Abstract
The 5-hydroxytryptamine (5-HT) 1D/1B receptors have gained particular interest as potential targets for treatment of migraine and depression. G-protein coupling and other intrinsic properties of the human 5-HT1D receptor were studied using a baculovirus-based expression system in Sf9 cells. Coexpression of the human 5-HT1D receptor with Gαi1, αi2, αi3, or Gαo-proteins and Gβ1γ2-subunits reconstituted a Gpp(NH)p-sensitive, high affinity binding of [3H]5-HT to this receptor, whereas the Gαqβ1γ2 heterotrimer was ineffective in this respect. Competition of [3H]5-HT binding by various compounds confirmed that coexpression of the human 5-HT1D receptor with Gαi/oβ1γ2 reconstitutes the receptor in a high affinity agonist binding state, having the same pharmacological profile as the receptor expressed in mammalian cells. Binding of the antagonist ocaperidone to the human 5-HT1Dreceptor in coupled or noncoupled state was analyzed. This compound competed with [3H]5-HT binding more potently on the human 5-HT1D receptor in the noncoupled state, showing its inverse agonistic character. Ocaperidone acted as a competitive inhibitor of [3H]5-HT binding when tested with the coupled receptor form but not so when tested with the noncoupled receptor preparation. Finally, [35S]GTPγS binding experiments using the inverse agonist ocaperidone revealed a high level of constitutive activity of the human 5-HT1D receptor. Taken together, the reconstitution of the human 5-HT1Dreceptor-G-protein coupling using baculovirus-infected Sf9 cells made possible the assessment of coupling specificity and the detection of different binding states of the receptor induced by G-protein coupling or ligand binding.
Footnotes
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Send reprint requests to: Josée Leysen, Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, Belgium. E-mail:jleysen2{at}janbe.jnj.com
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↵1 These authors contributed equally to the work.
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This work was supported in part by the IWT Grant 940232.
- Abbreviations:
- 5-HT
- 5-hydroxytryptamine
- R
- receptor
- Gαi/o
- combination of Gαi1, Gαi2, Gαi3, and Gαo
- GPCR
- G-protein-coupled receptor
- Gpp(NH)p
- guanosine 5′-(β,γ-imido)triphosphate
- GTPγS
- guanosine 5′-O-(3-thiotriphosphate)
- Sf9 cells
- Spodoptera frugiperda cells
- pIC50
- −log of the IC50 value
- pEC50
- −log of the EC50, value
- Received July 19, 1999.
- Accepted February 20, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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