The RGS14 GoLoco domain discriminates among Galphai isoforms.
Regulators of G protein signaling (RGS) modulate G protein activity by functioning as GTPase-activating proteins (GAPs) for alpha-subunits of heterotrimeric G proteins. RGS14 regulates G protein nucleotide exchange and hydrolysis by acting as a GAP through its RGS domain and as a guanine nucleotide dissociation inhibitor (GDI) through its GoLoco motif. RGS14 exerts GDI activity on Galphai1, but not Galphao. Selective interactions are mediated by contacts between the alphaA and alphaB helices of the Galphai1 helical domain and the GoLoco C terminus (Kimple, R. J., Kimple, M. E., Betts, L., Sondek, J., and Siderovski, D. P. (2002) Nature 416, 878-881). Three isoforms of Galphai exist in mammalian cells. In this study, we tested whether all three isoforms were subject to RGS14 GDI activity. We found that RGS14 inhibits guanine nucleotide exchange on Galphai1 and Galphai3 could, but not Galphai2. Galphai2 be rendered sensitive to RGS14 GDI activity by replacement of residues within the alpha-helical domain. In addition to the contact residues in the alphaA and alphaB helices previously identified, we found that the alphaA/alphaB and alphaB/alphaC loops are important determinants of Galphai selectivity. The striking selectivity observed for RGS14 GDI activity in vitro points to Galphai1 and Galphai3 as the likely targets of RGS14-GoLoco regulation in vivo.
Mittal
Vivek
V
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Linder
Maurine E
ME
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry*; D020690:GTPase-Activating Proteins / Q000737:chemistry*; D006147:Guanine / Q000737:chemistry; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry; D006868:Hydrolysis; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D020710:RGS Proteins / Q000737:chemistry* / Q000502:physiology; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013997:Time Factors
D017931:DNA Primers; D020690:GTPase-Activating Proteins; D020033:Protein Isoforms; D020710:RGS Proteins; C491400:RGS14 protein, human; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006147:Guanine; D006160:Guanosine Triphosphate; D019206:GTP-Binding Protein alpha Subunits, Gi-Go