Regulation of G Protein Activation and Effector Modulation by Fusion Proteins between the Human 5-Hydroxytryptamine1AReceptor and the α Subunit of Gi1α: Differences in Receptor-Constitutive Activity Imparted by Single Amino Acid Substitutions in Gi1Î

Fusion proteins were generated between the human 5-hydroxytryptamine (5-HT) 1A receptor and both wild-type (Cys 351 ) and pertussis toxin-resistant (Gly 351 and Ile 351 ) forms of G i1 . These were expressed stably. Pertussis toxin treatment substantially reduced basal high-affinity GTPase activity in clones expressing the 5-HT 1A receptor wild-type G i1 α construct but not in clones expressing 5-HT 1A receptor (Gly 351 )G i1 α or (Ile 351 )G i1 α. Spiperone functioned as an inverse agonist in membranes expressing the 5-HT 1A receptor wild-type G i1 α fusion protein and in those expressing 5-HT 1A receptor (Ile 351 )G i1 α but not the 5-HT 1A receptor (Gly 351 )G i1 α fusion protein. The effect of spiperone at the 5-HT 1A receptor wild-type G i1 α construct but not the 5-HT 1A receptor (Ile 351 )G i1 α construct was blocked by pertussis toxin treatment. By contrast, agonists functioned with equal effectiveness at the three fusion proteins and were unaffected by pertussis toxin treatment of the (Ile 351 )G i1 α- and (Gly 351 )G i1 α-containing constructs. 5-HT resulted in strong inhibition of forskolin-amplified adenylyl cyclase in intact cells expressing the isolated 5-HT 1A receptor. In fusion protein-expressing cells, 5-HT-mediated inhibition of adenylyl cyclase was also observed. Pertussis toxin treatment obliterated 5-HT-mediated inhibition in cells expressing the isolated receptor and the 5-HT 1A receptor wild-type G i1 α fusion protein but not in those expressing the 5-HT 1A receptor (Ile 351 ) or (Gly 351 )G i1 α fusion proteins. These studies demonstrate that alteration of a single amino acid in G i1 α located at a key contact site between the G protein and a G protein-coupled receptor can regulate agonist-independent constitutive activity of the G protein-coupled receptor and that fusion proteins can directly regulate adenylyl cyclase.