Stabilization of the GDP-bound Conformation of Giα by a Peptide Derived from the G-protein Regulatory Motif of AGS3*

Abstract

The G-protein regulatory (GPR) motif in AGS3 was recently identified as a region for protein binding to heterotrimeric G-protein α subunits. To define the properties of this ∼20-amino acid motif, we designed a GPR consensus peptide and determined its influence on the activation state of G-protein and receptor coupling to G-protein. The GPR peptide sequence (28 amino acids) encompassed the consensus sequence defined by the four GPR motifs conserved in the family of AGS3 proteins. The GPR consensus peptide effectively prevented the binding of AGS3 to Giα1,2 in protein interaction assays, inhibited guanosine 5′-O-(3-thiotriphosphate) binding to Giα, and stabilized the GDP-bound conformation of Giα. The GPR peptide had little effect on nucleotide binding to Goα and brain G-protein indicating selective regulation of Giα. Thus, the GPR peptide functions as a guanine nucleotide dissociation inhibitor for Giα. The GPR consensus peptide also blocked receptor coupling to Giαβγ indicating that although the AGS3-GPR peptide stabilized the GDP-bound conformation of Giα, this conformation of Giα_GDPwas not recognized by a G-protein coupled receptor. The AGS3-GPR motif presents an opportunity for selective control of Giα- and Gβγ−regulated effector systems, and the GPR motif allows for alternative modes of signal input to G-protein signaling systems.