Human 5-Hydroxytryptamine5A Receptors Activate Coexpressed Gi and Go Proteins inSpodoptera frugiperda 9 Cells
- Bart J. B. Francken1,
- Katty Josson1,
- Peter Lijnen1,
- Mirek Jurzak1,
- Walter H. M. L. Luyten2 and
- Josée E. Leysen1
- Departments of 1Biochemical Pharmacology (B.J.B.F., K.J., P.L., M.J., J.E.L.) and 2Functional Genomics (W.H.M.L.L.), Janssen Research Foundation, Beerse, Belgium
Abstract
The ability of the human 5-hydroxytryptamine serotonin type 5A (h5-ht5A) receptor to couple to G proteins from distinct families was investigated through the simultaneous infection ofSpodoptera frugiperda 9 insect cells with recombinant baculoviruses encoding the various proteins. Expression of G proteins was demonstrated in immunoblots. Receptor-G protein coupling was monitored by high-affinity agonist binding and agonist-induced stimulation of [35S]guanosine-5′-O-(3-thio) triphosphate binding to membranes. Receptors expressed alone displayed low-affinity agonist binding, and endogenous G proteins were only poorly stimulated on the addition of 5-hydroxytryptamine. When receptors were coexpressed with mammalian Gi/Go proteins (Gαi or Gαo plus Gβ1γ2), the coupled phenotype was achieved: agonists bound with high affinity in a guanosine-5′-(β,γ-imido)triphosphate-sensitive manner and stimulated [35S]guanosine-5′-O-(3-thio)triphosphate binding to high levels. These effects were not observed on coexpression with Gz/Gs/Gq/11/16 or G12/13. Various ligands were evaluated for their agonistic, antagonistic, or inverse agonistic behavior in both receptor binding and activation assays. Although Go displayed different receptor coupling characteristics than Gi proteins, no clear coupling preference was evident. Coexpression of receptors and Gαi subunits without Gβ1γ2produced increases in both agonist affinity and maximum G protein activation that were smaller than those in the presence of Gβ1γ2, suggesting that Gβ1γ2 coexpression improves receptor-G protein coupling. Similarly, coexpression of receptors with Gβ1γ2 alone resulted in an improved interaction with endogenous G proteins. Our results demonstrate that h5-ht5A receptors expressed in Spodoptera frugiperda 9 cells selectively and functionally couple to coexpressed mammalian Gi and Go proteins.
Footnotes
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Send reprint requests to: Dr. Josée E. Leysen, Janssen Pharmaceutica, Turnhoutseweg 30, B-2340 Beerse, Belgium. E-mail: jleysen2{at}janbe.jnj.com
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This work was supported by a grant from the IWT (Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie).
- Abbreviations:
- 5-HT
- 5-hydroxytryptamine (serotonin)
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- 5-CT
- 5-carboxamidotryptamine
- 5-MT
- 5-methoxytryptamine
- DHE
- dihydroergotamine
- Emax
- relative maximum stimulation
- G protein
- guanine nucleotide-binding protein
- Gi/o
- combination of Gi1, Gi2, Gi3, and Go proteins
- Gpp(NH)p
- guanosine-5′-(β,γ-imido)triphosphate
- Gα
- G protein α-subunit
- Gβ1γ2
- G protein β1γ2 dimer
- h5-ht5A
- human 5-hydroxytryptamine type 5A
- HEK
- human embryonic kidney
- IC50-corr
- corrected IC50
- Imax
- relative maximum inhibition
- LSD
- lysergic acid diethylamide
- m.o.i.
- multiplicity of infection
- Sf9
- Spodoptera frugiperda 9
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- Received July 12, 1999.
- Accepted January 6, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
