Identification and characterization of AGS4: a protein containing three G-protein regulatory motifs that regulate the activation state of Gialpha.
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.
Cao
Xiaoqing
X
The Guthrie Research Institute, Sayre, Pennsylvania 18840, USA.
Cismowski
Mary J
MJ
Sato
Motohiko
M
Blumer
Joe B
JB
Lanier
Stephen M
SM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation; D020555:GTP-Binding Protein Regulators / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014018:Tissue Distribution; D010738:Type C Phospholipases / Q000235:genetics
D029742:Caenorhabditis elegans Proteins; D020555:GTP-Binding Protein Regulators; D007295:Inositol Phosphates; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go