Coupling of canine serotonin 5-HT(1B) and 5-HT(1D) receptor subtypes to the formation of inositol phosphates by dual interactions with endogenous G(i/o) and recombinant G(alpha15) proteins.
Molecular cloning and expression of canine (ca) serotonin 5-HT(1B) and ca 5-HT(1D) receptor subtypes showed that besides the lower binding affinity of ketanserin for the ca 5-HT(1D) 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-HT(1D) receptor may partially account for the lower binding affinity of ketanserin. The coupling of ca 5-HT(1B) and ca 5-HT(1D) receptor subtypes to the phospholipase C pathway was analyzed by measuring stimulation of inositol phosphate formation in COS-7 cells. Zolmitriptan potently stimulated (EC(50) = 4.9 nM) the inositol phosphate formation at ca 5-HT(1D) receptors in a fully pertussis toxin (PTX)-dependent manner, whereas only a weak PTX-resistant inositol phosphate response (26-29% at 10 microM zolmitriptan) could be detected for the ca 5-HT(1B) receptor at a similar expression level. In contrast, both ca 5-HT(1B) and ca 5-HT(1D) receptor subtypes yielded a similar maximal magnitude of inositol phosphate formation (300-340% at 10 microM zolmitriptan) upon co-expression with a mouse (m) G(alpha15) protein. PTX treatment and co-expression with a beta-adrenergic receptor kinase C-terminal polypeptide partially (20-46%) abolished the m G(alpha15) protein-dependent ca 5-HT(1B) and ca 5-HT(1D) receptor-mediated stimulation of inositol phosphate formation. This study suggests both 5-HT receptor subtypes can activate betagamma subunits of endogenous G(i/o) proteins besides their coupling to recombinant m G(alpha15) protein.
Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, Castres, France.
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Wurch
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Pauwels
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D003001:Cloning, Molecular; D004285:Dogs; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism*; D007650:Ketanserin / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D044302:Receptor, Serotonin, 5-HT1B; D044346:Receptor, Serotonin, 5-HT1D; D011985:Receptors, Serotonin / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012701:Serotonin / Q000378:metabolism; D014162:Transfection
C470302:HTR1B protein, human; D007295:Inositol Phosphates; D044302:Receptor, Serotonin, 5-HT1B; D044346:Receptor, Serotonin, 5-HT1D; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D012701:Serotonin; D007650:Ketanserin; C112631:G protein alpha 16; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins