Cloning of serotonin 5-HT(1) receptor subtypes from the chimpanzee, gorilla and Rhesus monkey and their agonist-induced guanosine 5'gamma(35)S triphosphate binding.
5-HT(1) receptor subtypes ((1B), (1D) and (1F)) have been implicated in migraine pathophysiology and their ligands have been examined for pharmacological actions in various experimental animal models. Considerable divergences exist, however, in their primary sequences between experimental animals and human, and additional models closer to human, such as non-human primates seem to be useful for migraine research. Earlier, we cloned the 5-HT(1D), and here 5-HT(1B) and 5-HT(1F) receptors from the chimpanzee, gorilla and Rhesus monkey, via polymerase chain reactions with their genomic DNAs and primers designed from the corresponding human receptors. Direct sequencing of PCR products showed that the 5-HT(1B) receptors from the chimpanzee, gorilla and monkey differ from the human receptor by 0, 1 and 7 residues, respectively while 5-HT(1F) receptors differ by 0, 3 and 10 residues, respectively. These divergent residues are mostly conservatively substituted and also largely confined to the N-terminal region and the 3rd intracellular loop, away from transmembrane segments and intracellular loops near membrane which are critical for ligand binding and G protein coupling. The chimpanzee 5-HT(1D), 5-HT(1B) and monkey 5-HT(1F) receptors, as heterologously expressed in human embryonic kidney 293 cells, showed robust agonist-induced guanosine 5'gamma (35)S triphosphate (GTPgamma(35)S) binding through activation of G proteins containing Ggamma(i) subunits. Moreover, pronounced inhibition of basal GTPgamma(35)S binding by methiothepin (an antagonist), representing constitutively active receptors, was observed with only 5-HT(1D). Overall, ligand binding and GTPgamma(35)S binding profiles for these primate receptors are comparable to those for the human receptors and validate non-human primates as useful models for human migraine research.
Alberts
G L
GL
Biology II/Neurobiology, 7251-209-512, 301 Henrietta St., Kalamazoo, MI 49007, USA.
Pregenzer
J F
JF
Im
W B
WB
Slightom
J L
JL
D000818:Animals; D003001:Cloning, Molecular; D006071:Gorilla gorilla; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000493:pharmacokinetics*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008253:Macaca mulatta; D002679:Pan troglodytes; D044302:Receptor, Serotonin, 5-HT1B; D044346:Receptor, Serotonin, 5-HT1D; D011985:Receptors, Serotonin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017366:Serotonin Receptor Agonists / Q000494:pharmacology*; D013462:Sulfur Radioisotopes; D000096642:Receptor, Serotonin, 5-HT1F
D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008024:Ligands; D044302:Receptor, Serotonin, 5-HT1B; D044346:Receptor, Serotonin, 5-HT1D; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D017366:Serotonin Receptor Agonists; D013462:Sulfur Radioisotopes; D000096642:Receptor, Serotonin, 5-HT1F; C470302:HTR1B protein, human