title: Molecular cloning and pharmacological characterization of guinea pig 5-HT1B and 5-HT1D receptors.
abstract: Human 5-HT1B and 5-HT1D receptors have been implicated as molecular targets for the treatment of acute migraine based upon the pharmacological actions and clinical efficacy of sumatriptan, an agonist for human 5-HT1B/1D receptors. The guinea pig has served as an animal model to assess 5-HT1B/1D receptor function, most recently in evaluating 5-HT1B/1D receptor agonists as potential anti-migraine agents. Since two distinct, but closely-related receptors displaying "5-HT1D receptor pharmacology" have been cloned previously from most mammalian species, the genes encoding these receptors were isolated from a guinea pig liver genomic DNA library using oligonucleotide probes targeted to nonconserved regions of recombinant human 5-HT1B and 5-HT1D receptors. Sequence analysis indicates that guinea pig 5-HT1B and 5-HT1D receptors are comprised 390 and 378 amino acids, respectively. Comparison of the deduced amino acid sequences of guinea pig 5-HT1B and 5-HT1D receptor subtypes show that they display overall and transmembrane (TM) identities of 63% and 77%, respectively. Both clones contain a conserved threonine residue in TM7, a structural feature imparting "5-HT1D receptor pharmacology". Guinea pig 5-HT1B and 5-HT1D receptor genes were transiently expressed in Cos-7 cells and their binding properties were evaluated using [3H]5-HT. Both cloned receptor subtypes displayed "5-HT1D receptor pharmacology" with the following rank order of binding affinities: 5-CT > 5-HT > sumatriptan > 8-OH-DPAT > (-)-pindolol. Ketanserin displayed modest (five-fold) 5-HT1D receptor selectivity, while methiothepin exhibited a similar selectivity for the 5-HT1B subtype. In particular, ketanserin exhibits profound differences in 5-HT1D receptor affinity (and selectivity) across species. High correlations were observed between the binding affinities of serotonergic ligands for 5-HT1D binding sites measured in guinea pig cortical membranes and both cloned guinea pig 5-HT1B (r2 = 0.88) and 5-HT1D (r2 = 0.80) receptors, indicating that the development of subtype selective compounds (i.e. 5-HT1B versus 5-HT1D) using native tissues may be more difficult to achieve without the advantage of using recombinant receptor subtypes. Additionally, there is a good correspondence between binding profiles of recombinant guinea pig 5-HT1B and 5-HT1D receptor subtypes and to their respective cloned human homologs. However, species differences in binding affinities of a subset of compounds are evident. These data extend previous observations that subtype selective (i.e. 5-HT1D) compounds identified in one species may not discriminate between closely related receptors (i.e. 5-HT1B and 5-HT1D) in all animal model systems.
authors.affiliation: Synaptic Pharmaceutical Corporation, Paramus, NJ 07652, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Zgombick
authors.forename: J A
authors.initials: JA
authors.lastname: Bard
authors.forename: S A
authors.initials: SA
authors.lastname: Kucharewicz
authors.forename: D A
authors.initials: DA
authors.lastname: Urquhart
authors.forename: R L
authors.initials: RL
authors.lastname: Weinshank
authors.forename: T A
authors.initials: TA
authors.lastname: Branchek
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004285:Dogs; D006168:Guinea Pigs; D006801:Humans; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D011817:Rabbits; D051381:Rats; D011985:Receptors, Serotonin / Q000096:biosynthesis* / Q000187:drug effects; D018490:Serotonin Agents / Q000378:metabolism; D013045:Species Specificity
chemical_list: D008024:Ligands; D011985:Receptors, Serotonin; D018490:Serotonin Agents