Stabilization of the GDP-bound conformation of Giα by a peptide derived from the G-protein regulatory motif of AGS3

The G-protein regulatory (GPR) motif in AGS3 was recently identified as a region for protein binding to heterotrimeric G-protein alpha subunits. To define the properties of this similar to 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 Gi alpha1,2 in protein interaction assays, inhibited guanosine 5'-O-(3-thiotriphosphate) binding to Gi alpha, and stabilized the GDP-bound conformation of Gi alpha, The GPR peptide had little effect on nucleotide binding to Go alpha and brain G-protein indicating selective regulation of Gia. Thus, the GPR peptide functions as a guanine nucleotide dissociation inhibitor for Gi alpha. The GPR consensus peptide also blocked receptor coupling to Gi alpha beta gamma indicating that although the AGS3-GPR peptide stabilized the GDP-bound conformation of Gi alpha, this conformation of Gi alpha (GDP) was not recognized by a G-protein coupled receptor. The AGS3-GPR motif presents an opportunity for selective control of Gi alpha- and G beta gamma -regulated effector systems, and the GPR motif allows for alternative modes of signal input to G-protein signaling systems.