Stabilization of the GDP-bound conformation of Gi alpha 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 r egion for protein binding to heterotrimeric G-protein alpha subunits. To de fine the properties of this similar to 20-amino acid motif, we designed a G PR consensus peptide and determined its influence on the activation state o f G-protein and receptor coupling to G-protein. The GPR peptide sequence (2 8 amino acids) encompassed the consensus sequence defined by the four GPR m otifs conserved in the family of AGS3 proteins. The GPR consensus peptide e ffectively prevented the binding of AGS3 to Gi alpha1,2 in protein interact ion assays, inhibited guanosine 5'-O-(3-thiotriphosphate) binding to Gi alp ha, 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 ind icating selective regulation of Gia. Thus, the GPR peptide functions as a g uanine nucleotide dissociation inhibitor for Gi alpha. The GPR consensus pe ptide 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-protei n coupled receptor. The AGS3-GPR motif presents an opportunity for selectiv e control of Gi alpha- and G beta gamma -regulated effector systems, and th e GPR motif allows for alternative modes of signal input to G-protein signa ling systems.