Abstract
Like other biogenic amine G protein-coupled receptors, mutation of the conserved aspartatic residue into alanine at position 116 (D116A3.32) in the 5-hydroxytryptamine (5-HT)1A receptor greatly affects 5-HT binding and signal transduction. [3H]8-Hydroxy-2-dipropylaminotetralin (8-OH-DPAT) and [3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY100,635) are capable to bind the 5-HT1A-D116A mutant and, using these radioligands, we show here that this mutation dramatically reduces the affinities of the selective 5-HT1A 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-HT1A 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-HT1A 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-HT1A-D116A receptor to G protein activation as measured by Ca2+ mobilization and guanosine 5′-O-(3-[35S]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-HT1A antagonist WAY100,635 shows potent partial agonist activity at the 5-HT1A-D116A mutant, whereas spiperone maintains its inverse agonist properties. The pharmacological approach reported here re-evaluates the binding and functional properties of the 5-HT1A-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-HT1A receptor signaling in the central nervous system as well as Gi protein-mediated signaling pathways in other tissues.
- 5-HT, 5-hydroxytryptamine
- GPCR, G protein-coupled receptor
- RASSL, receptor activated solely by synthetic ligand
- 8-OH-DPAT, 8-hydroxy-2-(di-n-propylamino)tetralin
- WAY100, 635, [3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride
- 5-CT, 5-carboxamidotryptamine
- L694247, 2-[5-[3-(4-methylsulfonylamino)benzyl-1, 4-oxadiazol-5-yl]-1H-indole-3-yl]ethylamine
- F13640, N-(3-chloro-4-fluorobenzoyl)-4-fluoro-4-[(5-methylpyridin-2-yl)-methylamino methyl]piperidine
- F13714, 3-chloro-4-fluorophenyl-(4-fluorophenyl-4-{ [(5-methyl-6 methylamino-pyridin-2-ylmethyl)-amino]-methyl}-piperidin-1-yl-methanone
- LY228, 729, (−)-4-(dipropylamino)-1,3,4,5-tetrahydrobenz {c, d} indole-6-carboxamide
- S14506, 1-[2-(4-fluorobenzoylamino)ethyl]-4-(7-methoxynaphtyl) piperazine
- GIRK, G protein-coupled inward rectifier potassium
- h, human
- CHO, Chinese hamster ovary
- AFU, arbitrary fluorescence units
- [35S]GTPγS, guanosine-5′-O-(3-[35S]thio)-triphosphate.
Footnotes
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This work was supported by Pierre Fabre Médicament.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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ABBREVIATIONS:
- Received May 26, 2009.
- Accepted July 14, 2009.
- © 2009 by The American Society for Pharmacology and Experimental Therapeutics
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