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 guanine-nucleotide exchange by G(i)alpha. 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)alpha, and peptides derived from each of the G-protein regulatory motifs inhibited guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to purified G(i)alpha(1). AGS4/G18.1b was also complexed with G(i)alpha(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 Gbetagamma-regulated effector phospholipase C-beta2. 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)alpha independent of Gbetagamma participating in G-protein signaling events that are independent of classical G-protein-coupled receptors at the cell surface.
Highlights
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
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 Gi␣ independent of G␥ participating in G-protein signaling events that are independent of classical G-protein-coupled receptors at the cell surface
The yeast strain was further modified by replacing the yeast G␣ subunit Gpa1 with human Gi␣3 and rendering growth dependent upon activation of the pheromone response pathway. This yeast strain was used to screen a human prostate leiomyosarcoma cDNA library in a galactose-inducible vector pYES2 for entities that promoted growth in galactose-specific manner. cDNAs isolated in this screen that required the presence of G␥ for activity were termed activators of G-protein signaling (AGS) as described previously for AGS1–3 [6, 7]
Summary
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. Despite clear differences in key residues within the GPR consensus motif, protein interaction studies with a GST-AGS4/G18.1b fusion protein indicated direct binding of Gi␣1, and peptides derived from the GPR motifs inhibited GTP␥S binding to purified Gi␣1.
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