The Third Intracellular Loop of the Rat and Mouse Cholecystokinin-A Receptors Is Responsible for Different Patterns of Gene Activation
- Roya Poosti1,
- Laure di Malta1,
- Didier Gagne1,
- Nicole Bernad1,
- Jean-Claude Galleyrand1,
- Chantal Escrieut2,
- Sandrine Silvente-Poirot2,
- Daniel Fourmy2 and
- Jean Martinez1
- 1Laboratoire des Acides Aminés Peptides et Protéines, Centre National de la Recherche Scientifique, Unité Mixte de Recherche, 5810, Faculté de Pharmacie, Montpellier, France (R.P., L.d.M., D.G., N.B., J.-C.G., J.M.); and 2Institut National de la Santé et de la Recherche Médicale U151, Institut Louis Bugnard, Centre Hospitalier Universitaire Rangueuil, Toulouse, France (C.E., S.S.-P., D.F.)
Abstract
It has previously been reported that the cholecystokinin analog JMV-180 behaves differently on the rat and the mouse cholecystokinin-A receptor (CCK-AR). In mice this analog acts as an agonist on low- and high-affinity sites of the CCK-AR, whereas in rats this compound acts as an agonist on high-affinity sites and as an antagonist on low-affinity sites. In an attempt to understand why the same compound behaves differently on these two CCK-A receptors, we cloned the cDNA encoding the mouse CCK-AR. We then investigated a cellular model able to mimic the effect that was observed in rats and mice. HeLa cells were transiently cotransfected with plasmids leading to expression of the rat or mouse CCK-AR in the presence of pFos-Luc as reporter plasmid; such a plasmid placed the regulatory part of the humanc-Fos gene upstream from the firefly luciferase structural gene (Luc). We then observed that the two CCK-A receptors behaved differently, not only in the presence of compound JMV-180 but also in the presence of cholecystokinin or even in absence of ligand; the rat CCK-AR was 2 to 3 times more potent than the mouse CCK-AR in inducing the reporter protein, whatever the ligand studied. This result was confirmed using the same kind of experiment with the reporter plasmid p(TRE)3-tk-Luc. Using various mutated receptors, we investigated the role of the putative third intracellular loop. We concluded that both the primary structure of the receptor and the cellular context are in part responsible for the differential behavior of these CCK-A receptors.
Footnotes
-
Send reprint requests to: Pr. Jean Martinez, Laboratoire des Acides Aminés Peptides et Protéines (L.A.P.P.), CNRS-UMR 5810, Faculté de Pharmacie, 15 avenue Ch. Flahault, 34060 Montpellier cédex 2, France. E-mail:martinez{at}pharma.univ-montp1.fr
-
This work was supported in part by a grant from the Association de la Recherche Contre le Cancer (Grant ARC 9257). R.P. and L.d.M. contributed equally to this work.
- Abbreviations:
- CCK
- cholecystokinin
- CCK-AR
- cholecystokinin-A receptor
- CCK-BR
- cholecystokinin-B receptor
- CCK-8
- H-Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2
- BH-CCK-8
- Bolton-Hunter cholecystokinin (26–33) amide
- JMV-180
- butyloxycarbonyl-Tyr(SO3H)-Ahx-Gly-Trp-Ahx-Asp2-phenylethyl ester
- Luc
- luciferase
- TRE
- 12-O-tetradecanoylphorbol-13-acetate responsive element
- MAP
- mitogen-activated protein
- SAP
- stress-activated protein
- PCR
- polymerase chain reaction
-
- Received May 12, 2000.
- Accepted September 7, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
