Recently, evidence has accumulated in support of the heterologous expression of functional membrane proteins and their complexes on extracellular baculovirus particles (budded virus, BV). In this study, we attempted to apply this BV display system to detect G-protein-coupled receptor (GPCR) signaling. We infected Sf9 cells with a combination of four recombinant baculoviruses individually encoding the dopamine D1 receptor (DR-D1), G-protein alpha-subunit (Galpha(s)), G-protein beta(1)gamma(2) subunit dimer (Gbeta(1)gamma(2)), and adenylyl cyclase type VI (ACVI). The recovered BV fraction produced cAMP in response to the stimulation with dopamine. Co-expression of all three G-protein subunits in addition to receptor and ACVI led to a maximal response. BV co-expressing DR-D1, Galpha(s), Gbeta(1)gamma(2), and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other Galpha(s)-coupled receptors together with Galpha(s), Gbeta(1)gamma(2), and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, Galpha(s) and ACVI is reconstituted on BV. Since BV is essentially free of endogenous GPCRs, this BV co-display system should prove highly useful for the development of functional assay systems for GPCRs.
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
Previously, we reported that lysophosphatidic acid (LPA)-induced adenosine 3',5'-cyclic monophosphate (cAMP) production by human diploid fibroblasts depends on the age of the fibroblasts. In this study, we examined the role of A-kinase anchoring proteins (AKAP) in the regulation of LPA-stimulated cAMP production in senescent fibroblasts. We found that levels of protein kinase C (PKC)-dependent AKAPs, such as Gravin and AKAP79, were elevated in senescent cells. Co-immunoprecipitation experiments revealed that Gravin and AKAP79 do not associate with adenylyl cyclase type 2 (AC2) but bind to AC4/6, which interacts with calcium-dependent PKCs alpha/beta both in young and senescent fibroblasts. When the expression of Gravin and AKAP79 was blocked by small interference RNA transfection, the basal level of cAMP was greatly reduced and the cAMP status after LPA treatment was also reversed. Protein kinase A showed a similar pattern in terms of its basal activity and LPA-dependent modulation. These data suggest that Gravin and to a lesser extent, AKAP79, may play important roles in maintaining the basal AC activity and in coupling the AC systems to inhibitory signals such as Gialpha in young cells, and to stimulatory signals such as PKCs in senescent cells. This study also demonstrates that Gravin is especially important for the long-term activation of PKC by LPA in senescent cells. We conclude that LPA-dependent increased level of cAMP in senescent human diploid fibroblasts is associated with increases in Gravin levels resulting in its increased binding with and activation of calcium-dependent PKC alpha/beta and AC4/6.
Interacting selectively and non-covalently with a protein kinase, any enzyme that catalyzes the transfer of a phosphate group, usually from ATP, to a protein substrate.
Recently, evidence has accumulated in support of the heterologous expression of functional membrane proteins and their complexes on extracellular baculovirus particles (budded virus, BV). In this study, we attempted to apply this BV display system to detect G-protein-coupled receptor (GPCR) signaling. We infected Sf9 cells with a combination of four recombinant baculoviruses individually encoding the dopamine D1 receptor (DR-D1), G-protein alpha-subunit (Galpha(s)), G-protein beta(1)gamma(2) subunit dimer (Gbeta(1)gamma(2)), and adenylyl cyclase type VI (ACVI). The recovered BV fraction produced cAMP in response to the stimulation with dopamine. Co-expression of all three G-protein subunits in addition to receptor and ACVI led to a maximal response. BV co-expressing DR-D1, Galpha(s), Gbeta(1)gamma(2), and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other Galpha(s)-coupled receptors together with Galpha(s), Gbeta(1)gamma(2), and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, Galpha(s) and ACVI is reconstituted on BV. Since BV is essentially free of endogenous GPCRs, this BV co-display system should prove highly useful for the development of functional assay systems for GPCRs.
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 catecholamine stimulus. A catecholamine is any of a group of biogenic amines that includes 4-(2-aminoethyl)pyrocatechol [4-(2-aminoethyl)benzene-1,2-diol] and derivatives formed by substitution.
Recently, evidence has accumulated in support of the heterologous expression of functional membrane proteins and their complexes on extracellular baculovirus particles (budded virus, BV). In this study, we attempted to apply this BV display system to detect G-protein-coupled receptor (GPCR) signaling. We infected Sf9 cells with a combination of four recombinant baculoviruses individually encoding the dopamine D1 receptor (DR-D1), G-protein alpha-subunit (Galpha(s)), G-protein beta(1)gamma(2) subunit dimer (Gbeta(1)gamma(2)), and adenylyl cyclase type VI (ACVI). The recovered BV fraction produced cAMP in response to the stimulation with dopamine. Co-expression of all three G-protein subunits in addition to receptor and ACVI led to a maximal response. BV co-expressing DR-D1, Galpha(s), Gbeta(1)gamma(2), and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other Galpha(s)-coupled receptors together with Galpha(s), Gbeta(1)gamma(2), and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, Galpha(s) and ACVI is reconstituted on BV. Since BV is essentially free of endogenous GPCRs, this BV co-display system should prove highly useful for the development of functional assay systems for GPCRs.
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 prostagladin E stimulus.
Recently, evidence has accumulated in support of the heterologous expression of functional membrane proteins and their complexes on extracellular baculovirus particles (budded virus, BV). In this study, we attempted to apply this BV display system to detect G-protein-coupled receptor (GPCR) signaling. We infected Sf9 cells with a combination of four recombinant baculoviruses individually encoding the dopamine D1 receptor (DR-D1), G-protein alpha-subunit (Galpha(s)), G-protein beta(1)gamma(2) subunit dimer (Gbeta(1)gamma(2)), and adenylyl cyclase type VI (ACVI). The recovered BV fraction produced cAMP in response to the stimulation with dopamine. Co-expression of all three G-protein subunits in addition to receptor and ACVI led to a maximal response. BV co-expressing DR-D1, Galpha(s), Gbeta(1)gamma(2), and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other Galpha(s)-coupled receptors together with Galpha(s), Gbeta(1)gamma(2), and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, Galpha(s) and ACVI is reconstituted on BV. Since BV is essentially free of endogenous GPCRs, this BV co-display system should prove highly useful for the development of functional assay systems for GPCRs.
Recently, evidence has accumulated in support of the heterologous expression of functional membrane proteins and their complexes on extracellular baculovirus particles (budded virus, BV). In this study, we attempted to apply this BV display system to detect G-protein-coupled receptor (GPCR) signaling. We infected Sf9 cells with a combination of four recombinant baculoviruses individually encoding the dopamine D1 receptor (DR-D1), G-protein alpha-subunit (Galpha(s)), G-protein beta(1)gamma(2) subunit dimer (Gbeta(1)gamma(2)), and adenylyl cyclase type VI (ACVI). The recovered BV fraction produced cAMP in response to the stimulation with dopamine. Co-expression of all three G-protein subunits in addition to receptor and ACVI led to a maximal response. BV co-expressing DR-D1, Galpha(s), Gbeta(1)gamma(2), and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other Galpha(s)-coupled receptors together with Galpha(s), Gbeta(1)gamma(2), and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, Galpha(s) and ACVI is reconstituted on BV. Since BV is essentially free of endogenous GPCRs, this BV co-display system should prove highly useful for the development of functional assay systems for GPCRs.
The process in which a signal is passed on to downstream components within the cell, which become activated themselves to further propagate the signal and finally trigger a change in the function or state of the cell.
IEAInterPro 2 GO
Enzymatic activity
This protein acts as an enzyme. It is known to catalyze the following reaction
Adenylyl cyclases (AC), a family of enzymes that catalyze the synthesis of cyclic AMP, are critical regulators of cellular functions. The activity of adenylyl cyclase is stimulated by a range of hormone receptors, primarily via interactions with G-proteins; however, recently we identified an alternate mechanism by which growth factors sensitize adenylyl cyclase activation. We suggested that this mechanism might involve a Raf kinase-mediated serine phosphorylation of adenylyl cyclase. However, the direct involvement of a specific form of Raf kinase is yet to be demonstrated. Furthermore, whether this mechanism is generalized to other isoforms of adenylyl cyclase is unknown. In human embryonic kidney 293 cells, we now demonstrate that in reconstitution studies, c-Raf kinase can mediate phosphorylation of AC VI. Furthermore, AC VI coimmunoprecipitates with c-Raf. Raf kinase-dependent regulation of adenylyl cyclase VI is dependent on the integrity of Ser750 in the fourth intracellular loop of the enzyme and Ser603/Ser608 in the C1b region of the molecule. To examine how generalized this effect is, we studied representative isoforms of the major subfamilies of adenylyl cyclase viz., AC I, AC II, and AC V. Raf kinase-dependent sensitization/ phosphorylation of adenylyl cyclases is common to AC VI, AC V, and AC II isoforms but not AC I. In aggregate, these studies indicate that Raf kinase associates with adenylyl cyclases. Furthermore, Raf kinase regulation of adenylyl cyclase is isoform-selective. These functional interactions (as well as the physical association) between adenylyl cyclases and Raf kinases suggest an important but previously unrecognized interaction between these two key regulatory enzymes.
Enzyme that catalyzes the cleavage of C-C, C-O, C-S, C-N or other bonds by other means than by hydrolysis or oxidation, with two substrates in one reaction direction, and one in the other. In the latter direction, a molecule (of carbon dioxide, water, etc) is eliminated, thus creating a new double bond or a new ring.
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.
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