Elsevier

Cellular Signalling

Volume 14, Issue 7, July 2002, Pages 595-606
Cellular Signalling

Cloning and characterization of a novel regulator of G protein signalling in human platelets

https://doi.org/10.1016/S0898-6568(02)00012-8Get rights and content

Abstract

In an effort to understand the modulation of G protein-coupled receptor (GPCR)-mediated signalling in platelets, we sought to identify which regulators of G protein signalling proteins (RGSs) are present in human platelets. Using degenerate oligonucleotides, we performed RT-PCR with human platelet and megakaryocytic cell line RNA. In addition to confirming the presence of several known RGS transcripts, we found a novel RGS domain-containing transcript in platelet RNA. Northern blot analysis of multiple human tissues indicates that this transcript is most abundantly expressed in platelets compared to other tissues examined. Full-length cloning of this novel RGS, which we now term RGS18, demonstrates that this transcript is predicted to encode a 235-amino acid protein that is most closely related to RGS5 (46% identity) and that has ∼30–40% identity to other RGS proteins. RGS18 is expressed in platelet, leukocyte, and megakaryocyte cell lines and binds to endogenous Gαi1, Gαi2, Gαi3, and Gαq but not Gαz, Gαs or Gα12 in vitro.

Introduction

Signalling through G protein-coupled receptors (GPCRs), such as thrombin, thromboxane A2, and ADP, is in part responsible for platelet activation events such as fibrinogen receptor exposure, granule secretion, and aggregation [1]. Multiple intracellular signalling pathways have been implicated in platelet activation events, although the exact sequence of events and host of intracellular signalling molecules remains undefined. In an effort to identify proteins that might modulate upstream events in GPCR-mediated intracellular signalling in platelets, we sought to identify which members of a recently identified family of proteins known as regulators of G protein signalling (RGSs) are present in platelets. RGSs are a family of proteins that function to dampen signals generated upon stimulation of cell-surface G protein-coupled receptors (GPCRs). RGSs were first identified in genetic screens of yeast [2], [3] and the nematode Caenorhabditis elegans [4] based on their ability to modulate behavioural responses. Mammalian homologues of these lower eukaryotic RGSs were quickly identified [5], [6], [7], [8], [9]. The hallmark of this family is a highly homologous 120-amino acid region termed an RGS domain. Currently, there are more than 30 mammalian proteins or partial sequences that contain a putative RGS domain [10].

RGS proteins are thought to regulate GPCR signalling by interacting with the alpha subunits of heterotrimeric GTP-binding proteins. Heterotrimeric G proteins act as molecular switches in GPCR-mediated signal transduction controlling the rate and extent of activation of the effector (for a review of heterotrimeric G proteins, see Ref. [11]). RGS proteins attenuate signalling through GPCRs by acting as GTPase activating proteins (GAPs) for the alpha subunit [9], [12], [13]. Structural studies indicate that RGSs bind to the transition state of the alpha subunit thereby stabilizing it, accelerating GTP hydrolysis that limits the time the Gα subunit spends in its active state [14], [15]. So far, RGSs have been identified which interact with and activate members of the Gαi family (Gαi1, Gαi2, Gαi3, Gαz, Gαo, and Gαt), Gαq/11, and Gα12/13 but not Gαs [10]. In addition to their GAP activity, RGSs may also block signalling by acting as effector antagonists [16], [17]. RGS proteins have been identified in a variety of cell types and tissues that profoundly alter many GPCR-stimulated intracellular effectors, including regulation of adenylyl cyclase [18], MAP kinase activity [6], [17], inositol phosphate and Ca2+ signalling [16], [17], [18], [19], K+ channel conductance [20], and visual signal transduction [21], [22].

Since several of these signalling cascades are involved in platelet activation, we thought it likely that one or more members of the RGSs superfamily might be present in platelets and be responsible for regulating signalling pathways critical for platelet activation. In platelets, receptors for ADP, thromboxane A2, and thrombin couple to heterotrimeric GTP-binding proteins that transduce the signals to intracellular effectors, resulting in inhibition of adenylyl cyclase, activation of phospholipase C, and mobilization of intracellular calcium [1]. In an effort to understand better the regulation of G protein signalling in human platelets, in the present study we employed an RT-PCR strategy with degenerate oligonucleotides to amplify RGS transcripts from human platelet RNA and several megakaryocytic cell lines. In addition to identifying several known RGSs in these cells, we identified a novel RGS domain-containing protein in human platelets. This RGS, which we now call RGS18, is abundantly expressed in platelets, with much lower expression in other tissues, primarily those of the haematopoetic system. In vitro RGS18 binds to endogenous Gαi1/2/3 and Gαq but not to Gαz, Gαs or Gα12 in platelet lysates treated with GDP+AlF4 but not GDP alone. Since platelet aggregation requires activation of receptor coupled to Gαq and/or one or more forms of Gαi, RGS18 may be responsible in part for regulation of pathways important to platelet activation.

Section snippets

Miscellaneous

Reagents were obtained from Sigma (St. Louis, MO) unless otherwise noted. Oligonucleotides and peptides were produced by the Core Biotechnologies Department at Rhone Poulenc-Rorer Pharmaceuticals. Cell culture reagents were obtained from Gibco/BRL (Rockville, MD). Cell lines were obtained from ATCC (Manassas, VA).

Preparation of platelets, leukocytes, and cell lines

HEL and Meg-01 cells were maintained in RPMI supplemented with 10% foetal bovine serum, 0.3 mg/ml l-glutamine, and 100 U/ml penicillin G/100 μg/ml streptomycin sulfate. DAMI cells were

RT-PCR of RGSs from human platelets and megakaryocytic cell lines

A RT-PCR strategy was employed to identify which RGS family members are expressed in human platelets. We synthesized degenerate primers based on highly homologous regions in several of the previously identified RGS proteins [4] and used them to amplify total RNA from human platelets, and three megakaryocytic cell lines, DAMI, HEL, and MEG-01 cells. The resultant ∼240-bp PCR product was blunt-ligated to pCR2.1 and transformed into Escherichia coli. Fifty colonies from each PCR reaction were

Discussion

Recently, the complexity of G protein-coupled receptor signalling pathways, beyond the classical view of receptor, G protein, and effector, has been increased through the identification of additional mediators that modulate various aspects of the cascade, including RGSs that regulate the activity of heterotrimeric G proteins. In an effort to better understand GPCR-mediated signalling in human platelets, we initiated studies to examine which isoforms of the RGS family exist in platelets.

Acknowledgements

We acknowledge the contribution to these studies from several members of the Core Biotechnology Group at Aventis Pharmaceuticals. We thank Joseph Bruno and Amy Barber for automated plasmid purification and DNA sequencing and Richard Howk for oligonucleotides and peptide synthesis and conjugation to KLH.

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    Present address: Proteome, A Division of Incyte Genomics, Suite 435M, 100 Cummings Center, Beverly, MA 01915, USA.

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    Present address: Cardiovascular Department, Pfizer PGRD, 2800 Plymouth Road, Ann Arbor, MI 48105, USA.

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