Abstract

The mechanism by which receptors activate G proteins is unclear because a connection between the receptor and the nucleotide binding site has not been established. To investigate this mechanism, we evaluated the roles in receptor interaction of three potential receptor contact sites in α_s: the α2/β4, α3/β5, and α4/β6 loops. Substitutions of α_i2 homologs for α_sresidues in the α2/β4 loop and alanine substitutions of residues in the α4/β6 loop do not affect activation by the β₂-adrenergic receptor. However, replacement of five α_s residues in the α3/β5 loop region with the homologous α_i2 residues decreases receptor-mediated activation of α_s and increases the affinity of G_s for this receptor. The substitutions do not alter guanine nucleotide binding or hydrolysis, or activation by aluminum fluoride, indicating that the effects on receptor interaction are not due to a destabilization of the guanine-nucleotide bound state. In a model of the receptor-G protein complex, the α3/β5 loop maps near the second and third intracellular loops of the receptor. The effects of the α3/β5 substitutions suggest that the wild-type residues may be receptor contact sites that are optimized to ensure the reversibility of receptor-G protein interactions. Furthermore, the α3/β5 region corresponds to an exchange factor contact site in both EF-Tu and Ras, suggesting that the mechanisms by which seven-transmembrane receptors and exchange factors catalyze nucleotide exchange may share common elements.