Structural basis for lysophosphatidylserine recognition by GPR34.

GPR34 is a recently identified G-protein coupled receptor, which has an immunomodulatory role and recognizes lysophosphatidylserine (LysoPS) as a putative ligand. Here, we report cryo-electron microscopy structures of human GPR34-Gi complex bound with one of two ligands bound: either the LysoPS analogue S3E-LysoPS, or M1, a derivative of S3E-LysoPS in which oleic acid is substituted with a metabolically stable aromatic fatty acid surrogate. The ligand-binding pocket is laterally open toward the membrane, allowing lateral entry of lipidic agonists into the cavity. The amine and carboxylate groups of the serine moiety are recognized by the charged residue cluster. The acyl chain of S3E-LysoPS is bent and fits into the L-shaped hydrophobic pocket in TM4-5 gap, and the aromatic fatty acid surrogate of M1 fits more appropriately. Molecular dynamics simulations further account for the LysoPS-regioselectivity of GPR34. Thus, using a series of structural and physiological experiments, we provide evidence that chemically unstable 2-acyl LysoPS is the physiological ligand for GPR34. Overall, we anticipate the present structures will pave the way for development of novel anticancer drugs that specifically target GPR34.

Functional modulation of lysophosphatidic acid type 2 G-protein coupled receptor facilitates alveolar bone formation.

Lipid biosynthesis is recently studied its functions in a range of cellular physiology including differentiation and regeneration. However, it still remains to be elucidated in its precise function. To reveal this, we evaluated the roles of lysophosphatidic acid (LPA) signaling in alveolar bone formation using the LPA type 2 receptor (LPAR2) antagonist AMG-35 (Amgen Compound 35) using tooth loss without periodontal disease model which would be caused by trauma and usually requires a dental implant to restore masticatory function. In this study, in vitro cell culture experiments in osteoblasts and periodontal ligament fibroblasts revealed cell type-specific responses, with AMG-35 modulating osteogenic differentiation in osteoblasts in vitro. To confirm the in vivo results, we employed a mouse model of tooth loss without periodontal disease. Five to 10 days after tooth extraction, AMG-35 facilitated bone formation in the tooth root socket as measured by immunohistochemistry for differentiation markers KI67, Osteocalcin, Periostin, RUNX2, transforming growth factor beta 1 (TGF-ß1) and SMAD2/3. The increased expression and the localization of these proteins suggest that AMG-35 elicits osteoblast differentiation through TGF-ß1 and SMAD2/3 signaling. These results indicate that LPAR2/TGF-ß1/SMAD2/3 represents a new signaling pathway in alveolar bone formation and that local application of AMG-35 in traumatic tooth loss can be used to facilitate bone regeneration and healing for further clinical treatment.

Structural basis for ligand recognition and signaling of the lysophosphatidylserine receptors GPR34 and GPR174.

Lysophosphatidylserine (LysoPS) is a naturally occurring lipid mediator involved in various physiological and pathological processes especially those related to the immune system. GPR34, GPR174, and P2Y10 have been identified as the receptors for LysoPS, and its analogues have been developed as agonists or antagonists for these receptors. However, the lack of structural information hinders the drug development with novel characteristics, such as nonlipid ligands and allosteric modulators. Here, we determined the structures of human GPR34 and GPR174 in complex with LysoPS and G protein by cryo-EM. Combined with structural analysis and functional studies, we elucidated the lipid-binding modes of these receptors. By structural comparison, we identified the structural features of GPR34 and GPR174 in active state. Taken together, our findings provide insights into ligand recognition and signaling of LysoPS receptors and will facilitate the development of novel therapeutics for related inflammatory diseases and autoimmune diseases.

Isosteric Replacement of Ester Linkage of Lysophospholipids with Heteroaromatic Rings Retains Potency and Subtype Selectivity.

Our group has reported various derivatives of lysophosphatidylserine (LysoPS) as potent and subtype-selective agonists for G-protein-coupled receptors (GPCRs). However, the ester linkage between the glycerol moiety and fatty acid or fatty acid surrogate is present in all of them. In order to develop these LysoPS analogs as drug candidates, appropriate pharmacokinetic consideration is essential. Here, we found that the ester bond of LysoPS is highly susceptible to metabolic degradation in mouse blood. Accordingly, we examined isosteric replacement of the ester linkage with heteroaromatic rings. The resulting compounds showed excellent retention of potency and receptor subtype selectivity, as well as increased metabolic stability in vitro.

Exploration of LPS2 agonist binding modes using the combination of a new hydrophobic scaffold and homology modeling.

Lysophosphatidylserine (LysoPS) is an endogenous pan-agonist of three G-protein coupled receptors (GPCRs): LPS1/GPR34, LPS2/P2Y10, and LPS3/GPR174, and we previously reported a series of LysoPS-based agonists of these receptors. Interestingly, we found that LPS1 agonist activity was very sensitive to structural change at the hydrophobic fatty acid moiety, whereas LPS2 agonist activity was not. Here, to probe the molecular basis of LPS2 agonist binding, we developed a new class of hydrophobic fatty acid surrogates having a biphenyl-ether scaffold. The LPS2 agonist activity of these compounds proved sensitive to molecular modification of the hydrophobic skeleton. Thus, we next constructed an LPS2 model by homology modeling and docking/molecular dynamics (MD) simulation, and validated it by means of SAR studies together with point mutations of selected receptor amino-acid residues. The putative ligand-binding site of LPS2 is Γ-shaped, with a hydrophilic site horizontally embedded in the receptor transmembrane helix bundles and a perpendicular hydrophobic groove adjoining transmembrane domains 4 and 5 that is open to the membrane bilayer. The binding poses of LPS2 agonists to this site are consistent with easy incorporation of various kinds of fatty acid surrogates. Structural development based on this model afforded a series of potent and selective LPS2 full agonists, which showed enhanced in vitro actin stress fiber formation effect.

G-protein coupled receptor 34 regulates the proliferation and growth of LS174T cells through differential expression of PI3K subunits and PTEN.

PURPOSE: G-protein coupled receptor (GPR 34) has been found to play important roles in some cancers and regulates the proliferation, apoptosis, and migration of these cancer cells. However, the mechanisms underlying how GPR34 functions to regulate growth and proliferation of colorectal cancer cells remains to be clarified. METHODS: We employed stable GPR34 knockdown LS174T cell models, GPR34 Mab blocking, a CCK-8 kit, and a colony formation assay to characterize the effect of GPR34 on the proliferation of LS174T in vitro and xenograft tumor growth in vivo. The mRNA level of GPR34 was detected by RT-PCR in tumor tissues and adjacent normal tissues from 34 CRC patients. RESULTS: Based on RT-PCR results, GPR34 exhibited high level in tumor samples compared with adjacent normal samples. Increased expression of GPR34 is more associated with poor prognosis of CRC as shown in The Cancer Genome Atlas (TCGA) dataset by Kaplan-Meier survival analysis. Furthermore, we showed that GPR34 knockdown inhibited the proliferation of LS174T colon cancer cells and related xenograft tumor growth. Searching for the distinct molecular mechanism, we identified several contributors to proliferation of LS174T colon cancer cells: PI3K subunits/PTEN, PDK1/AKT, and Src/Raf/Ras/ERK. GPR34 knockdown inhibited the proliferation of LS174T cells by upregulating expression of PTEN, and downregulating expression of PI3K subunits p110-beta. CONCLUSION: Our findings provide direct evidence that GPR34 regulates the proliferation of LS174T cells and the growth of LS174T tumor xenografts by regulating different pathways. High expression of GPR34 mRNA could then be used to predict poor prognosis of CRC.

GPR34 activation potentially bridges lymphoepithelial lesions to genesis of salivary gland MALT lymphoma.

GPR34 translocation and mutation are specifically associated with salivary gland MALT lymphoma (SG-MALT-lymphoma). The majority of GPR34 mutations are clustered in its C-terminus, resulting in truncated proteins lacking the phosphorylation motif important for receptor desensitization. It is unclear why GPR34 genetic changes associate with SG-MALT-lymphoma and how these mutations contribute to the development of lymphoma. We generated isogenic Flp-InTRex293 cell lines that stably expressed a single copy of GPR34 or its various mutants and performed a range of in vitro assays. We found that the GPR34 Q340X truncation, but not the R84H and D151A mutants, conferred a significantly increased resistance to apoptosis and greater transforming potential than the GPR34 wild type. The GPR34 truncation mutant had a significantly delayed internalization compared with the wild type after ligand (lysophosphatidylserine) stimulation. Among the 9 signaling pathways examined, the GPR34 Q340X truncation, and to a lesser extent the D151A mutant, significantly activated CRE, NF-κB, and AP1 reporter activities, particularly in the presence of ligand stimulation. We further described the enhanced activities of phospholipase-A1/2 in the culture supernatant of Flp-InTRex293 cells that expressed the GPR34 Q340X mutant, as well as their potential to catalyze the synthesis of lysophosphatidylserine from phosphatidylserine. Importantly, phospholipase-A1 was abundantly expressed in the duct epithelium of salivary glands and those involved in lymphoepithelial lesions (LELs). Our findings advocate a model of paracrine stimulation of malignant B cells via GPR34, in which phospholipase A is released by LELs and hydrolyzes the phosphatidylserine exposed on apoptotic cells, generating lysophosphatidylserine, the ligand for GPR34. Thus, GPR34 activation potentially bridges LELs to genesis of SG-MALT-lymphoma.

MicroRNA-381 targets G protein-Coupled receptor 34 (GPR34) to regulate the growth, migration and invasion of human cervical cancer cells.

MicroRNAs (miRNAs) have emerged as the vital post-transcriptional regulators and control the growth and progression of different cancers types. The current study aimed at exploration of the role of microRNA-381 (miRNA-381) in human cervical cancer with emphasis on the evaluation of the underlying molecular mechanism. The results revealed a significant (P < 0.05) downregulation of miRNA-381 was found in cervical cancer tissues and cancer cell lines. Overexpression of miRNA-381 in cervical cancer cells significantly (P < 0.05) inhibited their proliferation through the induction of cell apoptosis which was accompanied by depletion of Bcl-2 and increase in Bax expression. Additionally, the cleavage of caspase-3 and 9 was also activated upon miRNA-381 overexpression. The Overexpression of miRNA-381 further inhibited the migration and invasion of cervical cancer cells. In silico analysis together with dual luciferase assay revealed G protein-Coupled receptor 34 (GPR34) to be the target of miRNA-381. The expression of GPR34 was significantly (P < 0.05) upregulated in the cervical cancer tissues and cell lines. Nonetheless, miRNA-381 overexpression caused a remarkable decrease in the expression of GPR34. The GPR34 knockdown and overexpression proved that the tumor-suppressive effects of miRNA-381 are mediated via GPR34. The study elucidated the essence of miRNA-381/GPR34 molecular regulatory axis in cervical cancer and unraveled the possibility of targeting this molecular axis as an important therapeutic approach against human cervical cancer.

Lysolipids in Vascular Development, Biology, and Disease.

Membrane phospholipid metabolism forms lysophospholipids, which possess unique biochemical and biophysical properties that influence membrane structure and dynamics. However, lysophospholipids also function as ligands for G-protein-coupled receptors that influence embryonic development, postnatal physiology, and disease. The 2 most well-studied species-lysophosphatidic acid and S1P (sphingosine 1-phosphate)-are particularly relevant to vascular development, physiology, and cardiovascular diseases. This review summarizes the role of lysophosphatidic acid and S1P in vascular developmental processes, endothelial cell biology, and their roles in cardiovascular disease processes. In addition, we also point out the apparent connections between lysophospholipid biology and the Wnt (int/wingless family) pathway, an evolutionarily conserved fundamental developmental signaling system. The discovery that components of the lysophospholipid signaling system are key genetic determinants of cardiovascular disease has warranted current and future research in this field. As pharmacological approaches to modulate lysophospholipid signaling have entered the clinical sphere, new findings in this field promise to influence novel therapeutic strategies in cardiovascular diseases.

Membrane Phospholipid Analogues as Molecular Rulers to Probe the Position of the Hydrophobic Contact Point of Lysophospholipid Ligands on the Surface of G-Protein-Coupled Receptor during Membrane Approach.

When lipid mediators bind to G-protein-coupled receptors (GPCRs), the ligand first enters the lipid bilayer, then diffuses laterally in the cell membrane to make hydrophobic contact with the receptor protein, and finally enters the receptor's binding pocket. In this process, the location of the hydrophobic contact point on the surface of the receptor has been little discussed even in cases in which the crystal structure has been determined, because the ligand binding pocket is buried inside the transmembrane (TM) domains. Here, we coupled an activator ligand to a series of membrane phospholipid surrogates, which constrain the depth of entry of the ligand into the lipid bilayer. Consequently, via measurement of the receptor-activating activity as a function of the depth of entry into the membrane, these surrogates can be used as molecular rulers to estimate the location of the hydrophobic contact point on the surface of GPCR. We focused on lysophosphatidylserine (LysoPS) receptor GPR34 and prepared a series of simplified membrane-lipid-surrogate-conjugated lysophospholipid analogues by attaching alkoxy amine chains of varying lengths to the hydrophobic tail of a potent GPR34 agonist. As expected, the activity of these lipid-conjugated LysoPS analogues was dependent on chain length. The predicted contact position matches the position of the terminal benzene ring of a nonlipidic ligand that protrudes between TMs 4 and 5 of the receptor. We further found that the nature of the terminal hydrophilic functional group of the conjugated membrane lipid surrogate strongly influences the activity, suggesting that lateral hydrophilic contact of LysoPS analogues with the receptor's surface is also crucial for ligand-GPCR binding.

GPR34 in spinal microglia exacerbates neuropathic pain in mice.

BACKGROUND: Neuropathic pain is caused by sensory nerve injury, but effective treatments are currently lacking. Microglia are activated in the spinal dorsal horn after sensory nerve injury and contribute to neuropathic pain. Accordingly, molecules expressed by these cells are considered potential targets for therapeutic strategies. Our previous gene screening study using a mouse model of motor nerve injury showed that the G-protein-coupled receptor 34 gene (GPR34) is induced by nerve injury. Because GPR34 is now considered a microglia-enriched gene, we explored the possibility that it might be involved in microglial activation in the dorsal horn in a mouse model of neuropathic pain. METHODS: mRNA expression of GPR34 and pro-inflammatory molecules was determined by quantitative real-time PCR in wild-type and GPR34-deficient mice with L4 spinal nerve injury. In situ hybridization was used to identify GPR34 expression in microglia, and immunohistochemistry with the microglial marker Iba1 was performed to examine microglial numbers and morphology. Mechanical sensitivity was evaluated by the von Frey hair test. Liquid chromatography-tandem mass spectrometry quantified expression of the ligand for GPR34, lysophosphatidylserine (LysoPS), in the dorsal horn, and a GPR34 antagonist was intrathecally administrated to examine the effect of inhibiting LysoPS-GPR34 signaling on mechanical sensitivity. RESULTS: GPR34 was predominantly expressed by microglia in the dorsal horn after L4 nerve injury. There were no histological differences in microglial numbers or morphology between WT and GPR34-deficient mice. However, nerve injury-induced pro-inflammatory cytokine expression levels in microglia and pain behaviors were significantly attenuated in GPR34-deficient mice. Furthermore, the intrathecal administration of the GPR34 antagonist reduced neuropathic pain. CONCLUSIONS: Inhibition of GPR34-mediated signal by GPR34 gene deletion reduced nerve injury-induced neuropathic pain by suppressing pro-inflammatory responses of microglia without affecting their morphology. Therefore, the suppression of GPR34 activity may have therapeutic potential for alleviating neuropathic pain.

'Crystal' Clear? Lysophospholipid Receptor Structure Insights and Controversies.

Lysophospholipids (LPLs), particularly sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA), are bioactive lipid modulators of cellular homeostasis and pathology. The discovery and characterization of five S1P- and six LPA-specific G protein-coupled receptors (GPCRs), S1P1-5 and LPA1-6, have expanded their known involvement in all mammalian physiological systems. Resolution of the S1P1, LPA1, and LPA6 crystal structures has fueled the growing interest in these receptors and their ligands as targets for pharmacological manipulation. In this review, we have attempted to provide an integrated overview of the three crystallized LPL GPCRs with biochemical and physiological structure-function data. Finally, we provide a novel discussion of how chaperones for LPLs may be considered when extrapolating crystallographic and computational data toward understanding actual biological interactions and phenotypes.

G protein-coupled receptor 34 in ovarian granulosa cells of cattle: changes during follicular development and potential functional implications.

Abundance of G protein-coupled receptor 34 (GPR34) mRNA is greater in granulosa cells (GCs) of cystic vs normal follicles of cattle. The present experiments were designed to determine if GPR34 mRNA in granulosa cell [GC] changes during selection and growth of dominant follicles in cattle as well as to investigate the hormonal regulation of GPR34 mRNA in bovine GC in vitro. In Exp. 1, estrous cycles of nonlactating cows were synchronized and then ovariectomized on either day 3-4 or 5-6 after ovulation. GPR34 mRNA abundance in GC was 2.8- to 3.8-fold greater (P < 0.05) in small (1-5 mm) and large (≥8 mm) estrogen-inactive dominant follicles than in large estrogen-active follicles. Also, GPR34 mRNA tended to be greater (P < 0.10) in F2 than F1 follicles on day 3-4 postovulation. In Exp. 2-7, ovaries were collected at an abattoir and GC were isolated and treated in vitro. Expression of GPR34 was increased (P < 0.05) 2.2-fold by IGF1. Tumor necrosis factor (TNF)-α decreased (P < 0.05) the IGF1-induced GPR34 mRNA abundance in small-follicle GC, whereas IGF1 decreased (P < 0.05) GPR34 expression by 45% in large-follicle GC. Treatment of small-follicle GC with either IL-2, prostaglandin E2 or angiogenin decreased (P < 0.05) GPR34 expression, whereas FSH, cortisol, wingless 3A, or hedgehog proteins did not affect (P > 0.10) GPR34 expression. In Exp. 6 and 7, 2 presumed ligands of GPR34, L-a-lysophosphatidylserine (LPPS) and LPP-ethanolamine, increased (P < 0.05) GC numbers and estradiol production by 2-fold or more in small-follicle GC, and this response was only observed in IGF1-treated GC. In conclusion, GPR34 is a developmentally and hormonally regulated gene in GC, and its presumed ligands enhance IGF1-induced proliferation and steroidogenesis of bovine GC.

Lysophospholipid Receptors, as Novel Conditional Danger Receptors and Homeostatic Receptors Modulate Inflammation-Novel Paradigm and Therapeutic Potential.

There are limitations in the current classification of danger-associated molecular patterns (DAMP) receptors. To overcome these limitations, we propose a new paradigm by using endogenous metabolites lysophospholipids (LPLs) as a prototype. By utilizing a data mining method we pioneered, we made the following findings: (1) endogenous metabolites such as LPLs at basal level have physiological functions; (2) under sterile inflammation, expression of some LPLs is elevated. These LPLs act as conditional DAMPs or anti-inflammatory homeostasis-associated molecular pattern molecules (HAMPs) for regulating the progression of inflammation or inhibition of inflammation, respectively; (3) receptors for conditional DAMPs and HAMPs are differentially expressed in human and mouse tissues; and (4) complex signaling mechanism exists between pro-inflammatory mediators and classical DAMPs that regulate the expression of conditional DAMPs and HAMPs. This novel insight will facilitate identification of novel conditional DAMPs and HAMPs, thus promote development of new therapeutic targets to treat inflammatory disorders.

Topogenesis and cell surface trafficking of GPR34 are facilitated by positive-inside rule that effects through a tri-basic motif in the first intracellular loop.

Protein folding, topogenesis and intracellular targeting of G protein-coupled receptors (GPCRs) must be precisely coordinated to ensure correct receptor localization. To elucidate how different steps of GPCR biosynthesis work together, we investigated the process of membrane topology determination and how it relates to the acquisition of cell surface trafficking competence in human GPR34. By monitoring a fused FLAG-tag and a conformation-sensitive native epitope during the expression of GPR34 mutant panel, a tri-basic motif in the first intracellular loop was identified as the key topogenic signal that dictates the orientation of transmembrane domain-1 (TM1). Charge disruption of the motif perturbed topogenic processes and resulted in the conformational epitope loss, post-translational processing alteration, and trafficking arrest in the Golgi. The placement of a cleavable N-terminal signal sequence as a surrogate topogenic determinant overcame the effects of tri-basic motif mutations and rectified the TM1 orientation; thereby restored the conformational epitope, post-translational modifications, and cell surface trafficking altogether. Progressive N-tail truncation and site-directed mutagenesis revealed that a proline-rich segment of the N-tail and all four cysteines individually located in the four separate extracellular regions must simultaneously reside in the ER lumen to muster the conformational epitope. Oxidation of all four cysteines was necessary for the epitope formation, but the cysteine residues themselves were not required for the trafficking event. The underlying biochemical properties of the conformational epitope was therefore the key to understand mechanistic processes propelled by positive-inside rule that simultaneously regulate the topogenesis and intracellular trafficking of GPR34.

The lysophosphatidylserine receptor GPR174 constrains regulatory T cell development and function.

Regulatory T cell (T reg cell) numbers and activities are tightly calibrated to maintain immune homeostasis, but the mechanisms involved are incompletely defined. Here, we report that the lysophosphatidylserine (LysoPS) receptor GPR174 is abundantly expressed in developing and mature T reg cells. In mice that lacked this X-linked gene, T reg cell generation in the thymus was intrinsically favored, and a higher fraction of peripheral T reg cells expressed CD103. LysoPS could act in vitro via GPR174 to suppress T cell proliferation and T reg cell generation. In vivo, LysoPS was detected in lymphoid organ and spinal cord tissues and was abundant in the colon. Gpr174(-/Y) mice were less susceptible to experimental autoimmune encephalomyelitis than wild-type mice, and GPR174 deficiency in T reg cells contributed to this phenotype. This study provides evidence that a bioactive lipid, LysoPS, negatively influences T reg cell accumulation and activity through GPR174. As such, GPR174 antagonists might have therapeutic potential for promoting immune regulation in the context of autoimmune disease.

G-protein coupled receptor 34 knockdown impairs the proliferation and migration of HGC-27 gastric cancer cells in vitro.

BACKGROUND: Overexpression of G-protein coupled receptor 34 (GPR34) affects the progression and prognosis of human gastric adenocarcinoma, however, the role of GPR34 in gastric cancer development and progression has not been well-determined. The current study aimed to investigate the effect of GPR34 knockdown on the proliferation, migration, and apoptosis of HGC-27 gastric cancer cells and the underlying mechanisms. METHODS: The expression of GPR34 in gastric cancer cell line HGC-27 was detected by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. HGC-27 cells were employed to construct the stable GPR34 knockdown cell model in this study. Real-time RT-PCR and Western blotting were applied to validate the effect of short hairpin RNA (ShRNA) on the expression of GPR34 in HGC-27 gastric cells. The proliferation, migration of these cells were examined by Cell Counting Kit-8 and transwell. We also measured expression profile of PI3K/PDK1/AKT and ERK using Western blotting. RESULTS: The ShRNA directed against GPR34 effectively inhibited both endogenous mRNA and protein expression levels of GPR34, and significantly down-regulated the expression of PIK3CB (P < 0.01), PIK3CD (P < 0.01), PDK1 (P < 0.01), phosphorylation of PDK1 (P < 0.01), Akt (P < 0.01), and ERK (P < 0.01). Furthermore, GPR34 knockdown resulted in an obvious reduction in HGC-27 cancer cell proliferation and migration activity (P < 0.01). CONCLUSIONS: GPR34 knockdown impairs the proliferation and migration of HGC-27 gastric cancer cells in vitro and provides a potential implication for therapy of gastric cancer.

G-protein coupled receptor 34 activates Erk and phosphatidylinositol 3-kinase/Akt pathways and functions as alternative pathway to mediate p185Bcr-Abl-induced transformation and leukemogenesis.

Tyrosine 177 and the Src homology 2 (SH2) domain play important roles in linking p185Bcr-Abl to downstream pathways critical for cell growth and survival. However, a mutant p185(Y177FR552L) (p185(YR)), in which tyrosine 177 and arginine 552 in the SH2 domain are mutated, is still capable of transforming hematopoietic cells in vitro. Transplant of these cells into syngeneic mice also leads to leukemogenesis, albeit with a phenotype distinct from that produced by wild-type p185Bcr-Abl (p185(wt))-transformed cells. Here we show that G-protein coupled receptor 34 (Gpr34) expression is markedly up-regulated in p185(YR)-transformed cells compared to those transformed by p185(wt). Knockdown of Gpr34 in p185(YR) cells is sufficient to suppress growth factor-independent proliferation and survival in vitro and attenuate leukemogenesis in vivo. The Erk and phosphatidylinositol 3-kinase/Akt pathways are activated in p185(YR) cells and the activation is dependent on Gpr34 expression. These studies identify Gpr34 as an alternative pathway that may mediate p185Bcr-Abl-induced transformation and leukemogenesis.