The recent identification of the Vzg-1/Edg2 protein as a functional G protein-coupled receptor for lysophosphatidic acid (LPA) has allowed a sequence-based search for new genes that may encode novel subtypes of LPA receptors. A human cDNA encoding a G protein-coupled receptor, designated Edg4, was identified by searching the GenBankTM for homologs of the human Edg2 LPA receptor. The Edg4 protein is 46% identical and 72% similar in amino acid sequence to human Edg2. When overexpressed in Jurkat T cells, the Edg4 protein mediated LPA-induced activation of a serum response element reporter gene with LPA concentration dependence (EC50 of 10 nM) and specificity. This LPA-induced reporter gene activation could be partially inhibited by pretreatment with pertussis toxin or C3 exoenzyme, suggesting requirements for both a Gi protein and Rho GTPase. Overexpression of Edg4 in Jurkat cells also led to increases in specific binding sites for [3H]LPA. Northern blots revealed that two edg4 mRNA transcripts of 1.8 and 8 kilobases are distributed very differently from edg2 mRNAs in adult human tissues and several cancer cell lines. The existence and distinctive tissue expression of structurally different subtypes of LPA receptors may provide one basis for tissue-specific functions and permit independent regulation of each subtype of LPA receptor.

The recent identification of the Vzg-1/Edg2 protein as a functional G protein-coupled receptor for lysophosphatidic acid (LPA) has allowed a sequence-based search for new genes that may encode novel subtypes of LPA receptors. A human cDNA encoding a G protein-coupled receptor, designated Edg4, was identified by searching the GenBankTM for homologs of the human Edg2 LPA receptor. The Edg4 protein is 46% identical and 72% similar in amino acid sequence to human Edg2. When overexpressed in Jurkat T cells, the Edg4 protein mediated LPA-induced activation of a serum response element reporter gene with LPA concentration dependence (EC50 of 10 nM) and specificity. This LPA-induced reporter gene activation could be partially inhibited by pretreatment with pertussis toxin or C3 exoenzyme, suggesting requirements for both a Gi protein and Rho GTPase. Overexpression of Edg4 in Jurkat cells also led to increases in specific binding sites for [3H]LPA. Northern blots revealed that two edg4 mRNA transcripts of 1.8 and 8 kilobases are distributed very differently from edg2 mRNAs in adult human tissues and several cancer cell lines. The existence and distinctive tissue expression of structurally different subtypes of LPA receptors may provide one basis for tissue-specific functions and permit independent regulation of each subtype of LPA receptor.

Lysophosphatidic acid (LPA) is a major constituent of blood and is involved in a variety of physiological and pathophysiological processes. LPA signals via the ubiquitously expressed G protein-coupled receptors (GPCRs), LPA(1) and LPA(2) that are specific for LPA. However, in large, the molecular mechanisms that regulate the signalling of these receptors are unknown. We show that the small GTPase RalA associates with both LPA(1) and LPA(2) in human embryonic kidney (HEK 293) cells and that stimulation of LPA(1) receptors with LPA triggers the activation of RalA. While RalA was not found to play a role in the endocytosis of LPA receptors, we reveal that LPA(1) receptor stimulation promoted Ral-dependent phospholipase C activity. Furthermore, we found that GRK2 is required for the desensitization of LPA(1) and LPA(2) and have identified a novel interaction between RalA and GRK2, which is promoted by LPA(1) receptor activity. Taken together, these results establish RalA and GRK2 as key regulators of LPA receptor signalling and demonstrate for the first time that LPA(1) activity facilitates the formation of a novel protein complex between these two proteins.

Lysophosphatidic acid (LPA) induces actin rearrangement, focal adhesion assembly, and cell migration through the activation of small G protein Rho and its downstream effectors. These diverse cellular responses are mediated by its associated G protein-coupled receptors. However, the mechanisms and specificity by which these LPA receptors mediate LPA actions are still poorly understood. Here we show that LPA stimulation promotes the interaction of the LPA(2) receptor with a focal adhesion molecule, TRIP6 (thyroid receptor interacting protein 6)/ZRP-1 (zyxin-related protein 1). TRIP6 directly binds to the carboxyl-terminal tail of the LPA(2) receptor through its LIM domains. LPA-dependent recruitment of TRIP6 to the plasma membrane promotes its targeting to focal adhesions and co-localization with actin stress fibers. In addition, TRIP6 associates with the components of focal complexes including paxillin, focal adhesion kinase, c-Src, and p130(cas) in an agonist-dependent manner. Overexpression of TRIP6 augments LPA-induced cell migration; in contrast, suppression of endogenous TRIP6 expression by a TRIP6-specific small interfering RNA reduces it in SKOV3 ovarian cancer cells. Strikingly, the association with TRIP6 is specific to the LPA(2) receptor but not LPA(1) or LPA(3) receptor, indicating a specific role for TRIP6 in regulating LPA(2) receptor-mediated signaling. Taken together, our results suggest that TRIP6 functions at a point of convergence between the activated LPA(2) receptor and downstream signals involved in cell adhesion and migration.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells that line the airway, gut, and exocrine glands; it is well established that CFTR plays a pivotal role in cholera toxin (CTX)-induced secretory diarrhea. Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in blood and foods, has been reported to play a vital role in a variety of conditions involving gastrointestinal wound repair, apoptosis, inflammatory bowel disease, and diarrhea. Here we show, for the first time, that type 2 LPA receptors (LPA2) are expressed at the apical surface of intestinal epithelial cells, where they form a macromolecular complex with Na+/H+ exchanger regulatory factor-2 and CFTR through a PSD95/Dlg/ZO-1-based interaction. LPA inhibited CFTR-dependent iodide efflux through LPA2-mediated Gi pathway, and LPA inhibited CFTR-mediated short-circuit currents in a compartmentalized fashion. CFTR-dependent intestinal fluid secretion induced by CTX in mice was reduced substantially by LPA administration; disruption of this complex using a cell-permeant LPA2-specific peptide reversed LPA2-mediated inhibition. Thus, LPA-rich foods may represent an alternative method of treating certain forms of diarrhea.

Mobilization of intracellular Ca2+ is a critical cellular response to lysophosphatidic acid (LPA) in many cell types. Recent identification of endothelial differentiation gene (Edg) 2 and Edg4 as subtypes of G protein-coupled receptors for LPA allowed examination of the Ca2+ mobilization mediated specifically by each subtype. To reduce endogenous background levels while enhancing recombinant receptor-specific signals, the aequorin luminescence method was used to quantify cytoplasmic Ca2+ levels. In TAg-Jurkat T cells transiently co-transfected with apoaequorin and human Edg2 or Edg4 cDNA, LPA dose-dependently increased light emission triggered by increased Ca2+ bound to aequorin. N-Palmitoyl-L-serine-phosphoric acid and N-palmitoyl-L-tyrosine-phosphoric acid, which had been previously shown to be antagonists for Xenopus laevis LPA receptors, did not antagonize the Ca2+-mobilizing effects of Edg2 and Edg4. Surprisingly, they acted as agonists or partial agonists for Edg2 and Edg4. The Ca2+ mobilization by Edg2 and Edg4 was further characterized in stable transfectants of rat HTC4 hepatoma cells. By using the fura-2 fluorescence method, a difference in the kinetics of Ca2+ flux with Edg2 and Edg4 was observed. With Edg2, but not Edg4, the initial increase in the Ca2+ concentration was followed by a sustained influx of extracellular Ca2+. The coincident production of inositol phosphates and the inhibition of Ca2+ mobilization by the phospholipase C inhibitor U73122 strongly suggested that Edg2 and Edg4 mobilize Ca2+ through inositol trisphosphate generated by phospholipase C activation. Pertussis toxin almost completely blocked LPA-induced Ca2+ mobilization by Edg2 but only partially blocked that by Edg4, which suggests that Edg2 transduces Ca2+ mobilization largely through pertussis toxin-sensitive Gi proteins, whereas Edg4 requires both Gi and Gq.

Mobilization of intracellular Ca2+ is a critical cellular response to lysophosphatidic acid (LPA) in many cell types. Recent identification of endothelial differentiation gene (Edg) 2 and Edg4 as subtypes of G protein-coupled receptors for LPA allowed examination of the Ca2+ mobilization mediated specifically by each subtype. To reduce endogenous background levels while enhancing recombinant receptor-specific signals, the aequorin luminescence method was used to quantify cytoplasmic Ca2+ levels. In TAg-Jurkat T cells transiently co-transfected with apoaequorin and human Edg2 or Edg4 cDNA, LPA dose-dependently increased light emission triggered by increased Ca2+ bound to aequorin. N-Palmitoyl-L-serine-phosphoric acid and N-palmitoyl-L-tyrosine-phosphoric acid, which had been previously shown to be antagonists for Xenopus laevis LPA receptors, did not antagonize the Ca2+-mobilizing effects of Edg2 and Edg4. Surprisingly, they acted as agonists or partial agonists for Edg2 and Edg4. The Ca2+ mobilization by Edg2 and Edg4 was further characterized in stable transfectants of rat HTC4 hepatoma cells. By using the fura-2 fluorescence method, a difference in the kinetics of Ca2+ flux with Edg2 and Edg4 was observed. With Edg2, but not Edg4, the initial increase in the Ca2+ concentration was followed by a sustained influx of extracellular Ca2+. The coincident production of inositol phosphates and the inhibition of Ca2+ mobilization by the phospholipase C inhibitor U73122 strongly suggested that Edg2 and Edg4 mobilize Ca2+ through inositol trisphosphate generated by phospholipase C activation. Pertussis toxin almost completely blocked LPA-induced Ca2+ mobilization by Edg2 but only partially blocked that by Edg4, which suggests that Edg2 transduces Ca2+ mobilization largely through pertussis toxin-sensitive Gi proteins, whereas Edg4 requires both Gi and Gq.

The recent identification of the Vzg-1/Edg2 protein as a functional G protein-coupled receptor for lysophosphatidic acid (LPA) has allowed a sequence-based search for new genes that may encode novel subtypes of LPA receptors. A human cDNA encoding a G protein-coupled receptor, designated Edg4, was identified by searching the GenBankTM for homologs of the human Edg2 LPA receptor. The Edg4 protein is 46% identical and 72% similar in amino acid sequence to human Edg2. When overexpressed in Jurkat T cells, the Edg4 protein mediated LPA-induced activation of a serum response element reporter gene with LPA concentration dependence (EC50 of 10 nM) and specificity. This LPA-induced reporter gene activation could be partially inhibited by pretreatment with pertussis toxin or C3 exoenzyme, suggesting requirements for both a Gi protein and Rho GTPase. Overexpression of Edg4 in Jurkat cells also led to increases in specific binding sites for [3H]LPA. Northern blots revealed that two edg4 mRNA transcripts of 1.8 and 8 kilobases are distributed very differently from edg2 mRNAs in adult human tissues and several cancer cell lines. The existence and distinctive tissue expression of structurally different subtypes of LPA receptors may provide one basis for tissue-specific functions and permit independent regulation of each subtype of LPA receptor.

The recent identification of the Vzg-1/Edg2 protein as a functional G protein-coupled receptor for lysophosphatidic acid (LPA) has allowed a sequence-based search for new genes that may encode novel subtypes of LPA receptors. A human cDNA encoding a G protein-coupled receptor, designated Edg4, was identified by searching the GenBankTM for homologs of the human Edg2 LPA receptor. The Edg4 protein is 46% identical and 72% similar in amino acid sequence to human Edg2. When overexpressed in Jurkat T cells, the Edg4 protein mediated LPA-induced activation of a serum response element reporter gene with LPA concentration dependence (EC50 of 10 nM) and specificity. This LPA-induced reporter gene activation could be partially inhibited by pretreatment with pertussis toxin or C3 exoenzyme, suggesting requirements for both a Gi protein and Rho GTPase. Overexpression of Edg4 in Jurkat cells also led to increases in specific binding sites for [3H]LPA. Northern blots revealed that two edg4 mRNA transcripts of 1.8 and 8 kilobases are distributed very differently from edg2 mRNAs in adult human tissues and several cancer cell lines. The existence and distinctive tissue expression of structurally different subtypes of LPA receptors may provide one basis for tissue-specific functions and permit independent regulation of each subtype of LPA receptor.