title: Structural basis for the selectivity of the RGS protein, GAIP, for Galphai family members. Identification of a single amino acid determinant for selective interaction of Galphai subunits with GAIP.
abstract: GAIP is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by some G protein alpha subunits. In the present studies, we have examined the structural basis for the ability of GAIP to discriminate among members of the Galphai family. Galphai1, Galphai3, and Galphao interacted strongly with GAIP, whereas Galphai2 interacted weakly and Galphas did not interact at all. A chimeric G protein composed of a Galphai2 N terminus and a Galphai1 C terminus interacted as strongly with GAIP as native Galphai1, whereas a chimeric N-terminal Galphai1 with a Galphai2 C terminus did not interact. These results suggest that the determinants responsible for GAIP selectivity between these two Galphais reside within the C-terminal GTPase domain of the G protein. To further localize residues contributing to G protein-GAIP selectivity, a panel of 15 site-directed Galphai1 and Galphai2 mutants were assayed. Of the Galphai1 mutants tested, only that containing a mutation at aspartate 229 located at the N terminus of Switch 3 did not interact with GAIP. Furthermore, the only Galphai2 variant that interacted strongly with GAIP contained a replacement of the corresponding Galphai2 Switch 3 residue (Ala230) with aspartate. To determine whether GAIP showed functional preferences for Galpha subunits that correlate with the binding data, the ability of GAIP to enhance the GTPase activity of purified alpha subunits was tested. GAIP catalyzed a 3-5-fold increase in the rate of GTP hydrolysis by Galphai1 and Galphai2(A230D) but no increase in the rate of Galphai2 and less than a 2-fold increase in the rate of Galphai1(D229A) under the same conditions. Thus, GAIP was able to discriminate between Galphai1 and Galphai2 in both binding and functional assays, and in both cases residue 229/230 played a critical role in selective recognition.
authors.affiliation: Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: D S
authors.initials: DS
authors.lastname: Woulfe
authors.forename: J M
authors.initials: JM
authors.lastname: Stadel
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D020558:GTP Phosphohydrolases / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D020710:RGS Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae
chemical_list: D020690:GTPase-Activating Proteins; D009711:Nucleotides; D010750:Phosphoproteins; D011506:Proteins; D020710:RGS Proteins; D011993:Recombinant Fusion Proteins; C097187:regulator of G-protein signalling 19; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins