A novel regulator of G-protein signaling bearing GAP activity for Gαi and Gαq in megakaryocytes

Abstract

The regulator of G-protein signaling (RGS) negatively regulates the ␣ subunit of G proteins by accelerating their intrinsic guanosine triphosphatase (GTPase) activity. Here are reported the isolation and characterization of a novel mouse RGS, termed RGS18, which is a new member of RGS subfamily B. Northern blot analysis showed that RGS18 messenger RNA was detected predominantly in spleen and hematopoietic cells, and immunohistochemical studies demonstrated that RGS18 was expressed in megakaryocytes, platelets, granulocytes/monocytes, and, weakly, in hematopoietic stem cells, but not in lymphocytes or erythrocytes. Although various subcellular localizations of RGS have been reported, RGS18 was found to be localized in cytoplasm in megakaryocytes. In vitro binding assays of RGS18 with megakaryocyte cell lysates with or without AlF 4 ؊ treatment demonstrated that RGS18 specifically binds to 2 ␣ subunits of the G protein, G␣i and G␣q. Furthermore, RGS18 clearly exhibited GTPase-activating protein (GAP) activity for G␣i and G␣q but not for G␣s or G␣12. In addition, chemokine stromal-derived factor 1 (SDF-1), which has been reported to stimulate megakaryocyte colony formation in the presence of thrombopoietin, affected the binding of RGS18 to G␣i but not to G␣q. Therefore, the newly isolated RGS18 turned out to be a new member of the RGS family bearing GAP activity for G␣i, which might be stimulated by SDF-1 in megakaryocytes, as well as for G␣q. Thus, RGS18 may play an important role in proliferation, differentiation, and/or migration of megakaryocytes. IntroductionThe guanosine triphosphate (GTP)-binding protein (G-protein) signaling pathway is one of the most important signaling cascades used to relay extracellular signals and sensory stimuli to eukaryotic cells. 1 Heterotrimeric G proteins, which couple heptahelical receptors to effectors in the signal transduction pathway, are composed of ␣, ␤, and ␥ subunits. 2,3 Each subunit has multiple isoforms; the ␣ subunit isoforms are grouped into 4 subfamilies: G␣i, G␣s, G␣q, and G␣12. 4 The guanosine diphosphate (GDP)-bound heterotrimer is an inactive form of the G protein. Ligand-bound activated receptors catalyze the exchange of GDP by GTP on the ␣ subunit, leading to dissociation of the ␣ from the ␤␥ dimer and signal transduction by the separated G-protein subunits (G␣ and G␤␥). The large body of research examining G-protein-mediated signaling pathways has focused mainly on the involvement of receptor phosphorylation and sequestration in the desensitization process.Recently, a new family of proteins, the regulator of the G-protein-signaling (RGS) family, has been identified. 5-7 Genetic screenings for negative regulators for the pheromone response pathway in yeast identified a protein, Sst2. 8 Further analyses revealed that Sst2 interacted directly with the G-protein ␣ subunit. 9 To date, at least 26 different RGS proteins have been described in mammals. They all contain a conserved, characteristic domain (termed RGS domain) that interacts specifically with the ac...

A newly identified complex of spinophilin and the tyrosine phosphatase, SHP-1, modulates platelet activation by regulating G protein–dependent signaling

“…RNA transcripts encoding as many as 10 RGS proteins have been reported in platelets. [17][18][19][20][21][22] However, we were able to detect only 2 (RGS10 and RGS18; Figure 1B), which is consistent with transcriptome data. 23 Although not reported previously, the following observations indicate that SPL can bind to both of these RGS proteins: in resting platelets and serum-starved CHO cells, immunoprecipitating RGS10 or RGS18 coprecipitated SPL ( Figure 1C-D).…”

Regulator of G-protein signaling 18 integrates activating and inhibitory signaling in platelets

“…8,9 First identified in 2001, RGS18 is abundantly expressed in platelets and megakaryocytes. [10][11][12][13] Interaction partners include G-␣-q (Gq) and Gi1,2,3, but not Gz, Gs, or G12. 10,11 Platelet activation by GPCR ligands requires both Gq and Gi signaling.…”

“…[10][11][12][13] Interaction partners include G-␣-q (Gq) and Gi1,2,3, but not Gz, Gs, or G12. 10,11 Platelet activation by GPCR ligands requires both Gq and Gi signaling. 14 Gq activates PLC-␤, leading to the production of inositol-1,4,5-trisphosphate (IP 3 ) and the release of Ca 2ϩ from intracellular stores.…”

Modulating platelet reactivity through control of RGS18 availability

“…[8][9][10][11] Both proteins are relatively small, consisting primarily of a characteristic RGS domain that interacts with Ga. 12 Each can serve as GTPase-accelerating proteins for Gia and Gqa, but not Gsa. [13][14][15][16][17] RGS18 is primarily expressed in hematopoietic cells 14,16,[18][19][20] whereas RGS10 is widely expressed. [21][22][23] In addition to being expressed in platelets, there is a small, but growing, body of evidence that RGS proteins are biologically relevant regulators of platelet activation.…”