Structural requirements for G(o) activation by receptor-derived peptides: activation and modulation domains of the...

Synthetic peptides are important tools for understanding the sites and mechanisms of receptor/G protein interactions. We examined the structural determinants of receptor-fragment peptides for G protein binding and activation. A dimer of peptides from the carboxyl-terminal (i3c) and amino-terminal (i3n) regions of the alpha 2A-adrenergic receptor is most potent in stimulating guanine-nucleotide exchange of any peptides studied. Stimulation of GTPase by i3n is partially blocked by pertussis toxin treatment, whereas stimulation by i3c is not, which is consistent with action of i3c at the amino terminus of Gi. Both peptides inhibit adenylyl cyclase in Chinese hamster ovary cell membranes, but only the i3c effect is consistent with a pure Gi stimulation. We also examined the mechanism and defined a minimal structural subset of i3c required for G protein activation. Residues 361-365 from the receptor were essential for GTPase stimulation, whereas determinants in the region 368-373 modulated that activity. A specific role for arginines is defined beyond just their positive charge. Complex effects of modifications of Thr373 suggest a regulatory or conformational role of that residue in the previously defined constitutive activation of the alpha 2-adrenergic receptor [J. Biol. Chem. 268:16483-16487 (1993)]. Thus, our data plus recent mutagenesis results support a role for hydrophobicity in the i3n region and a positively charged/arginine-rich region approximately 15-20 residues from the sixth transmembrane span in G protein activation. In contrast, the immediate perimembrane region of i3c seems to have largely conformational effects in producing constitutive activation of the receptors.