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.
Subject(s)
Receptors, Serotonin/biosynthesis , Amino Acid Sequence , Animals , Cloning, Molecular , Dogs , Guinea Pigs , Humans , Ligands , Mice , Molecular Sequence Data , Rabbits , Rats , Receptors, Serotonin/drug effects , Serotonin Agents/metabolism , Species SpecificityABSTRACT
A cDNA for a member of the G protein-coupled receptor family was isolated from Drosophila using a probe derived from a human beta 2-adrenergic receptor cDNA. This Drosophila receptor gene is localized at 99A10-B1 on the right arm of chromosome 3 and is preferentially expressed in Drosophila heads. The insect octopamine receptor has been permanently expressed in mammalian cells, where it mediates the attenuation of adenylate cyclase activity and exhibits a pharmacological profile consistent with an octopamine type 1 receptor. Sequence and pharmacological comparisons indicate that the octopamine receptor is unique but closely related to mammalian adrenergic receptors, perhaps as an evolutionary precursor.
Subject(s)
Drosophila/genetics , Gene Expression , Genes , Receptors, Adrenergic/genetics , Receptors, Biogenic Amine , Amino Acid Sequence , Animals , Base Sequence , Brain/metabolism , Cell Line , Chromosome Mapping , Cloning, Molecular/methods , DNA/genetics , DNA/isolation & purification , GTP-Binding Proteins/genetics , Genomic Library , Humans , Molecular Sequence Data , Octopamine/metabolism , Oligonucleotide Probes , Protein Conformation , Receptors, Adrenergic, beta/genetics , Restriction Mapping , Sequence Homology, Nucleic Acid , TransfectionABSTRACT
Two cDNA clones (3.7 kb and 4.8 kb) encoding a Drosophila muscarinic acetylcholine receptor were isolated from a Drosophila head cDNA library and characterized by automated DNA sequence analysis. The Drosophila muscarinic receptor contains 788 amino acids with a calculated Mr of 84,807 and displays greater than 60% homology with mammalian muscarinic receptors. The muscarinic receptor maps to the tip of the right arm of the second chromosome of the Drosophila genome.
