Abstract

Fusion proteins were generated between the human 5-hydroxytryptamine (5-HT)_1A receptor and both wild-type (Cys³⁵¹) and pertussis toxin-resistant (Gly³⁵¹ and Ile³⁵¹) 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_i1α construct but not in clones expressing 5-HT_1A receptor (Gly³⁵¹)G_i1α or (Ile³⁵¹)G_i1α. Spiperone functioned as an inverse agonist in membranes expressing the 5-HT_1A receptor wild-type G_i1α fusion protein and in those expressing 5-HT_1A receptor (Ile³⁵¹)G_i1α but not the 5-HT_1A receptor (Gly³⁵¹)G_i1α fusion protein. The effect of spiperone at the 5-HT_1A receptor wild-type G_i1α construct but not the 5-HT_1A receptor (Ile³⁵¹)G_i1α 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 (Ile³⁵¹)G_i1α- and (Gly³⁵¹)G_i1α-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_i1α fusion protein but not in those expressing the 5-HT_1A receptor (Ile³⁵¹) or (Gly³⁵¹)G_i1α fusion proteins. These studies demonstrate that alteration of a single amino acid in G_i1α 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.