Dual regulation of lysophosphatidic acid (LPA1) receptor signalling by Ral and GRK

doi:10.1016/j.cellsig.2009.03.011

Cellular Signalling

Volume 21, Issue 7, July 2009, Pages 1207-1217

https://doi.org/10.1016/j.cellsig.2009.03.011 Get rights and content

Abstract

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), LPA₁ and LPA₂ 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 LPA₁ and LPA₂ in human embryonic kidney (HEK 293) cells and that stimulation of LPA₁ 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 LPA₁ receptor stimulation promoted Ral-dependent phospholipase C activity. Furthermore, we found that GRK2 is required for the desensitization of LPA₁ and LPA₂ and have identified a novel interaction between RalA and GRK2, which is promoted by LPA₁ receptor activity. Taken together, these results establish RalA and GRK2 as key regulators of LPA receptor signalling and demonstrate for the first time that LPA₁ activity facilitates the formation of a novel protein complex between these two proteins.

Introduction

The bioactive lipid molecule lysophosphatidic acid (LPA) is a known mediator of diverse biological processes including cell survival, cell differentiation and proliferation, gene transcription, olfaction, as well as cardiovascular function, reproduction and brain development. LPA also mediates cell migration that is required for processes such as wound healing, tissue remodelling, and neurogenesis [1]. A role for LPA has also been implicated in various pathophysiological processes such as cancer, myocardial injury, hypertension, atherosclerosis, and neurodegenerative and psychiatric diseases [2], [3], [4], [5], [6], [7], [8]. LPA is detected in blood, and in humans, LPA concentration in serum is high (1–5 μM), with major sources being activated platelets, fibroblasts and adipocytes [1]. LPA evokes its multiple effects through G protein-coupled receptors (GPCRs) that belong to the endothelial differentiation gene (Edg) family. LPA₁/Edg2, LPA₂/Edg4 and LPA₃/Edg7, are specific, high-affinity receptors for LPA and share approximately 50% sequence identity [7], [9]. LPA₁ and LPA₂ are ubiquitously expressed, whereas LPA₃ is highly expressed in the brain, lung and ovary. Four other structurally distinct LPA receptors, LPA_4–7 have been recognized that belong to the P2Y family of nucleotide receptors, but less is known about these receptors [10]. LPA₁ and LPA₂ activate diverse signalling pathways by coupling to multiple heterotrimeric G proteins, namely G_q to activate the enzyme phospholipase C (PLC), G_i and G_s to modulate adenylate cyclase activity, and G_12/13 activating Rho GTPases [11].

LPA activity is believed to be regulated at the level of receptor desensitization, internalization or sequestration, and degradation. To date, very little has been directly reported on these early regulatory events at the molecular level. Homologous desensitization of GPCRs is regulated by G protein-coupled receptor kinases (GRKs) that constitute a family of seven serine/threonine protein kinases. GRKs exclusively phosphorylate agonist-occupied GPCRs leading to the uncoupling the receptor from heterotrimeric G proteins [12], [13]. Thus far, only one study has implicated the ubiquitously expressed GRK2 in the desensitization of endogenous LPA receptors in rat thyroid cells, although it is unknown which specific LPA receptor(s) were desensitized [14]. While LPA₁ has been shown to internalize via β-arrestin- and clathrin-dependent pathways [15], [16], to date there are no published studies on the regulation of LPA₂.

Several studies have implicated a role for the small GTP-binding protein Ral in receptor-mediated endocytosis (reviewed, [13], [17]). Ral GTPases are members of the Ras superfamily and consist of two isoforms, RalA and RalB, which share 85% sequence identity [18]. Ral is primarily localized to the plasma membrane but is also observed in endocytic vesicles [18]. In addition to its role in cell transformation, differentiation, and gene transcription, Ral also regulates the cytoskeleton, cell migration, and cell adhesion [18], [19]. In addition, Ral can regulate the endocytosis of certain GPCRs such as the metabotropic glutamate receptors (mGluR1a and mGluR5) [20], as well as tyrosine kinase receptors epidermal growth factor and insulin receptors, acting via its downstream effectors phospholipase D and Ral-binding protein 1 (RalBP1) [21], [22], [23]. However, a role for Ral in GPCR function is largely unknown. This study reports for the first time that RalA interacts with both LPA₁ and LPA₂. Although RalA does not appear to be necessary for LPA receptor endocytosis, this small GTPase critically regulates LPA-induced inositol phosphate formation. Furthermore, we report a novel interaction between RalA and GRK2 in response to LPA activation, and that these proteins determine the outcome of LPA receptor activation.

Section snippets

Materials

The HEK 293 cell line was purchased from ATCC (Manassas, VA). Lysophosphatidic acid was purchased from Avanti Polar Lipids (Albaster, AL). Protein G-Sepharose beads were from Amersham Biosciences (Oakville, Ontario, Canada). The anti-Flag M2 agarose affinity beads, polycolonal antibodies and all other biochemical reagents were purchased from Sigma (St. Louis, MO). Flag-tagged LPA₁ and LPA₂ receptors were obtained from Dr. G. Mills (MD Anderson Cancer Institute, Houston, TX).

Cell culture and transfections

HEK 293 cells were

RalA interacts and colocalizes with LPA₁ and LPA₂ receptors

The small Ras-like G proteins of the Ral subfamily are activated downstream of certain receptor tyrosine kinases and GPCRs [24] and can play a role in receptor endocytosis [20]. Previously, for example, it was shown that metabotropic mGLUR1 glutamate receptors can activate RalA, which in turn promotes phospholipase D-dependent internalization of this GPCR [20]. We found that both Flag–LPA₁ and Flag–LPA₂ could constitutively associate with GFP–RalA when co-expressed in HEK 293 cells by

Discussion

The present results reveal several novel and unexpected properties of LPA₁ and LPA₂ function. Our findings show that the small GTPase RalA binds to both receptors and that LPA triggers RalA activation through LPA₁. While Ral GTPases are known to be activated in response to GPCR stimulation, and evidence exists that activated Ral can promote phospholipase C function [26], [27], to our knowledge the present study is the first to show Ral-dependent phospholipase C stimulation in response to the

Conclusions

•
Ral associates and colocalizes with LPA₁ and LPA₂ but does not play a role in their internalization.
•
GRK2 desensitizes LPA₁ and LPA₂ and this appears to occur in a phosphorylation-independent manner.
•
Ral mediates the activation of phospholipase C signalling by LPA₁.
•
Ral mediates the signalling of LPA₁ by associating with GRK2. Ral and GRK2 compete for binding to LPA₁ and hence affect the signalling properties of the receptor.

Acknowledgments

Grant support: this study was funded by a grant from Natural Sciences and Engineering Research Council of Canada (NSERC). Dr. Moshmi Bhattacharya is a recipient of an NSERC University Faculty Salary Award. Dr. Andy Babwah holds a Canadian Institutes of Health Research New Investigator Award, and Dr. Peter Chidiac holds a Career Investigator Award from the Heart and Stroke Foundation of Ontario.

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In the last decades a growing body of evidence showed that GRK2 may also regulate downstream activity of GPCRs independently of receptor phosphorylation. Desensitization of GPCRs signaling independently of kinase activity has been described for adenosine type 1 receptor (A1R) and μ-opioid receptor (μOR) [12], metabotropic glutamate receptor 5 (mGluR5) [13], histamine H1 and H2 receptors (H1R and H2R) [14,15], lysophosphatidic acid (LPA1) receptor [16], endothelin receptor type B (ETb) [8], follicle-stimulating hormone receptor [17] and D2 dopamine receptor [18] among others. Even more, direct interaction of GRKs with signaling molecules downstream of GPCRs has been described [19] and in some cases expression of the RH domain of GRK2 resulted sufficient for attenuating receptor signaling through Gαq activation [20,21].
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Dual regulation of lysophosphatidic acid (LPA1) receptor signalling by Ral and GRK

Abstract

Introduction

Section snippets

Materials

Cell culture and transfections

RalA interacts and colocalizes with LPA1 and LPA2 receptors

Discussion

Conclusions

Acknowledgments

Ann. Oncol.

Biochim. Biophys. Acta

Atherosclerosis

Mol. Cell Neurosci.

J. Biol. Chem.

Int. J. Biochem. Cell Biol.

Trends. Cell Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Lab. Invest.

Biochim. Biophys. Acta

J. Biol. Chem.

Methods Enzymol.

J. Biol. Chem.

Biochim. Biophys. Acta

Bioessays

J. Cell Biochem.

Nat. Rev. Cancer

Neuroreport

Mol. Pharmacol.

Curr. Opin. Pharmacol.

Annu. Rev. Biochem.

Biochem. Soc. Trans.

Dual regulation of lysophosphatidic acid (LPA₁) receptor signalling by Ral and GRK

RalA interacts and colocalizes with LPA₁ and LPA₂ receptors