Mutant 5-hydroxytryptamine 1A receptor D116A is a receptor activated solely by synthetic ligands with a rich pharmacology.
Like other biogenic amine G protein-coupled receptors, mutation of the conserved aspartatic residue into alanine at position 116 (D116A(3.32)) in the 5-hydroxytryptamine (5-HT)(1A) receptor greatly affects 5-HT binding and signal transduction. [(3)H]8-Hydroxy-2-dipropylaminotetralin (8-OH-DPAT) and [(3)H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY100,635) are capable to bind the 5-HT(1A)-D116A mutant and, using these radioligands, we show here that this mutation dramatically reduces the affinities of the selective 5-HT(1A) agonists N-(3-chloro-4-fluorobenzoyl)-4-fluoro-4-[(5-methylpyridin-2-yl)-methylamino methyl]piperidine (F13640), 3-chloro-4-fluorophenyl-(4-fluorophenyl-4-{[(5-methyl-6 methylamino-pyridin-2-ylmethyl)-amino]-methyl}-piperidin-1-yl-methanone (F13714), and 2-[5-[3-(4-methylsulfonylamino)benzyl-1,4-oxadiazol-5-yl]-1H-indole-3-yl]ethylamine (L694247) and that of 5-carboxamidotryptamine. Although to a lesser extent, the binding of buspirone, (+)-flesinoxan, (-)-pindolol, and (-)-8-OH-DPAT are also highly decreased. In contrast, affinities of the 5-HT(1A) ligands WAY100,635, spiperone, (-)-4-(dipropylamino)-1,3,4,5-tetrahydrobenz {c,d}indole-6-carboxamide (LY228,729), and 1-[2-(4-fluorobenzoylamino)ethyl]-4-(7-methoxynaphtyl) piperazine (S14506) and the prototypical 5-HT(1A) agonist (+)-8-OH-DPAT are only slightly affected by the mutation, suggesting a moderate contribution of Asp116 to the binding pocket for these latter. Furthermore, LY228,729, S14506, and (+)-8-OH-DPAT induce a potent and efficacious coupling of the 5-HT(1A)-D116A receptor to G protein activation as measured by Ca(2+) mobilization and guanosine 5'-O-(3-[(35)S]thio)triphosphate binding in Chinese hamster ovary cells as well as by G protein-coupled inwardly rectifying potassium channel current activation in Xenopus laevis oocytes. It is interesting that the selective 5-HT(1A) antagonist WAY100,635 shows potent partial agonist activity at the 5-HT(1A)-D116A mutant, whereas spiperone maintains its inverse agonist properties. The pharmacological approach reported here re-evaluates the binding and functional properties of the 5-HT(1A)-D116A receptor and describes for the first time this mutant as a receptor activated solely by synthetic ligands (RASSL), with a rich pharmacology. By bioengineering animal models incorporating this RASSL, one may further explore the role of 5-HT(1A) receptor signaling in the central nervous system as well as G(i) protein-mediated signaling pathways in other tissues.
Cussac
Didier
D
Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, Castres, France. didier.cussac@pierre-fabre.com
Palmier
Christiane
C
Finana
Frédéric
F
De Vries
Luc
L
Tardif
Stéphanie
S
Léger
Céline
C
Bernois
Sophie
S
Heusler
Peter
P
D017371:8-Hydroxy-2-(di-n-propylamino)tetralin / Q000378:metabolism / Q000494:pharmacology; D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006801:Humans; D008024:Ligands; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010879:Piperazines / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D011725:Pyridines / Q000378:metabolism / Q000494:pharmacology; D044282:Receptor, Serotonin, 5-HT1A / Q000235:genetics* / Q000378:metabolism*; D017366:Serotonin Receptor Agonists / Q000378:metabolism / Q000494:pharmacology; D014982:Xenopus laevis
D008024:Ligands; D010879:Piperazines; D011725:Pyridines; D017366:Serotonin Receptor Agonists; D044282:Receptor, Serotonin, 5-HT1A; D001224:Aspartic Acid; C090413:N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; D000409:Alanine