Site-directed mutations in the third intracellular loop of the serotonin 5-HT1A receptor alter G protein coupling from G(i) to G(s) in a ligand-dependent manner

The effect of mutations (V344E and T343A/V344E) in the third intracellular loop of the serotonin 5-HT1A receptor expressed transiently in human embryo nic kidney 293 cells have been examined in terms of receptor/G protein inte raction and signaling. Serotonin, (R)-8-hydroxy-2-dipropylaminotetralin [(R )-8-OH-DPAT], and buspirone inhibited cyclic AMP production in cells expres sing native and mutant 5-HT1A receptors. Serotonin, however, produced inver se bell-shaped cyclic AMP concentration-response curves at native and mutan t 5-HT1A receptors, indicating coupling not only to G(i)/G(o), but also to G(s). (R)-8-OH-DPAT, however, induced stimulation of cyclic AMP production only after inactivation of G(i)/G(o) proteins by pertussis toxin and only a t the mutant receptors. The partial agonist buspirone was unable to induce coupling to G(s) at any of the receptors, even after pertussis toxin treatm ent. The basal activities of native and mutant 5-HT1A receptors in suppress ing cyclic AMP levels were not found to be significantly different. The rec eptor binding characteristics of the native and mutant receptors were inves tigated using the novel 5-HT1A receptor antagonist [H-3]NAD-299. For other receptors, analogous mutations have produced constitutive activation. This does not occur for the 5-HT1A receptor, and for this receptor the mutations seem to alter receptor/G protein coupling, allowing ligand-dependent coupl ing of receptor to G(s) in addition to G(i)/G(o) proteins.