The G protein-coupled receptor GPR4 suppresses ERK activation in a ligand-independent manner

The lysophospholipids, lysophosphatidic acid, sphingosine-1-phosphate, and sphingosylphosphorylcholine (SPC), are bioactive lipid molecules that regulate diverse biological processes. Although the specific G protein-coupled receptors for lysophosphatidic acid and sphingosine-1-phosphate have been well-characterized, much less is known of the SPC receptors. It has been reported that ovarian cancer G protein-coupled receptor 1 (OGR1) is a high affinity receptor for SPC, and its closely related homologue GPR4 is a high affinity receptor for SPC with low affinity for lysophosphatidylcholine (LPC). However, in a functional assay to examine the specificity of ligand binding, we found that neither SPC nor LPC, or other related lysophospholipids, induced internalization of GPR4 from the plasma membrane. In agreement, these lysolipids also did not induce translocation of beta-arrestin2-GFP from the cytosol to the plasma membrane in GPR4 expressing cells. However, when these cells were cotransfected with G protein-coupled receptor kinase 2, in the absence of added ligands, beta-arrestin2-GFP accumulated in cytoplasmic vesicles, reminiscent of vesicular labeling usually observed after agonist stimulation of GPCRs. In addition, neither SPC nor LPC stimulated the binding of GTPgammaS to membranes prepared from GPR4 expressing cells and did not activate ERK1/2. Surprisingly, enforced expression of GPR4 inhibited activation of ERK1/2 induced by several stimuli, including SPC, sphingosine-1-phosphate, and even EGF. Collectively, our results suggest that SPC and LPC are not the ligands for GPR4 and that this receptor may constitutively inhibit ERK1/2 activation.     

Sphingosylphosphorylcholine and lysophosphatidylcholine: G protein-coupled receptors and receptor-mediated signal transduction

In recent years, certain lysophospholipids (lyso-PLs) have been recognized as important cell signaling molecules. Among them, two phosphorylcholine-containing lyso-PLs, sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC), have been shown to be involved in many cellular processes and are produced under physiological and pathological conditions. Although signaling properties of SPC and LPC have been studied in a variety of cellular systems, specific cell membrane receptors for SPC and LPC have not been identified previously. Recently, ovarian cancer G protein-coupled receptor 1 (OGR1, also known as GPR68), G protein-coupled receptor 4 (GPR4), and G2A have been identified as receptors for SPC and LPC. The signaling and ligand-binding properties of these receptors are reviewed here. These discoveries provide an intriguing opportunity and a novel approach in studying the pathophysiological roles of SPC and LPC and their receptors.     

Receptors for protons or lipid messengers or both?

The subfamily of G protein-coupled receptors comprising GPR4, OGR1, TDAG8, and G2A was originally characterized as a group of proteins mediating biological responses to the lipid messengers sphingosylphosphorylcholine (SPC), lysophosphatidylcholine (LPC), and psychosine. We challenged this view by reporting that OGR1 and GPR4 sense acidic pH and that this process is not affected by concentrations of SPC or LPC previously reported as agonistic. The original publications describing GPR4, OGR1, and G2A as receptors for LPC or SPC have now been retracted, and the first studies exploring receptors of this family as pH sensors in physiology have appeared. Here we review the status of this field and we confirm that GPR4, OGR1, and TDAG8 should be considered as proton-sensing receptors. Negative regulation of these receptors by high micromolar concentrations of lipids appears not specific in our experiments.     

Receptors for Protons or Lipid Messengers or Both?

The subfamily of G protein-coupled receptors comprising GPR4, OGR1, TDAG8, and G2A was originally characterized as a group of proteins mediating biological responses to the lipid messengers sphingosylphosphorylcholine (SPC), lysophosphatidylcholine (LPC), and psychosine. We challenged this view by reporting that OGR1 and GPR4 sense acidic pH and that this process is not affected by concentrations of SPC or LPC previously reported as agonistic. The original publications describing GPR4, OGR1, and G2A as receptors for LPC or SPC have now been retracted, and the first studies exploring receptors of this family as pH sensors in physiology have appeared. Here we review the status of this field and we confirm that GPR4, OGR1, and TDAG8 should be considered as proton-sensing receptors. Negative regulation of these receptors by high micromolar concentrations of lipids appears not specific in our experiments.     

G2A is a proton-sensing G-protein-coupled receptor antagonized by lysophosphatidylcholine

G2A (from G2 accumulation) is a G-protein-coupled receptor (GPCR) that regulates the cell cycle, proliferation, oncogenesis, and immunity. G2A shares significant homology with three GPCRs including ovarian cancer GPCR (OGR1/GPR68), GPR4, and T cell death-associated gene 8 (TDAG8). Lysophosphatidylcholine (LPC) and sphingosylphosphorylcholine (SPC) were reported as ligands for G2A and GPR4 and for OGR1 (SPC only), and a glycosphingolipid psychosine was reported as ligand for TDAG8. As OGR1 and GPR4 were reported as proton-sensing GPCRs (Ludwig, M. G., Vanek, M., Guerini, D., Gasser, J. A., Jones, C. E., Junker, U., Hofstetter, H., Wolf, R. M., and Seuwen, K. (2003) Nature 425, 93-98), we evaluated the proton-sensing function of G2A. Transient expression of G2A caused significant activation of the zif 268 promoter and inositol phosphate (IP) accumulation at pH 7.6, and lowering extracellular pH augmented the activation only in G2A-expressing cells. LPC inhibited the pH-dependent activation of G2A in a dose-dependent manner in these assays. Thus, G2A is another proton-sensing GPCR, and LPC functions as an antagonist, not as an agonist, and regulates the proton-dependent activation of G2A.     

Novel clusters of receptors for sphingosine-1-phosphate, sphingosylphosphorylcholine, and (lyso)-phosphatidic acid: new receptors for "old" ligands

The (lyso)phospholipid mediators sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), sphingosylphosphorylcholine (SPC), and phosphatidic acid (PA) regulate diverse cellular responses such as proliferation, survival and death, cytoskeletal rearrangements, cell motility, and differentiation among many others. Signaling is complex and many signaling events are mediated through the activation of cell surface seven transmembrane (7TM) G protein coupled receptors. Five high affinity receptors for S1P have been identified so far and named S1P(1, 2,3,4,5) (formerly referred to as endothelial differentiation gene (edg)1, 5, 3, 6, 8). Recently, the orphan receptor GPR63 was identified a low affinity S1P receptor structurally distant from the S1P(1-5) family. The orphan GPR3, 6, 12 cluster, phylogenetically related to the edg and melanocortin receptors appears to be subject to modulation by S1P and SPC although all three receptors are strong constitutive stimulators of the Galphas-adenylyl cyclase (AC) pathway and would not require additional ligand stimulation but rather inverse agonism to control activity. Ovarian cancer G protein coupled receptor 1 (OGR1) and GPR4, two structurally closely related receptors were assigned in functional and binding studies as high affinity molecular targets for SPC. Very recently, however, both OGR1 and GPR4 were described as receptors endowed with the ability to signal cells in response to protons. LPA exerts its biological effects through the activation of G protein coupled LPA(1-3) receptors (formerly referred to as edg2, 4, 7). A fourth high affinity LPA receptor has been identified: P2Y9 (GPR23) structurally related to nucleotide receptors and phylogenetically quite distant from the high affinity LPA(1-3) cluster. This review attempts to give an overview about the existing families of lysophosholipid receptors and the spectrum of lipid agonists they use as high or low affinity ligands to relay extracellular signals into intracellular responses. Recently deorphaned lipid receptors, within and outside the known lipid receptor clusters will receive particular attention.     

质子感知受体与肿瘤发生和肿瘤转移之间的关系

G蛋白偶联受体家族卵巢癌G蛋白偶联受体1（ovarian cancer G protein-coupled receptor 1, OGR1）亚家族的OGR1、T细胞死亡偶联基因8（T－cell death associated gene 8, TDAG8）、G 蛋白偶联受体4（G protein－coupled receptor 4, GPR4）及诱导细胞停滞于G2/M期的G蛋白偶联受体G2A（from G2 accumulation）4 种受体是最新发现的一类质子感知受体.除了质子,体内又有它们各自特定的脂质分 子配体活化这些受体来调节细胞机能.该类受体广泛分布于人的各种正常组织和肿瘤 组织细胞中,在肿瘤的发生与转移、细胞骨架重组等生理病理过程中发挥双重作用. 正常表达时它们有一定的抑制肿瘤作用,但这些受体的异常表达或过表达使某些组织 和细胞恶性转化,导致肿瘤的发生.本文综述了在肿瘤组织的酸性微环境中,细胞表 达的质子（pH）感知受体对肿瘤发生与肿瘤转移的调节作用及其相关的信号通路.     

Sphingosylphosphorylcholine enhances calcium entry in thyroid FRO cells by a mechanism dependent on protein kinase C

Several sphingolipid derivatives, including sphingosylphosphorylcholine (SPC), regulate a multitude of biological processes. In the present study we show that both human thyroid cancer cells (FRO cells) and normal human thyroid cells express G protein-coupled receptor 4 (GPR4) and ovarian cancer G protein-coupled receptor 1 (OGR1), putative SPC-specific receptors. In FRO cells SPC evoked a concentration-dependent increase in intracellular free calcium concentration ([Ca2+]i) in a calcium containing, but not in a calcium-free buffer. Sphingosine 1-phosphate (S1P) evoked an increase in [Ca2+]i in both a calcium containing and a calcium-free buffer. The phospholipase C (PLC) inhibitor U 73122 potently attenuated the effect of SPC, suggesting that effects of SPC were mediated by a G protein coupled receptor. Overnight pretreatment of the cells with pertussis toxin did not affect the SPC-evoked response. Interestingly, SPC did not evoke an increase in inositol phosphates, although S1P did so. Furthermore, in cells pretreated with thapsigargin to deplete intracellular calcium stores, SPC still evoked an increase in [Ca2+]i, suggesting that SPC mainly evoked entry of extracellular calcium. When the cells were pretreated with the protein kinase C (PKC) inhibitor GF 109203X, or when the cells were pretreated with PMA for 24 h, the SPC-evoked calcium entry was attenuated. Thus, the SPC-evoked calcium entry was apparently dependent on PKC. In sharp contrast, the increase in [Ca2+]i evoked by S1P was not sensitive to GF 109203X. Furthermore, the calcium entry evoked by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol was not inhibited by GF 109203X. In addition, SPC decreased the incorporation of 3H-thymidine in a concentration-dependent manner in FRO cells. Taken together, SPC may be an important factor regulating thyroid cancer cell function.     

Lysophosphatidylcholine impairs endothelial barrier function through the G protein-coupled receptor GPR4

Abundant evidence indicates that lysophosphatidylcholine (LPC) is proinflammatory and atherogenic. In the vascular endothelium, LPC increases permeability and expression of proinflammatory molecules such as adhesion molecules and cytokines. Yet, mechanisms by which LPC mediates these activities remain unclear and controversial. Recent evidence implicates involvement of a novel subfamily of G protein-coupled receptors (GPR4, G2A, OGR1, and TDAG8) that are sensitive to lysolipids and protons. We previously reported that one of these receptors, GPR4, is selectively expressed by a variety of endothelial cells and therefore hypothesize that the LPC-stimulated endothelial barrier dysfunction is mediated through GPR4. We developed a peptide Ab against GPR4 that detected GPR4 expression in transfected COS 7 cells and endogenous GPR4 expression in endothelial cells by Western blot. Endothelial cells infected with a retrovirus containing small interference RNA (siRNA) to GPR4 resulted in 40-50% decreased GPR4 expression, which corresponded with partial prevention of the LPC-induced 1) decrease in transendothelial resistance, 2) stress fiber formation, and 3) activation of RhoA. Furthermore, coexpression of the siRNA-GPR4 with a siRNA-resistant mutant GPR4 fully restored the LPC-induced resistance decrease. However, extracellular pH of <7.4 did not alter baseline or LPC-stimulated resistances. The results provide strong evidence that the LPC-mediated endothelial barrier dysfunction is regulated by endogenous GPR4 in endothelial cells and suggest that GPR4 may play a critical role in the inflammatory responses activated by LPC.     

Lysophospholipid receptors: signalling, pharmacology and regulation by lysophospholipid metabolism

The lysophospholipids, sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC), activate diverse groups of G-protein-coupled receptors that are widely expressed and regulate decisive cellular functions. Receptors of the endothelial differentiation gene family are activated by S1P (S1P(1-5)) or LPA (LPA(1-3)); two more distantly related receptors are activated by LPA (LPA(4/5)); the GPR(3/6/12) receptors have a high constitutive activity but are further activated by S1P and/or SPC; and receptors of the OGR1 cluster (OGR1, GPR4, G2A, TDAG8) appear to be activated by SPC, LPC, psychosine and/or protons. G-protein-coupled lysophospholipid receptors regulate cellular Ca(2+) homoeostasis and the cytoskeleton, proliferation and survival, migration and adhesion. They have been implicated in development, regulation of the cardiovascular, immune and nervous systems, inflammation, arteriosclerosis and cancer. The availability of S1P and LPA at their G-protein-coupled receptors is regulated by enzymes that generate or metabolize these lysophospholipids, and localization plays an important role in this process. Besides FTY720, which is phosphorylated by sphingosine kinase-2 and then acts on four of the five S1P receptors of the endothelial differentiation gene family, other compounds have been identified that interact with more ore less selectivity with lysophospholipid receptors.     

Prostaglandin I(2) production and cAMP accumulation in response to acidic extracellular pH through OGR1 in human aortic smooth muscle cells

Ovarian cancer G-protein-coupled receptor 1 (OGR1) and GPR4 have recently been identified as proton-sensing or extracellular pH-responsive G-protein-coupled receptors stimulating inositol phosphate production and cAMP accumulation, respectively. In the present study, we found that OGR1 and GPR4 mRNAs were expressed in human aortic smooth muscle cells (AoSMCs). Acidic extracellular pH induced inositol phosphate production, a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), and cAMP accumulation in these cells. When small interfering RNAs (siRNAs) targeted for OGR1 and GPR4 were transfected to the cells, the acid-induced inositol phosphate production and [Ca(2+)](i) increase were markedly inhibited by the OGR1 siRNA but not by the GPR4 siRNA. Unexpectedly, the acid-induced cAMP accumulation was also largely inhibited by OGR1 siRNA but only slightly by GPR4 siRNA. Acidic extracellular pH also stimulated prostaglandin I2 (PGI(2)) production, which was again inhibited by OGR1 siRNA. The specific inhibitors for extracellular signal-regulated kinase kinase and cyclooxygenase attenuated the acid-induced PGI(2) production and cAMP accumulation without changes in the inositol phosphate production. A specific inhibitor of phospholipase C also inhibited the acid-induced cAMP accumulation. In conclusion, OGR1 is a major receptor involved in the extracellular acid-induced stimulation of PGI(2) production and cAMP accumulation in AoSMCs. The cAMP accumulation may occur through OGR1-mediated stimulation of the phospholipase C/cyclooxygenase/PGI(2) pathway.     

Inflammatory stress increases receptor for lysophosphatidylcholine in human microvascular endothelial cells

The atherogenic serum lysophosphatidylcholine (LPC) is known to mediate vascular endothelial responses ranging from upregulation of adhesion molecules and growth factors to secretion of chemokines and superoxide anion. We investigated whether endothelial cells express receptors for LPC, which may account for their actions. Human brain microvascular (HBMEC) and dermal microvascular endothelial cells (HMEC) were prepared for RT-PCR analysis for possible expression of the G protein-coupled receptors, GPR4 and G2A, which are believed to be specific LPC receptors. Results indicated that HBMEC expressed low basal GPR4 mRNA, but stimulation with tumor necrosis factor-alpha (TNF-alpha) (100 U/ml) or H2O2 (50 micromol/l) for 2 h or overnight upregulated expression severalfold. In contrast, HMEC expressed high basal GPR4 mRNA, which was not further increased by either TNF-alpha or H2O2 stimulation. Another LPC receptor, G2A, was not detected in either endothelial cell type. Competition binding studies were made to evaluate specific binding of [3H]LPC to the intact endothelial cell monolayer. Basal specific [3H]LPC binding in HBMEC was approximately eight times lower than in HMEC; however, TNF-alpha or H2O2 stimulation increased [3H]LPC binding on HMBEC but not HMEC. The results indicated that GPR4 expression was consistent with specific [3H]LPC binding. Overall, we report that endothelial cells selectively expressed GPR4, a specific LPC receptor. Furthermore, GPR4 expression by HBMEC, but not HMEC, was increased by inflammatory stresses. We conclude that endogenous GPR4 in endothelial cells may be a potential G protein-coupled receptor by which LPC signals proinflammatory activities.     

Sphingosylphosphorylcholine induces proliferation of human adipose tissue-derived mesenchymal stem cells via activation of JNK

Sphingosylphosphorylcholine (SPC) has been implicated in a variety of cellular responses, including proliferation and differentiation. In this study, we demonstrate that d-erythro-SPC, but not l-threo-SPC, stereoselectively stimulated the proliferation of human adipose tissue-derived mesenchymal stem cells (hADSCs), with a maximal increase at 5 microM, and increased the intracellular concentration of Ca(2+) ([Ca(2+)](i)) in hADSCs, which do not express known SPC receptors (i.e., OGR1, GPR4, G2A, and GPR12). The SPC-induced proliferation and increase in [Ca(2+)](i) were sensitive to pertussis toxin (PTX) and the phospholipase C (PLC) inhibitor U73122, suggesting that PTX-sensitive G proteins, Gi or Go, and PLC are involved in SPC-induced proliferation. In addition, SPC treatment induced the phosphorylation of c-Jun and extracellular signal-regulated kinase, and SPC-induced proliferation was completely prevented by pretreatment with the c-Jun N-terminal kinase (JNK)-specific inhibitor SP600125 but not with the MEK-specific inhibitor U0126. Furthermore, the SPC-induced proliferation and JNK activation were completely attenuated by overexpression of a dominant negative mutant of JNK2, and the SPC-induced activation of JNK was inhibited by pretreatment with PTX or U73122. Treatment of hADSCs with lysophosphatidic acid (LPA) receptor antagonist, Ki16425, had no impact on the SPC-induced increase in [Ca(2+)](i). However, SPC-induced proliferation was partially, but significantly, attenuated by pretreatment of the cells with Ki16425.These results indicate that SPC stimulates the proliferation of hADSCs through the Gi/Go-PLC-JNK pathway and that LPA receptors may be responsible in part for the SPC-induced proliferation.     

Lysophospholipid receptors: Signalling, pharmacology and regulation by lysophospholipid metabolism

The lysophospholipids, sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC), activate diverse groups of G-protein-coupled receptors that are widely expressed and regulate decisive cellular functions. Receptors of the endothelial differentiation gene family are activated by S1P (S1P_1-5) or LPA (LPA_1-3); two more distantly related receptors are activated by LPA (LPA_4/5); the GPR_3/6/12 receptors have a high constitutive activity but are further activated by S1P and/or SPC; and receptors of the OGR1 cluster (OGR1, GPR4, G2A, TDAG8) appear to be activated by SPC, LPC, psychosine and/or protons. G-protein-coupled lysophospholipid receptors regulate cellular Ca²⁺ homoeostasis and the cytoskeleton, proliferation and survival, migration and adhesion. They have been implicated in development, regulation of the cardiovascular, immune and nervous systems, inflammation, arteriosclerosis and cancer. The availability of S1P and LPA at their G-protein-coupled receptors is regulated by enzymes that generate or metabolize these lysophospholipids, and localization plays an important role in this process. Besides FTY720, which is phosphorylated by sphingosine kinase-2 and then acts on four of the five S1P receptors of the endothelial differentiation gene family, other compounds have been identified that interact with more ore less selectivity with lysophospholipid receptors.     

Sphingosylphosphorylcholine is a ligand for ovarian cancer G-protein-coupled receptor 1

Sphingosylphosphorylcholine (SPC) is a bioactive lipid that acts as an intracellular and extracellular signalling molecule in numerous biological processes. Many of the cellular actions of SPC are believed to be mediated by the activation of unidentified G-protein-coupled receptors. Here we show that SPC is a high-affinity ligand for an orphan receptor, ovarian cancer G-protein-coupled receptor 1 (OGR1). In OGR1-transfected cells, SPC binds to OGR1 with high affinity (Kd = 33.3 nM) and high specificity and transiently increases intracellular calcium. The specific binding of SPC to OGR1 also activates p42/44 mitogen-activated protein kinases (MAP kinases) and inhibits cell proliferation. In addition, SPC causes internalization of OGR1 in a structurally specific manner.     

The proton-activated G protein coupled receptor OGR1 acutely regulates the activity of epithelial proton transport proteins

The Ovarian cancer G protein-coupled Receptor 1 (OGR1; GPR68) is proton-sensitive in the pH range of 6.8 - 7.8. However, its physiological function is not defined to date. OGR1 signals via inositol trisphosphate and intracellular calcium, albeit downstream events are unclear. To elucidate OGR1 function further, we transfected HEK293 cells with active OGR1 receptor or a mutant lacking 5 histidine residues (H5Phe-OGR1). An acute switch of extracellular pH from 8 to 7.1 (10 nmol/l vs 90 nmol/l protons) stimulated NHE and H(+)-ATPase activity in OGR1-transfected cells, but not in H5Phe-OGR1-transfected cells. ZnCl(2) and CuCl(2) that both inhibit OGR1 reduced the stimulatory effect. The activity was blocked by chelerythrine, whereas the ERK1/2 inhibitor PD 098059 had no inhibitory effect. OGR1 activation increased intracellular calcium in transfected HEK293 cells. We next isolated proximal tubules from kidneys of wild-type and OGR1-deficient mice and measured the effect of extracellular pH on NHE activity in vitro. Deletion of OGR1 affected the pH-dependent proton extrusion, however, in the opposite direction as expected from cell culture experiments. Upregulated expression of the pH-sensitive kinase Pyk2 in OGR1 KO mouse proximal tubule cells may compensate for the loss of OGR1. Thus, we present the first evidence that OGR1 modulates the activity of two major plasma membrane proton transport systems. OGR1 may be involved in the regulation of plasma membrane transport proteins and intra- and/or extracellular pH.     

Homo- and hetero-dimerization of LPA/S1P receptors, OGR1 and GPR4

G protein coupled receptors (GPCRs) form homo- and hetero-dimers or -oligomers, which are functionally important. Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are bioactive lysophopholipids involved in diverse biological processes. We have examined homo- and hetero-dimerization among three major LPA receptors (LPA(1-3)), three major S1P receptors (S1P(1-3)), as well as OGR1 and GPR4. Using LacZ complementation assays, we have shown that LPA receptors form homo- and hetero-dimers within the LPA receptor subgroup and hetero-dimers with other receptors (S1P(1-3) and GPR4). In addition, we have found that although GPR4 and OGR1 share more than 50% homology, GPR4 forms strong homo- and hetero-dimers with LPA and S1P receptors, but OGR1 forms very weak homo-dimer and relatively weak hetero-dimers with other receptors. Using chimeric receptors between GPR4 and OGR1, we have shown that different domains of GPR4 receptor are involved in its dimerization with different GPCRs and more than one domain may be involved in some of the complex formation. Our results suggest that when studying a signal transduction induced by a stimulus, not only is the expression and activation of its own receptor(s), but also the status of the interacting receptors should be taken into consideration.     

Oleate promotes the proliferation of breast cancer cells via the G protein-coupled receptor GPR40

Evidence from epidemiological studies and animal models suggests a link between high levels of dietary fat intake and risk of breast cancer. In addition, obesity, in which circulating lipids are elevated, is associated with increased risk of various cancers. Relative to this point, we previously showed that oleate stimulates the proliferation of breast cancer cells and that phosphatidylinositol 3-kinase plays a role in this process. Nonetheless, questions remain regarding the precise mechanism(s) by which oleate promotes breast cancer cell growth. Pharmacological inhibitors of the GTP-binding proteins G(i)/G(o), phospholipase C, Src, and mitogenic-extracellular signal-regulated kinase 1/2 (MEK 1/2) decreased oleate-induced [3H]thymidine incorporation in the breast cancer cell line MDA-MB-231. In addition, oleate caused a rapid and transient rise in cytosolic Ca2+ and an increase in protein kinase B phosphorylation. Overexpressing in these cells the G protein-coupled receptor GPR40, a fatty acid receptor, amplified oleate-induced proliferation, whereas silencing the GPR40 gene using RNA interference decreased it. Overexpressing GPR40 in T47D and MCF-7 breast cancer cells that are poorly responsive to oleate allowed a robust proliferative action of oleate. The data indicate that the phospholipase C, MEK 1/2, Src, and phosphatidylinositol 3-kinase/protein kinase B signaling pathways are implicated in the proliferative signal induced by oleate and that these effects are mediated at least in part via the G protein-coupled receptor GPR40. The results suggest that GPR40 is implicated in the control of breast cancer cell growth by fatty acids and that GPR40 may provide a link between fat and cancer.