Molecular Cloning and Characterization of a Novel Regulator of G-protein Signaling from Mouse Hematopoietic Stem Cells

Abstract

A novel regulator of G-protein signaling (RGS) has been isolated from a highly purified population of mouse long-term hematopoietic stem cells, and designated RGS18. It has 234 amino acids consisting of a central RGS box and short divergent NH 2 and COOH termini. The calculated molecular weight of RGS18 is 27,610 and the isoelectric point is 8.63. Mouse RGS18 is expressed from a single gene and shows tissue specific distribution. It is most highly expressed in bone marrow followed by fetal liver, spleen, and then lung. In bone marrow, RGS18 level is highest in long-term and shortterm hematopoietic stem cells, and is decreased as they differentiate into more committed multiple progenitors. The human RGS18 ortholog has a tissue-specific expression pattern similar to that of mouse RGS18. Purified RGS18 interacts with the ␣ subunit of both G i and G q subfamilies. The results of in vitro GTPase single-turnover assays using G␣ i indicated that RGS18 accelerates the intrinsic GTPase activity of G␣ i . Transient overexpression of RGS18 attenuated inositol phosphates production via angiotensin receptor and transcriptional activation through cAMP-responsive element via M1 muscarinic receptor. This suggests RGS18 can act on G q -mediated signaling pathways in vivo.A large number of extracellular stimuli act via cell surface receptors coupled to G-proteins (1). Inactive G-proteins are heterotrimeric proteins consisting of ␣, ␤, and ␥ subunits. Upon binding of a specific ligand to the G-protein-coupled receptor, the receptor promotes the exchange of GDP to GTP in the ␣ subunit, resulting in dissociation of the ␣ subunit from the ␤␥ subunits. The ␤␥ subunits are tightly associated and do not dissociate under physiological conditions. The free ␣ subunit and ␤␥ subunits then transmit signals through various signal transduction pathways. The activated ␣ subunit has slow intrinsic GTPase activity. When the ␣ subunit is in the GDPbound form, it re-associates with the ␤␥ subunits, leading to an inactive form. The duration of the G-protein signal depends on the rate of GTP hydrolysis and the rate of subunit re-association. For small GTP-binding proteins such as ras, there are GAP 1 proteins (GTPase activating protein), which increase the GTP hydrolysis rate. Recently, functional homologs of the ras-GAP have been identified for the heterotrimeric G-protein.These are called RGS (regulator of G-protein signaling) proteins. The first RGS identified, Sst2 (supersensitivity to pheromone) in yeast, is a negative regulator of pheromone signaling (2). Later, the SST2 gene product was shown to function as a GAP for Gpa1, a molecule involved in pheromone desensitization (3). So far ϳ20 RGS have been identified (4 -12), and more could be anticipated. All RGS proteins have a highly conserved domain consisting of 120 amino acid residues, the RGS box, with varying lengths of NH 2 and COOH termini. RGS4 can be expressed in bacteria, and it has been co-crystallized with G␣ i1 as the GDP-AlF4 Ϫ -bound form (13). It was shown that RGS binds to G␣ i t...

Reciprocal regulation of alternative lineages by Rgs18 and its transcriptional repressor Gfi1b

“…This signaling molecule is highly expressed in megakaryocytes, platelets and in hematopoietic progenitors of the myeloerythroid lineage (Gagnon et al, 2002;Louwette et al, 2012;Park et al, 2001;Yowe et al, 2001). Although Rgs18 is the most abundantly expressed Rgs protein in platelets, other family members, such as Rgs6, Rgs10 and Rgs16, are also known to be expressed in this lineage (Kim et al, 2006).…”

Differential gene expression profiling of adult murine hematopoietic stem cells

“…Isolation of a population of Lin Ϫ/lo Thy-1.1 lo c-kit ϩ Sca-1 ϩ HSCs, RNA extraction, cDNA synthesis, and polymerase chain reaction (PCR) amplification have been described previously. 15 There were 7.5 ϫ 10 6 clones in the original library.…”

“…28 Some of the genes we identified are specific to hematopoietic tissues; for example, RGS18 is expressed in LT-HSCs and ST-HSCs as well as monocytes. 15 Among unknown genes, there are genes that code for a variety of important functional domains and several genes with no obvious homology to any known functional domains. Some of the other unknown genes were analyzed by Northern hybridization.…”

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.…”