title: Characterization of the 5-HT6 receptor coupled to Ca2+ signaling using an enabling chimeric G-protein.
abstract: We examined the feasibility of coupling the 5-HT(6) receptor to a Ca(2+) signaling read-out using a chimeric G-protein, comprising of G(alphaq) with the C-terminal five amino acids from G(alphas), to facilitate assays on the fluorometric imaging plate reader (FLIPR). Using a transient transfection assay in human embryonic kidney (HEK) cells, Ca(2+) signaling in response to serotonin (5-HT) was facilitated by co-transfection of the 5-HT(6) receptor with the G(alphaq)/G(alphas) chimera, but not with the 5-HT(6) receptor alone or with a similar chimera incorporating the C-terminal five amino acids of G(alphai3). A series of agonist concentration-response curves were constructed using the 5-HT(6)-G(alphaq)/G(alphas) signaling assay generating the following rank order of agonist potency; 5-methoxytryptamine (EC(50), 9 nM)=5-HT (12 nM)=2-methyl 5-HT (13 nM)>tryptamine (86 nM)=5-carboxamidotryptamine (5-CT) (119 nM)>>lisuride (>1 microM). In comparison, essentially identical EC(50) values were observed for the stimulation of cAMP accumulation with the same compounds; 5-methoxytryptamine (EC(50), 6 nM)=5-HT (6 nM)=2-methyl 5-HT (15 nM)>tryptamine (91 nM)=5-CT (153 nM)>lisuride (>350 nM). Clozapine and SB 271046 both produced a concentration-dependent antagonism of the 5-HT-stimulated Ca(2+) response with IC(50) values of 45 and 11 nM, respectively. In contrast, aripiprazole, a recently launched atypical anti-psychotic with a novel mechanism of action described as a dopamine/serotonin stabilizer, was essentially devoid of 5-HT(6) receptor antagonist activity. Our results demonstrate that a FLIPR-based Ca(2+) signaling assay is a feasible approach to the functional characterization of 5-HT(6) receptor ligands. Moreover, the equivalent coupling efficiency, as indexed by agonist potency, observed using this system compared with the native coupling assay to cAMP suggests that the C-terminal five amino acids of G(alphas) are the major determinant for the receptor/G-protein interaction of the 5-HT(6) receptor subtype.
authors.affiliation: Neuroscience Discovery Research, Wyeth Research, CN-8000, Princeton, NJ 08543-8000, USA.
authors.forename: Jean Y
authors.initials: JY
authors.lastname: Zhang
authors.forename: Stanley
authors.initials: S
authors.lastname: Nawoschik
authors.forename: Dianne
authors.initials: D
authors.lastname: Kowal
authors.forename: Deborah
authors.initials: D
authors.lastname: Smith
authors.forename: Taylor
authors.initials: T
authors.lastname: Spangler
authors.forename: Rafal
authors.initials: R
authors.lastname: Ochalski
authors.forename: Lee
authors.initials: L
authors.lastname: Schechter
authors.forename: John
authors.initials: J
authors.lastname: Dunlop
mesh_terms: D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D006801:Humans; D011985:Receptors, Serotonin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012702:Serotonin Antagonists / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D014162:Transfection
chemical_list: D011985:Receptors, Serotonin; D011993:Recombinant Fusion Proteins; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; C097216:serotonin 6 receptor; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium