Coupling of canine serotonin 5-HT1B and 5-HT1D receptor subtypes to the formation of inositol phosphates by dual interactions with endogenous G(i/o) and recombinant G(alpha 15) proteins

Molecular cloning and expression of canine (ca) serotonin 5-HT1B and ca 5-HT1D receptor subtypes showed that besides the lower binding affinity of ketanserin for the ca 5-HT1D receptor, the ligand binding profiles were similar to their human homologues. Site-directed mutagenesis studies suggest that a Gln(189) residue in the second extracellular loop of the ca 5-HT1D receptor may partially account for the lower binding affinity of ketanserin. The coupling of ca 5-HT1B and ca 5-HT1D receptor subtypes to the phospholipase C pathway was analyzed by measuring stimulation of inositol phosphate formation in COS-7 cells. Zolmitriptan potently stimulated (EC50 = 4.9 nM) the inositol phosphate formation at ca 5-HT1D receptors in a fully pertussis toxin (PTX)-dependent manner, whereas only a weak PTX-resistant inositol phosphate response (26-29% at 10 mu M zolmitriptan) could be detected for the ca 5-HT1B receptor at a similar expression level. In contrast, both ca 5-HT1B and ca 5-HT1D receptor subtypes yielded a similar maximal magnitude of inositol phosphate formation (300-340% at 10 mu M zolmitriptan) upon co-expression with a mouse (m) G(alpha 15) protein. PTX treatment and co-expression with a beta-adrenergic receptor kinase C-terminal polypeptide partially (20-46%) abolished the m G(alpha 15) protein-dependent ca 5-HT1B and ca 5-HT1D receptor-mediated stimulation of inositol phosphate formation. This study suggests both 5-HT receptor subtypes can activate beta gamma subunits of endogenous G(i/o) proteins besides their coupling to recombinant m G(alpha 15) protein.