KSIMM cell line. To examine whether the IGF pathway is also involved in KSHV-mediated transformation of ECs, we examined the expression and function of the IGF system in KSHV-infected, immortalized dermal microvascular EC (E-DMVEC). The expression of the insulin receptor (IR) was strongly induced in latently infected E-DMVEC, whereas the expression levels of the IGF-IR remained unchanged. Gene knockdown of IR, but not IGF-IR, prevented the characteristic focus formation seen in KSHV-infected E-DMVEC. Similarly, treatment with the IR-specific small-molecule inhibitor HNMPA-(AM(3)) inhibited postconfluent growth. These data suggest a role for the IR, but not the IGF-IR, in KSHV-induced transformation of vascular ECs.
Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCzeta and PKClambda) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKClambda in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKClambda (lambdaKD or lambdaDeltaNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKClambda, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by lambdaKD or lambdaDeltaNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKClambda was approximately 50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKClambda mutant that lacks the pseudosubstrate domain (lambdaDeltaPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of lambdaDeltaPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKClambda. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKClambda pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.
In the present study, insulin is shown to rapidly stimulate by 8- to 12-fold the enzymatic activity of RAC-PK alpha, a pleckstrin homology domain containing ser/thr kinase. In contrast, activation of protein kinase C by phorbol esters had almost no effect on the enzymatic activity of RAC-PK alpha. Insulin activation was accompanied by a shift in molecular weight of the RAC-PK alpha protein, and the activated kinase was deactivated by treatment with a phosphatase, indicating that insulin activated the enzyme by stimulating its phosphorylation. This insulin-induced shift in RAC-PK was also observed in primary rat epididymal adipocytes, as well as in a muscle cell line called C2C12 cells. The insulin-stimulated increase in RAC-PK alpha activity was inhibited by wortmannin (an inhibitor of phosphatidylinositol 3-kinase) in a dose-dependent manner with a half-maximal inhibition of 10 nM, but not by 20 ng/ml of rapamycin. Activation of RAC-PK alpha activity was also observed in a variant RAC lacking the pleckstrin homology domain. These results indicate that RAC-PK alpha activity can be regulated by the insulin receptor. RAC-PK alpha may therefore play a general role in intracellular signaling mediated by receptor tyrosine kinases.
The chemical reactions and pathways involving carbohydrates, any of a group of organic compounds based of the general formula Cx(H2O)y. Includes the formation of carbohydrate derivatives by the addition of a carbohydrate residue to another molecule.
Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a growth factor stimulus.
Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of an insulin stimulus. Insulin is a polypeptide hormone produced by the islets of Langerhans of the pancreas in mammals, and by the homologous organs of other organisms.
Tissue-specific alternative splicing of exon 11 of the insulin receptor gene results in 2 mRNAs that differ by 36 nucleotides within the coding region. The 2 transcripts encode 2 protein isoforms with (Ex11+) or without (Ex11-) 12 additional amino acids at the carboxy-terminus of the receptor alpha-subunit. Previous studies of the 2 isoforms of the human insulin receptor expressed in mammalian cell transfectants have revealed small functional differences at the levels of equilibrium insulin binding affinity and acute ligand-induced receptor internalization. In the present study, we determined the biochemical basis for differential insulin binding affinity. Further functional characterization of the 2 receptor isoforms was also performed. The results obtained were as follows. 1) Studies of ligand association demonstrated a faster (1.8-fold) "on rate" for Ex11- receptors than for Ex11+ receptors, as determined by the kinetics of [125I]insulin binding to transfected cells. In addition, dissociation of prebound [125I]insulin from Ex11- receptors was characterized by an accelerated "off rate" relative to that of Ex11+ receptors. 2) Using both intact Chinese hamster ovary (CHO) cells and partially purified solubilized insulin receptors, the ability of insulin-like growth factor-I to compete for [125I]insulin binding to either isoform differed markedly. The mean IC50 for Ex11- was 40 nM vs. 350 nM for Ex11+. 3) Both Ex11- and Ex11+ receptors were equally capable of hybrid formation with endogenous CHO cell insulin-like growth factor-I receptors. 4) The relative abilities of 2 inhibitory polyclonal antiinsulin receptor antisera to displace [125I]insulin binding did not differ between the two isoforms. 5) Studies of insulin-induced (300 nM) receptor down-regulation in CHO cell transfectants suggested preferential down-regulation of Ex11- receptors; however, no down-regulation difference was observed when Rat 1 cell transfectants expressing the two splice variants were studied. These findings further support the idea that the 2 isoforms of the insulin receptor are functionally distinct in important ways.
The process whose specific outcome is the progression of the exocrine pancreas over time, from its formation to the mature structure. The exocrine pancreas produces and store zymogens of digestive enzymes, such as chymotrypsinogen and trypsinogen in the acinar cells.
A series of molecular signals that proceeds with an activated receptor promoting the exchange of GDP for GTP on the alpha-subunit of an associated heterotrimeric G-protein complex. The GTP-bound activated alpha-G-protein then dissociates from the beta- and gamma-subunits to further transmit the signal within the cell. The pathway begins with receptor-ligand interaction, or for basal GPCR signaling the pathway begins with the receptor activating its G protein in the absence of an agonist, and ends with regulation of a downstream cellular process, e.g. transcription.
J. Biol. Chem. 272, 10135-10143 (1997)[PubMed:9092559]
Human fat cells possess a multireceptor-linked H2O2-generating system that is activated by insulin. Previous studies revealed that manganese was the sole cofactor required for a hormonal regulation of NADPH-dependent H2O2 generation in vitro. In this report it is shown that the synergistic activation of NADPH-dependent H2O2 generation by Mn2+ and insulin was blocked by GDPbetaS (guanosine 5'-O-(2-thiodiphosphate)), pertussis toxin and COOH-terminal anti-Galphai1-2 or the corresponding peptide. Consistently, manganese could be replaced by micromolar concentrations of GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), which increased NADPH-dependent H2O2 generation by 20-40%. Insulin shifted the dose response curve for GTPgammaS to the left (>10-fold) and increased the maximal response. In the presence of 10 microM GTPgammaS, the hormone was active at picomolar concentrations, indicating that insulin acted via its cognate receptor. The insulin receptor and Gi were co-adsorbed on anti-Galphai and anti-insulin receptor beta-subunit (anti-IRbeta) affinity columns. Partially purified insulin receptor preparations contained Galphas, Galphai2, and Gbetagamma (but no Galphai1 or Galphai3). The functional nature of the insulin receptor-Gi2 complex was made evident by insulin's ability to modulate labeling of Gi by bacterial toxins. Insulin action was mimicked by activated Galphai, but not by Galphao or Gbetagamma, indicating that insulin's signal was transduced via Galphai2. Thus, NADPH oxidase is the first example of an effector system that is coupled to the insulin receptor via a heterotrimeric G protein.
Homozygous inactivation of a gene, as is frequently performed to generate mouse models, provides an opportunity to elucidate the role that the gene plays in normal physiology. However, studies of human disease provide direct insight into the effect of inactivating mutations in man. In this investigation, we have identified a one year-old boy from a consanguineous pedigree who is homozygous for deletion of the insulin receptor gene resulting in leprechaunism. Contrary to previous predictions, the complete deletion of the insulin receptor gene is compatible with life.
The developmental process in which the heart is generated and organized. The heart is a hollow, muscular organ, which, by contracting rhythmically, keeps up the circulation of the blood.
Homozygous inactivation of a gene, as is frequently performed to generate mouse models, provides an opportunity to elucidate the role that the gene plays in normal physiology. However, studies of human disease provide direct insight into the effect of inactivating mutations in man. In this investigation, we have identified a one year-old boy from a consanguineous pedigree who is homozygous for deletion of the insulin receptor gene resulting in leprechaunism. Contrary to previous predictions, the complete deletion of the insulin receptor gene is compatible with life.
Highly purified preparations of insulin receptor catalyzed the phosphorylation of the 95,000-dalton subunit of the insulin receptor. This subunit of the insulin receptor was also labeled with [alpha-32P]8-azidoadenosine 5'-triphosphate, a photoaffinity label for adenosine triphosphate binding sites. The identity of the 95,000-dalton band was confirmed in both cases by precipitation with a monoclonal antibody to the insulin receptor. These results suggest that the insulin receptor is itself a protein kinase.
Tissue-specific alternative splicing of exon 11 of the insulin receptor gene results in 2 mRNAs that differ by 36 nucleotides within the coding region. The 2 transcripts encode 2 protein isoforms with (Ex11+) or without (Ex11-) 12 additional amino acids at the carboxy-terminus of the receptor alpha-subunit. Previous studies of the 2 isoforms of the human insulin receptor expressed in mammalian cell transfectants have revealed small functional differences at the levels of equilibrium insulin binding affinity and acute ligand-induced receptor internalization. In the present study, we determined the biochemical basis for differential insulin binding affinity. Further functional characterization of the 2 receptor isoforms was also performed. The results obtained were as follows. 1) Studies of ligand association demonstrated a faster (1.8-fold) "on rate" for Ex11- receptors than for Ex11+ receptors, as determined by the kinetics of [125I]insulin binding to transfected cells. In addition, dissociation of prebound [125I]insulin from Ex11- receptors was characterized by an accelerated "off rate" relative to that of Ex11+ receptors. 2) Using both intact Chinese hamster ovary (CHO) cells and partially purified solubilized insulin receptors, the ability of insulin-like growth factor-I to compete for [125I]insulin binding to either isoform differed markedly. The mean IC50 for Ex11- was 40 nM vs. 350 nM for Ex11+. 3) Both Ex11- and Ex11+ receptors were equally capable of hybrid formation with endogenous CHO cell insulin-like growth factor-I receptors. 4) The relative abilities of 2 inhibitory polyclonal antiinsulin receptor antisera to displace [125I]insulin binding did not differ between the two isoforms. 5) Studies of insulin-induced (300 nM) receptor down-regulation in CHO cell transfectants suggested preferential down-regulation of Ex11- receptors; however, no down-regulation difference was observed when Rat 1 cell transfectants expressing the two splice variants were studied. These findings further support the idea that the 2 isoforms of the insulin receptor are functionally distinct in important ways.
J. Biol. Chem. 268, 11256-11264 (1993)[PubMed:8496180]
Insulin-stimulated autophosphorylation of the cytoplasmic juxtamembrane region of the human insulin receptor was examined by Tricine/SDS-PAGE. Various mutant receptor molecules were used to identify two tryptic phosphopeptides associated with the juxtamembrane region which accounts for 15% of the autophosphorylation of partially purified insulin receptor. These phosphopeptides were immunoprecipitated with an antipeptide antibody against the juxtamembrane sequence and were phosphorylated exclusively on tyrosine. Substitution of both Tyr960 and Tyr953 with alanine eliminated insulin-stimulated phosphorylation of the juxtamembrane region without affecting tyrosine autophosphorylation in the C terminus or regulatory regions. Monosubstitution of Tyr960 with phenylalanine or alanine reduced phosphorylation in the juxtamembrane region by more than 50%, and manual Edman degradation indicated that Tyr960 was phosphorylated in wild-type receptor. In vivo, phosphorylation of the juxtamembrane region accounts for one-third of the insulin receptor phosphorylation and contains both phosphoserine and phosphotyrosine. Deletion of Tyr960 and 11 adjacent amino acids eliminated insulin-stimulated phosphorylation of the juxtamembrane region. Substitution of Tyr960 reduced this phosphorylation by more than 50%. The insulin receptor also undergoes serine phosphorylation outside of the juxtamembrane region which depends on the presence of Tyr1151. Together with our previous studies, this report suggests that phosphorylation of Tyr960 may play an important role in signal transduction by the insulin receptor.
The insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) have a highly homologous structure, but different biological effects. Insulin and IGF-I half-receptors can heterodimerize, leading to the formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) that bind IGF-I with high affinity. As the IR exists in two isoforms (IR-A and IR-B), we evaluated whether the assembly of the IGF-IR with either IR-A or IR-B moieties may differently affect Hybrid-R signaling and biological role. Three different models were studied: (a) 3T3-like mouse fibroblasts with a disrupted IGF-IR gene (R(-) cells) cotransfected with the human IGF-IR and with either the IR-A or IR-B cDNA; (b) a panel of human cell lines variably expressing the two IR isoforms; and (c) HepG2 human hepatoblastoma cells predominantly expressing either IR-A or IR-B, depending on their differentiation state. We found that Hybrid-Rs containing IR-A (Hybrid-Rs(A)) bound to and were activated by IGF-I, IGF-II, and insulin. By binding to Hybrid-Rs(A), insulin activated the IGF-I half-receptor beta-subunit and the IGF-IR-specific substrate CrkII. In contrast, Hybrid-Rs(B) bound to and were activated with high affinity by IGF-I, with low affinity by IGF-II, and insignificantly by insulin. As a consequence, cell proliferation and migration in response to both insulin and IGFs were more effectively stimulated in Hybrid-R(A)-containing cells than in Hybrid-R(B)-containing cells. The relative abundance of IR isoforms therefore affects IGF system activation through Hybrid-Rs, with important consequences for tissue-specific responses to both insulin and IGFs.
J. Cell. Biochem. 82, 610-618 (2001)[PubMed:11500939]
Insulin-like growth factor II (IGF-II) plays a key role in mitogenesis during development and tumorigenesis and is believed to exert its mitogenic functions mainly through the IGF-I receptor. Recently, we identified the insulin receptor isoform A (IR(A)) as an additional high affinity receptor for IGF-II in both fetal and cancer cells. Here we investigated the mitogenic signaling of IGF-II via the Akt/Glycogen synthase kinase 3 (Gsk3) axis employing R-IR(A) cells that are IGF-I receptor null mouse embryonic fibroblasts expressing the human IR(A). IGF-II induced activation of the proto-oncogenic serine kinase Akt, reaching maximal at 5-10 min. IGF-II also caused the rapid and sustained deactivation of glycogen synthase kinase 3-beta (Gsk3beta), reaching maximal at 1-3 min, shortly preceding, therefore, maximal activation of Akt. Under our conditions, IGF-II and insulin induced 70-80% inhibition of Gsk3betaactivity. In these cells IGF-II also deactivated Gsk3alpha although less effectively than Gsk3beta. In parallel experiments, we found that IGF-II induced transient activation of extracellular-signal-regulated kinases (Erk) reaching maximal at 5-10 min and decreasing thereafter. Time courses and potencies of regulation of both mitogenic pathways (Akt/Gsk3beta and Erk) by IGF-II via IR(A) were similar to those of insulin. Furthermore, IGF-II like insulin effectively stimulated cell cycle progression from the G0/G1 to the S and G2/M phases. Interestingly, AP-1-mediated gene expression, that was reported to be negatively regulated by Gsk3beta was only weakly increased after IGF-II stimulation. Our present data suggest that the coordinated activation or deactivation of Akt, Gsk3beta, and Erk may account for IGF-II mitogenic effects and support an active role for IR(A) in IGF-II action.
Recently, single chain peptides have been designed that target the insulin receptor and mimic insulin action. The aim of this study is to explore if activation of the insulin receptor with such an optimized peptide (S597) leads to the same activation of signaling pathways and biological endpoints i.e. stimulation of glycogen synthesis and cell proliferation as stimulation with insulin. We find that surface activation of the insulin receptor A-isoform with S597 leads to activation of protein kinase B (PKB) and glycogen synthesis comparable to activation by insulin, even though the level of insulin receptor phosphorylation is lower. In contrast, both Src homology 2/alpha collagen-related (Shc) and extracellular signal-regulated kinase (ERK) 2 activation are virtually absent upon stimulation with S597. Cell proliferation is only stimulated slightly by S597, suggesting that it depends on signals from Shc and ERK. The differences in signaling response could explain both the earlier reported differences in gene expression, and the reported differences in cell proliferation and glycogen synthesis induced by insulin and S597. In conclusion, despite binding equipotency, insulin, and S597 initiate different signaling and biological responses through the same insulin receptor isoform. We show for the first time that it is possible to design insulin receptor ligand mimetics with metabolic equipotency but low mitogenicity.
Homozygous inactivation of a gene, as is frequently performed to generate mouse models, provides an opportunity to elucidate the role that the gene plays in normal physiology. However, studies of human disease provide direct insight into the effect of inactivating mutations in man. In this investigation, we have identified a one year-old boy from a consanguineous pedigree who is homozygous for deletion of the insulin receptor gene resulting in leprechaunism. Contrary to previous predictions, the complete deletion of the insulin receptor gene is compatible with life.
The insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) have a highly homologous structure, but different biological effects. Insulin and IGF-I half-receptors can heterodimerize, leading to the formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) that bind IGF-I with high affinity. As the IR exists in two isoforms (IR-A and IR-B), we evaluated whether the assembly of the IGF-IR with either IR-A or IR-B moieties may differently affect Hybrid-R signaling and biological role. Three different models were studied: (a) 3T3-like mouse fibroblasts with a disrupted IGF-IR gene (R(-) cells) cotransfected with the human IGF-IR and with either the IR-A or IR-B cDNA; (b) a panel of human cell lines variably expressing the two IR isoforms; and (c) HepG2 human hepatoblastoma cells predominantly expressing either IR-A or IR-B, depending on their differentiation state. We found that Hybrid-Rs containing IR-A (Hybrid-Rs(A)) bound to and were activated by IGF-I, IGF-II, and insulin. By binding to Hybrid-Rs(A), insulin activated the IGF-I half-receptor beta-subunit and the IGF-IR-specific substrate CrkII. In contrast, Hybrid-Rs(B) bound to and were activated with high affinity by IGF-I, with low affinity by IGF-II, and insignificantly by insulin. As a consequence, cell proliferation and migration in response to both insulin and IGFs were more effectively stimulated in Hybrid-R(A)-containing cells than in Hybrid-R(B)-containing cells. The relative abundance of IR isoforms therefore affects IGF system activation through Hybrid-Rs, with important consequences for tissue-specific responses to both insulin and IGFs.
Insulin stimulates the autophosphorylation of tyrosine residues of the beta subunit of the insulin receptor (IR); this modified insulin-independent kinase has increased activity toward exogenous substrates in vitro. We show here that replacement of one or both of the twin tyrosines (residues 1162 and 1163) with phenylalanine results in a dramatic reduction in or loss of insulin-activated autophosphorylation and kinase activity in vitro. In vivo, these mutations not only result in a substantial decrease in insulin-stimulated IR autophosphorylation but also in a parallel decrease in the insulin-activated uptake of 2-deoxyglucose. Furthermore, a truncated IR protein (lacking the last 112 amino acids) has an unstable beta subunit; this mutant has no kinase activity in vitro or in vivo and does not mediate insulin-stimulated uptake of 2-deoxyglucose. IR autophosphorylation is thus implicated in the regulation of IR activities, with tyrosines 1162 and 1163 as major sites of this regulation.
Insulin stimulates the transport of glucose into fat and muscle cells. Although the precise molecular mechanisms involved in this process remain uncertain, insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates. One such substrate is the Cbl proto-oncogene product. Cbl is recruited to the insulin receptor by interaction with the adapter protein CAP, through one of three adjacent SH3 domains in the carboxy terminus of CAP. Upon phosphorylation of Cbl, the CAP-Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched, triton-insoluble membrane fraction. Here, to identify a molecular mechanism underlying this subcellular redistribution, we screened a yeast two-hybrid library using the amino-terminal region of CAP and identified the caveolar protein flotillin. Flotillin forms a ternary complex with CAP and Cbl, directing the localization of the CAP-Cbl complex to a lipid raft subdomain of the plasma membrane. Expression of the N-terminal domain of CAP in 3T3-L1 adipocytes blocks the stimulation of glucose transport by insulin, without affecting signalling events that depend on phosphatidylinositol-3-OH kinase. Thus, localization of the Cbl-CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake.
Recently, single chain peptides have been designed that target the insulin receptor and mimic insulin action. The aim of this study is to explore if activation of the insulin receptor with such an optimized peptide (S597) leads to the same activation of signaling pathways and biological endpoints i.e. stimulation of glycogen synthesis and cell proliferation as stimulation with insulin. We find that surface activation of the insulin receptor A-isoform with S597 leads to activation of protein kinase B (PKB) and glycogen synthesis comparable to activation by insulin, even though the level of insulin receptor phosphorylation is lower. In contrast, both Src homology 2/alpha collagen-related (Shc) and extracellular signal-regulated kinase (ERK) 2 activation are virtually absent upon stimulation with S597. Cell proliferation is only stimulated slightly by S597, suggesting that it depends on signals from Shc and ERK. The differences in signaling response could explain both the earlier reported differences in gene expression, and the reported differences in cell proliferation and glycogen synthesis induced by insulin and S597. In conclusion, despite binding equipotency, insulin, and S597 initiate different signaling and biological responses through the same insulin receptor isoform. We show for the first time that it is possible to design insulin receptor ligand mimetics with metabolic equipotency but low mitogenicity.
Homozygous inactivation of a gene, as is frequently performed to generate mouse models, provides an opportunity to elucidate the role that the gene plays in normal physiology. However, studies of human disease provide direct insight into the effect of inactivating mutations in man. In this investigation, we have identified a one year-old boy from a consanguineous pedigree who is homozygous for deletion of the insulin receptor gene resulting in leprechaunism. Contrary to previous predictions, the complete deletion of the insulin receptor gene is compatible with life.
J. Cell. Biochem. 82, 610-618 (2001)[PubMed:11500939]
Insulin-like growth factor II (IGF-II) plays a key role in mitogenesis during development and tumorigenesis and is believed to exert its mitogenic functions mainly through the IGF-I receptor. Recently, we identified the insulin receptor isoform A (IR(A)) as an additional high affinity receptor for IGF-II in both fetal and cancer cells. Here we investigated the mitogenic signaling of IGF-II via the Akt/Glycogen synthase kinase 3 (Gsk3) axis employing R-IR(A) cells that are IGF-I receptor null mouse embryonic fibroblasts expressing the human IR(A). IGF-II induced activation of the proto-oncogenic serine kinase Akt, reaching maximal at 5-10 min. IGF-II also caused the rapid and sustained deactivation of glycogen synthase kinase 3-beta (Gsk3beta), reaching maximal at 1-3 min, shortly preceding, therefore, maximal activation of Akt. Under our conditions, IGF-II and insulin induced 70-80% inhibition of Gsk3betaactivity. In these cells IGF-II also deactivated Gsk3alpha although less effectively than Gsk3beta. In parallel experiments, we found that IGF-II induced transient activation of extracellular-signal-regulated kinases (Erk) reaching maximal at 5-10 min and decreasing thereafter. Time courses and potencies of regulation of both mitogenic pathways (Akt/Gsk3beta and Erk) by IGF-II via IR(A) were similar to those of insulin. Furthermore, IGF-II like insulin effectively stimulated cell cycle progression from the G0/G1 to the S and G2/M phases. Interestingly, AP-1-mediated gene expression, that was reported to be negatively regulated by Gsk3beta was only weakly increased after IGF-II stimulation. Our present data suggest that the coordinated activation or deactivation of Akt, Gsk3beta, and Erk may account for IGF-II mitogenic effects and support an active role for IR(A) in IGF-II action.
J. Cell. Biochem. 82, 610-618 (2001)[PubMed:11500939]
Insulin-like growth factor II (IGF-II) plays a key role in mitogenesis during development and tumorigenesis and is believed to exert its mitogenic functions mainly through the IGF-I receptor. Recently, we identified the insulin receptor isoform A (IR(A)) as an additional high affinity receptor for IGF-II in both fetal and cancer cells. Here we investigated the mitogenic signaling of IGF-II via the Akt/Glycogen synthase kinase 3 (Gsk3) axis employing R-IR(A) cells that are IGF-I receptor null mouse embryonic fibroblasts expressing the human IR(A). IGF-II induced activation of the proto-oncogenic serine kinase Akt, reaching maximal at 5-10 min. IGF-II also caused the rapid and sustained deactivation of glycogen synthase kinase 3-beta (Gsk3beta), reaching maximal at 1-3 min, shortly preceding, therefore, maximal activation of Akt. Under our conditions, IGF-II and insulin induced 70-80% inhibition of Gsk3betaactivity. In these cells IGF-II also deactivated Gsk3alpha although less effectively than Gsk3beta. In parallel experiments, we found that IGF-II induced transient activation of extracellular-signal-regulated kinases (Erk) reaching maximal at 5-10 min and decreasing thereafter. Time courses and potencies of regulation of both mitogenic pathways (Akt/Gsk3beta and Erk) by IGF-II via IR(A) were similar to those of insulin. Furthermore, IGF-II like insulin effectively stimulated cell cycle progression from the G0/G1 to the S and G2/M phases. Interestingly, AP-1-mediated gene expression, that was reported to be negatively regulated by Gsk3beta was only weakly increased after IGF-II stimulation. Our present data suggest that the coordinated activation or deactivation of Akt, Gsk3beta, and Erk may account for IGF-II mitogenic effects and support an active role for IR(A) in IGF-II action.
Any process that activates or increases the frequency, rate or extent of the chemical reactions and pathways resulting in the formation of nitric oxide.
BACKGROUND: Previously, we demonstrated that insulin stimulates production of nitric oxide (NO) in endothelial cells. However, specific insulin-signaling pathways mediating production of NO have not been elucidated. METHODS AND RESULTS: We developed methods for transfection of human umbilical vein endothelial cells (HUVECs) and direct measurement of NO to begin defining insulin-signaling pathways related to NO production. HUVECs were cotransfected with enhanced Green Fluorescent Protein (eGFP) and another gene of interest. Transfection efficiencies >95% were obtained by selecting cells expressing eGFP. Overexpression of insulin receptors in HUVECs resulted in an approximately 3-fold increase in production of NO in response to insulin. In contrast, HUVECs overexpressing a tyrosine kinase-deficient mutant insulin receptor had a dose-response curve similar to that of control cells. Overexpression of inhibitory mutants of either phosphatidylinositol 3-kinase (PI3K) or Akt resulted in nearly complete inhibition of insulin-stimulated production of NO. Overexpression of an inhibitory mutant of Ras had a much smaller effect. CONCLUSIONS: Receptor kinase activity is necessary to mediate production of NO through the insulin receptor. Both PI3K and Akt contribute importantly to this process, whereas the contribution of Ras is small.
Any process that activates or increases the frequency, rate or extent of the protein kinase B signaling cascade, a series of reactions mediated by the intracellular serine/threonine kinase protein kinase B.
J. Cell. Biochem. 82, 610-618 (2001)[PubMed:11500939]
Insulin-like growth factor II (IGF-II) plays a key role in mitogenesis during development and tumorigenesis and is believed to exert its mitogenic functions mainly through the IGF-I receptor. Recently, we identified the insulin receptor isoform A (IR(A)) as an additional high affinity receptor for IGF-II in both fetal and cancer cells. Here we investigated the mitogenic signaling of IGF-II via the Akt/Glycogen synthase kinase 3 (Gsk3) axis employing R-IR(A) cells that are IGF-I receptor null mouse embryonic fibroblasts expressing the human IR(A). IGF-II induced activation of the proto-oncogenic serine kinase Akt, reaching maximal at 5-10 min. IGF-II also caused the rapid and sustained deactivation of glycogen synthase kinase 3-beta (Gsk3beta), reaching maximal at 1-3 min, shortly preceding, therefore, maximal activation of Akt. Under our conditions, IGF-II and insulin induced 70-80% inhibition of Gsk3betaactivity. In these cells IGF-II also deactivated Gsk3alpha although less effectively than Gsk3beta. In parallel experiments, we found that IGF-II induced transient activation of extracellular-signal-regulated kinases (Erk) reaching maximal at 5-10 min and decreasing thereafter. Time courses and potencies of regulation of both mitogenic pathways (Akt/Gsk3beta and Erk) by IGF-II via IR(A) were similar to those of insulin. Furthermore, IGF-II like insulin effectively stimulated cell cycle progression from the G0/G1 to the S and G2/M phases. Interestingly, AP-1-mediated gene expression, that was reported to be negatively regulated by Gsk3beta was only weakly increased after IGF-II stimulation. Our present data suggest that the coordinated activation or deactivation of Akt, Gsk3beta, and Erk may account for IGF-II mitogenic effects and support an active role for IR(A) in IGF-II action.
In the present study, insulin is shown to rapidly stimulate by 8- to 12-fold the enzymatic activity of RAC-PK alpha, a pleckstrin homology domain containing ser/thr kinase. In contrast, activation of protein kinase C by phorbol esters had almost no effect on the enzymatic activity of RAC-PK alpha. Insulin activation was accompanied by a shift in molecular weight of the RAC-PK alpha protein, and the activated kinase was deactivated by treatment with a phosphatase, indicating that insulin activated the enzyme by stimulating its phosphorylation. This insulin-induced shift in RAC-PK was also observed in primary rat epididymal adipocytes, as well as in a muscle cell line called C2C12 cells. The insulin-stimulated increase in RAC-PK alpha activity was inhibited by wortmannin (an inhibitor of phosphatidylinositol 3-kinase) in a dose-dependent manner with a half-maximal inhibition of 10 nM, but not by 20 ng/ml of rapamycin. Activation of RAC-PK alpha activity was also observed in a variant RAC lacking the pleckstrin homology domain. These results indicate that RAC-PK alpha activity can be regulated by the insulin receptor. RAC-PK alpha may therefore play a general role in intracellular signaling mediated by receptor tyrosine kinases.
Any process that increases the rate frequency or extent of a phase of elevated metabolic activity, during which oxygen consumption increases; this leads to the production, by an NADH dependent system, of hydrogen peroxide (H2O2), superoxide anions and hydroxyl radicals.
J. Biol. Chem. 272, 10135-10143 (1997)[PubMed:9092559]
Human fat cells possess a multireceptor-linked H2O2-generating system that is activated by insulin. Previous studies revealed that manganese was the sole cofactor required for a hormonal regulation of NADPH-dependent H2O2 generation in vitro. In this report it is shown that the synergistic activation of NADPH-dependent H2O2 generation by Mn2+ and insulin was blocked by GDPbetaS (guanosine 5'-O-(2-thiodiphosphate)), pertussis toxin and COOH-terminal anti-Galphai1-2 or the corresponding peptide. Consistently, manganese could be replaced by micromolar concentrations of GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), which increased NADPH-dependent H2O2 generation by 20-40%. Insulin shifted the dose response curve for GTPgammaS to the left (>10-fold) and increased the maximal response. In the presence of 10 microM GTPgammaS, the hormone was active at picomolar concentrations, indicating that insulin acted via its cognate receptor. The insulin receptor and Gi were co-adsorbed on anti-Galphai and anti-insulin receptor beta-subunit (anti-IRbeta) affinity columns. Partially purified insulin receptor preparations contained Galphas, Galphai2, and Gbetagamma (but no Galphai1 or Galphai3). The functional nature of the insulin receptor-Gi2 complex was made evident by insulin's ability to modulate labeling of Gi by bacterial toxins. Insulin action was mimicked by activated Galphai, but not by Galphao or Gbetagamma, indicating that insulin's signal was transduced via Galphai2. Thus, NADPH oxidase is the first example of an effector system that is coupled to the insulin receptor via a heterotrimeric G protein.
J. Biol. Chem. 268, 11256-11264 (1993)[PubMed:8496180]
Insulin-stimulated autophosphorylation of the cytoplasmic juxtamembrane region of the human insulin receptor was examined by Tricine/SDS-PAGE. Various mutant receptor molecules were used to identify two tryptic phosphopeptides associated with the juxtamembrane region which accounts for 15% of the autophosphorylation of partially purified insulin receptor. These phosphopeptides were immunoprecipitated with an antipeptide antibody against the juxtamembrane sequence and were phosphorylated exclusively on tyrosine. Substitution of both Tyr960 and Tyr953 with alanine eliminated insulin-stimulated phosphorylation of the juxtamembrane region without affecting tyrosine autophosphorylation in the C terminus or regulatory regions. Monosubstitution of Tyr960 with phenylalanine or alanine reduced phosphorylation in the juxtamembrane region by more than 50%, and manual Edman degradation indicated that Tyr960 was phosphorylated in wild-type receptor. In vivo, phosphorylation of the juxtamembrane region accounts for one-third of the insulin receptor phosphorylation and contains both phosphoserine and phosphotyrosine. Deletion of Tyr960 and 11 adjacent amino acids eliminated insulin-stimulated phosphorylation of the juxtamembrane region. Substitution of Tyr960 reduced this phosphorylation by more than 50%. The insulin receptor also undergoes serine phosphorylation outside of the juxtamembrane region which depends on the presence of Tyr1151. Together with our previous studies, this report suggests that phosphorylation of Tyr960 may play an important role in signal transduction by the insulin receptor.
Evidence
2:
Inferred from Mutant PhenotypeUniProtKB
The SHC proteins have been implicated in insulin receptor (IR) signaling. In this study, we used the sensitive two-hybrid assay of protein-protein interaction to demonstrate that SHC interacts directly with the IR. The interaction is mediated by SHC amino acids 1 to 238 and is therefore independent of the Src homology 2 domain. The interaction is dependent upon IR autophosphorylation, since the interaction is eliminated by mutation of the IR ATP-binding site. In addition, mutational analysis of the Asn-Pro-Glu-Tyr (NPEY) motif within the juxtamembrane domain of the IR showed the importance of the Asn, Pro, and Tyr residues to both SHC and IR substrate 1 (IRS-1) binding. We conclude that SHC interacts directly with the IR and that phosphorylation of Tyr-960 within the IR juxtamembrane domain is necessary for efficient interaction. This interaction is highly reminiscent of that of IRS-1 with the IR, and we show that the SHC IR-binding domain can substitute for that of IRS-1 in yeast and COS cells. We identify a homologous region within the IR-binding domains of SHC and IRS-1, which we term the SAIN (SHC and IRS-1 NPXY-binding) domain, which may explain the basis of these interactions. The SAIN domain appears to represent a novel motif which is able to interact with autophosphorylated receptors such as the IR.
Highly purified preparations of insulin receptor catalyzed the phosphorylation of the 95,000-dalton subunit of the insulin receptor. This subunit of the insulin receptor was also labeled with [alpha-32P]8-azidoadenosine 5'-triphosphate, a photoaffinity label for adenosine triphosphate binding sites. The identity of the 95,000-dalton band was confirmed in both cases by precipitation with a monoclonal antibody to the insulin receptor. These results suggest that the insulin receptor is itself a protein kinase.
The formation of a protein heterotetramer, a macromolecular structure consisting of four noncovalently associated subunits, of which not all are identical.
The native subunit structure of the insulin receptor was reinvestigated by two-dimensional nonreducing/reducing gel electrophoresis. Human insulin receptor expressed in murine fibroblasts was found to be a single oligomer, the alpha 2 beta 2 heterotetramer. The structure was assessed using receptor metabolically labeled with [35S]methionine, and using receptor autophosphorylation at two levels of purification: the insulin affinity-purified receptor and the more commonly used wheat germ agglutinin-Sepharose-enriched fraction from whole membrane extracts. Lower molecular weight oligomers and free subunits were observed only upon heating the sample prior to electrophoresis. This artifact of sample handling was dependent upon three factors: (i) temperature, (ii) time of heating, and (iii) impurities typically present in partially purified receptor preparations. We conclude that the alpha 2 beta 2 tetramer is the only insulin receptor subunit structure native in intact cells and subsequently isolated from cell membranes.
Homozygous inactivation of a gene, as is frequently performed to generate mouse models, provides an opportunity to elucidate the role that the gene plays in normal physiology. However, studies of human disease provide direct insight into the effect of inactivating mutations in man. In this investigation, we have identified a one year-old boy from a consanguineous pedigree who is homozygous for deletion of the insulin receptor gene resulting in leprechaunism. Contrary to previous predictions, the complete deletion of the insulin receptor gene is compatible with life.
The human insulin receptor (IR) exists in two isoforms (IR-A and IR-B). IR-A is a short isoform, generated by the skipping of exon 11, a small exon encoding for 12 amino acid residues at the carboxyl terminus of the IR alpha-subunit. Recently, we found that IR-A is the predominant isoform in fetal tissues and malignant cells and binds with a high affinity not only insulin but also insulin-like growth factor-II (IGF-II). To investigate whether the activation of IR-A by the two ligands differentially activate post-receptor molecular mechanisms, we studied gene expression in response to IR-A activation by either insulin or IGF-II, using microarray technology. To avoid the interfering effect of the IGF-IR, IGF-II binding to the IR-A was studied in IGF-IR-deficient murine fibroblasts (R- cells) transfected with the human IR-A cDNA (R-/IR-A cells). Gene expression was studied at 0.5, 3, and 8 h. We found that 214 transcripts were similarly regulated by insulin and IGF-II, whereas 45 genes were differentially transcribed. Eighteen of these differentially regulated genes were responsive to only one of the two ligands (12 to insulin and 6 to IGF-II). Twenty-seven transcripts were regulated by both insulin and IGF-II, but a significant difference between the two ligands was present at least in one time point. Interestingly, IGF-II was a more potent and/or persistent regulator than insulin for these genes. Results were validated by measuring the expression of 12 genes by quantitative real-time reverse transcriptase-PCR. In conclusion, we show that insulin and IGF-II, acting via the same receptor, may differentially affect gene expression in cells. These studies provide a molecular basis for understanding some of the biological differences between the two ligands and may help to clarify the biological role of IR-A in embryonic/fetal growth and the selective biological advantage that malignant cells producing IGF-II may acquire via IR-A overexpression.
Highly purified preparations of insulin receptor catalyzed the phosphorylation of the 95,000-dalton subunit of the insulin receptor. This subunit of the insulin receptor was also labeled with [alpha-32P]8-azidoadenosine 5'-triphosphate, a photoaffinity label for adenosine triphosphate binding sites. The identity of the 95,000-dalton band was confirmed in both cases by precipitation with a monoclonal antibody to the insulin receptor. These results suggest that the insulin receptor is itself a protein kinase.
Kaposi sarcoma (KS), a multifocal neoplasm of the skin that can spread to visceral organs, is the most prevalent malignant tumor in acquired immuno deficiency syndrome (AIDS) patients. KS-associated herpesvirus (KSHV or HHV8) is considered the primary etiological factor of this malignancy, as well as of primary effusion lymphoma and multicentric Castleman's disease. KS lesions are characterized by proliferating spindle cells of endothelial cell (EC) origin. The action of the insulin-like growth factor (IGF) system has been implicated in many malignancies, and recent data have demonstrated that the IGF-I receptor (IGF-IR) is required for in vitro growth of the KS-derived KSIMM cell line. To examine whether the IGF pathway is also involved in KSHV-mediated transformation of ECs, we examined the expression and function of the IGF system in KSHV-infected, immortalized dermal microvascular EC (E-DMVEC). The expression of the insulin receptor (IR) was strongly induced in latently infected E-DMVEC, whereas the expression levels of the IGF-IR remained unchanged. Gene knockdown of IR, but not IGF-IR, prevented the characteristic focus formation seen in KSHV-infected E-DMVEC. Similarly, treatment with the IR-specific small-molecule inhibitor HNMPA-(AM(3)) inhibited postconfluent growth. These data suggest a role for the IR, but not the IGF-IR, in KSHV-induced transformation of vascular ECs.
This protein acts as an enzyme. It is known to catalyze the following reaction
EC 2.7.10.1: ATP + a [protein]-L-tyrosine ⇄ ADP + a [protein]-L-tyrosine phosphate.
CuratedUniProtKB
It is regulated in the following manner
Activated in response to insulin. Autophosphorylation activates the kinase activity. PTP1B dephosphorylates important tyrosine residues, thereby reducing INSR activity. Inhibited by ENPP1. GRB10 and GRB14 inhibit the catalytic activity of the INSR, they block access of substrates to the activated receptor. SOCS1 and SOCS3 act as negative regulators of INSR activity, they bind to the activated INRS and interfere with the phosphorylation of INSR substrates.
Plasma cell membrane glycoprotein-1 (PC-1) inhibits insulin receptor (IR) tyrosine kinase activity and subsequent cellular signaling. PC-1 content is elevated in fibroblasts, muscle, and adipose tissue from insulin-resistant subjects, and its elevation correlates with in vivo insulin resistance. In vitro, when PC-1 is transfected and overexpressed in cultured cells, it inhibits IR tyrosine kinase activity. To determine the mechanism whereby PC-1 regulates the IR, we studied how PC-1 interacts with this protein. Overexpression of PC-1 in MCF-7 cells inhibited tyrosine kinase activity of the IR, but not of the IGF-I receptor. When the IR was immunocaptured by specific IR monoclonal antibodies, PC-1 was associated with this receptor. In contrast, after specific immunocapture, PC-1 was not associated with the IGF-I receptor. We next studied HTC cells that were overexpressing an IR alpha-subunit mutant. This IR mutant binds insulin but has a deletion in the tyrosine kinase regulatory domain located in amino acids 485-599. In contrast to normal IRs, PC-1 did not associate with this mutant and did not affect tyrosine kinase activity. To determine whether decreasing PC-1 expression would reverse the inhibition of tyrosine kinase activity, we treated MCF-7 cells overexpressing PC-1 with a monoclonal antibody to PC-1. This treatment decreased PC-1 levels; concomitantly, IR tyrosine kinase activity increased. In contrast, IGF-I receptor tyrosine kinase activity was not increased. These studies indicate, therefore, that PC-1 may inhibit the IR by interacting directly with a specific region in the IR alpha-subunit. These studies also raise the possibility that monoclonal antibodies to PC-1 could be a new treatment for insulin resistance.
J. Biol. Chem. 265, 18673-18681 (1990)[PubMed:2211730]
Human placental insulin receptor contains 47 Cys per an alpha beta dimer. Most of the 94 Cys in an intact alpha 2 beta 2 receptor are expected to form interchain or intrachain disulfide bonds, since there appears to be only one free cysteine residue in each beta subunit. In order to gain more insight into the three-dimensional organization of the insulin receptor, we have used limited trypsin digestion, SDS-PAGE, and protein microsequencing. The present study revealed the following; major tryptic cleavages occurred at alpha 164, alpha 270, alpha 582, and beta 1115, generating Mr 175,000, 130,000, 100,000, 70,000, and 55,000 disulfide-linked complexes. Under reducing conditions, tryptic fragments of Mr values = 30,000, 70,000, 20,000, 55,000, and 20,000 were identified to be alpha(1-164), alpha(165-582), alpha(165-270), alpha(271-582), and alpha(583-C-terminal), respectively. The major beta subunit tryptic fragment of Mr = 55,000 was assumed to have beta(724-1115) or beta(N-terminal-392). The Mr 175,000 complex appeared to contain two alpha(1-164) and two alpha(165-582), whereas the Mr 70,000 complex contained alpha(583-C-terminal) and beta(724-1115). Tryptic cleavage at alpha 582 apparently produced one Mr 175,000 and two Mr 70,000 complexes, suggesting that the alpha(583-C-terminal) domain interacts with the extracellular domain of the beta subunit by disulfide bonds. Tryptic cleavage at alpha 270 resulting in a formation of one Mr 100,000 complex consisting of two alpha(1-270) and two Mr 130,000 complexes consisting of alpha(271-C-terminal) and beta(724-1115) suggests that Cys residues involved with disulfide bonds between the two alpha subunits are located in the alpha(1-270) domain. The identification of the Mr 55,000 complex consisting of small tryptic fragments between alpha(122-270) indicates that 40 Cys residues in the two alpha(122-270) domains are inter- and intramolecularly associated by disulfide bonds. The alpha(1-121) domain does not appear to be linked to any other domains by disulfide bonds. These results are consistent with the structural model that the N-terminal domains of alpha subunits (122-270) are disulfide-linked together while the C-terminal domain (583-C-terminal) of the alpha subunit is linked to the N-terminal domain of the beta subunit by disulfide bonds.
Grb10 has been proposed to inhibit or activate insulin signaling, depending on cellular context. We have investigated the mechanism by which full-length hGrb10gamma inhibits signaling through the insulin receptor substrate (IRS) proteins. Overexpression of hGrb10gamma in CHO/IR cells and in differentiated adipocytes significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. Inhibition occurred rapidly and was sustained for 60 min during insulin stimulation. In agreement with inhibited signaling through the IRS/PI 3-kinase pathway, we found hGrb10gamma to both delay and reduce phosphorylation of Akt at Thr(308) and Ser(473) in response to insulin stimulation. Decreased phosphorylation of IRS-1/2 may arise from impaired catalytic activity of the receptor, since hGrb10gamma directly associates with the IR kinase regulatory loop. However, yeast tri-hybrid studies indicated that full-length Grb10 blocks association between IRS proteins and IR, and that this requires the SH2 domain of Grb10. In cells, hGrb10gamma inhibited insulin-stimulated IRS-1 tyrosine phosphorylation in a dose-dependent manner, but did not affect IR catalytic activity toward Tyr(972) in the juxtamembrane region and Tyr(1158/1162/1163) in the regulatory domain. We conclude that binding of hGrb10gamma to IR decreases signaling through the IRS/PI 3-kinase/AKT pathway by physically blocking IRS access to IR.
Grb14 belongs to the Grb7 family of adapters and was recently identified as a partner of the insulin receptor (IR). Here we show that Grb14 inhibits in vitro IR substrate phosphorylation. Grb14 does not alter the K(m) for ATP and behaves as an uncompetitive inhibitor for the IR substrate. Similar experiments performed with other members of the Grb7 family, Grb7 and Grb10, and with IGF-1 receptor argue in favor of a specific inhibition of the IR catalytic activity by Grb14. The IR-interacting domain of Grb14, the PIR, is sufficient for the inhibitory effect of Grb14, whereas the SH2 domain has no effect on IR catalytic activity. In Chinese hamster ovary (CHO) cells overexpressing both IR and Grb14, Grb14 binds to the IR as early as 1 min after insulin stimulation, and the two proteins remain associated. When interacting with Grb14, the IR is protected against tyrosine phosphatases action and therefore maintained under a phosphorylated state. However, the binding of Grb14 to the IR induces an early delay in the activation of Akt and ERK1/2 in CHO-IR cells, and ERK1/2 are less efficiently phosphorylated. These findings show that Grb14 is a direct inhibitor of the IR catalytic activity and could be considered as a modulator of insulin signaling.
Protein participating in biochemical reactions in which carbohydrates are involved. Carbohydrate is a general term for sugars and related compounds with the general formula Cn(H2O)n. The smallest are monosaccharides (e.g. glucose); polysaccharides (e.g. starch, cellulose, glycogen) can be large and vary in length.
Enzyme which catalyzes the transfer of the terminal phosphate of ATP to a specific tyrosine residue on its target protein. Many of these kinases play significant roles in development and cell division. Tyrosine-protein kinases can be divided into two subfamilies: receptor tyrosine kinases, which have an intracellular tyrosine kinase domain, a transmembrane domain and an extracellular ligand-binding domain; and non-receptor (cytoplasmic) tyrosine kinases, which are soluble, cytoplasmic kinases.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
My favorites 
My labels 
Login
