Identification and Characterization of AGS4

Abstract

Activators of G-protein signaling 1-3 (AGS1-3) were identified in a functional screen of mammalian cDNAs that activated G-protein signaling in the absence of a receptor. We report the isolation and characterization of an additional AGS protein (AGS4) from a human prostate leiomyosarcoma cDNA library. AGS4 is identical to G18.1b, which is encoded by a gene within the major histocompatibility class III region of chromosome 6. The activity of AGS4 in the yeast-based functional screen was selective for G i2 /G i3 and independent of guaninenucleotide exchange by G i ␣. RNA blots indicated enrichment of AGS4/G18.1b mRNA in heart, placenta, lung, and liver. Immunocytochemistry with AGS4/G18.1b-specific antisera indicated a predominant nonhomogeneous, extranuclear distribution within the cell following expression in COS7 or Chinese hamster ovary cells. AGS4/ G18.1b contains three G-protein regulatory motifs downstream of an amino terminus domain with multiple prolines. Glutathione S-transferase (GST)-AGS4/G18.1b fusion proteins interacted with purified G i ␣, and peptides derived from each of the G-protein regulatory motifs inhibited guanosine 5 -3-O-(thio)triphosphate (GTP␥S) binding to purified G i ␣ 1 . AGS4/G18.1b was also complexed with G i ␣ 3 in COS7 cell lysates following cell transfection. However, AGS4/G18.1b did not alter the generation of inositol phosphates in COS7 cells cotransfected with the G␤␥-regulated effector phospholipase C-␤2. These data suggest either that an additional signal is required to position AGS4/G18.1b in the proper cellular location where it can access heterotrimer and promote subunit dissociation or that AGS4 serves as an alternative binding partner for G i ␣ independent of G␤␥ participating in G-protein signaling events that are independent of classical G-protein-coupled receptors at the cell surface.

The Proline-rich N-terminal Domain of G18 Exhibits a Novel G Protein Regulatory Function

“…We found that G18 was highly expressed in spleen and lung and moderately expressed in heart, kidney, liver, brain, and adipose tissue. These results are consistent with a previous report by Cao et (3,24). To extend these findings, we tested the binding between purified G18 and purified G␣ i1 or G␣ o in both their inactive GDP-bound and fluoroaluminateactivated states.…”

“…2) that contain an N-terminal truncation (⌬NG18), inactivating point substitutions within each GoLoco motif (G18-mGL), or a combination of both modifications (⌬NG18-mGL). Consistent with previous studies (3,24), G18wt and ⌬NG18 interacted with G␣ i1 -GDP, whereas G18-mGL and ⌬NG18-mGL did not (Fig. 3).…”

“…G18 is a 160-amino acid protein that is composed of three tandem GoLoco motifs interspersed through its middle and C-terminal area plus an uncharacterized N-terminal segment that is rich in proline (14 of 60 residues). Previous biochemical analyses have shown that at least two GoLoco motifs of G18 can interact with GDP-bound G␣ i1 both in vitro and in overexpressed cell systems (3,24). However, the overall physiological function of G18 still remains unknown.…”

“…The GoLoco/GPR motif was originally identified in the Drosophila RGS12 homologue, Loco (3)(4)(5). The GoLoco motif is a 19-amino acid sequence that can bind to the G␣ subunit of G i/o proteins in their inactive state (G␣-GDP) to inhibit the exchange of GDP for GTP.…”

Identification of a receptor-independent activator of G protein signaling (AGS8) in ischemic heart and its interaction with Gβγ

“…In such epistasis experiments, nine of the cDNAs required G␤␥ for their activity and thus were consistent with the definition of a receptorindependent activator of G protein signaling ( Table 1). Three of the nine cDNAs encoded the previously characterized AGS1, AGS3, and AGS4 (12,13,20). AGS5 and AGS6 encoded truncated versions of the previously characterized proteins LGN and RGS12 respectively and these truncated versions each contained the G protein regulatory (GPR) motif(s) found in these two proteins.…”