Agonist efficacy at the alpha2A-adrenoceptor:G alpha15 fusion protein: an analysis based on Ca2+ responses.
A fusion protein was constructed between the recombinant human alpha2A-adrenoceptor and a mouse G alpha15 protein to measure the efficacy of agonist-induced Ca2+ responses at a receptor:G alpha15 protein stoichiometry of 1. Activation of this fusion protein in CHO-K1 cells by (-)-adrenaline induced a time- and concentration-dependent (pEC50: 7.28+/-0.04) increase in the intracellular Ca2+ concentration. The magnitude of the Ca2+ response was related to the amount of the fusion protein and the number of surface alpha2A-adrenoceptor binding sites as estimated by [3H]RX 821002 binding. Whereas UK 14304 was as efficacious as (-)-adrenaline, the following ligands displayed partial agonist properties [Emax in percentage vs. (-)-adrenaline: d-medetomidine (76+/-3) > BHT 920 (53+/-3) > clonidine (39+/-4) >> oxymetazoline (10+/-1)]. This ligand activation profile was not affected over a 30-fold range of expression of the fusion protein in contrast to the observed enhancement of the partial agonists' maximal responses by co-expression of the alpha2A-adrenoceptor with increasing amounts of the G alpha15 protein. In conclusion, the fusion protein approach opens perspectives to quantify intrinsic activities of ligands under controlled experimental conditions of a fixed receptor:G alpha15 protein ratio of 1.
Pauwels
P J
PJ
Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, Castres, France. peter.pauwels@pierre-fabre.com
Finana
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Colpaert
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Wurch
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D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000818:Animals; D000068438:Brimonidine Tartrate; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D004837:Epinephrine / Q000031:analogs & derivatives / Q000494:pharmacology*; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D019329:Idazoxan / Q000031:analogs & derivatives* / Q000378:metabolism; D008024:Ligands; D051379:Mice; D011810:Quinoxalines / Q000494:pharmacology; D011869:Radioligand Assay; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
C547768:ADRA2A protein, human; C547769:Adra2a protein, mouse; D000316:Adrenergic alpha-Agonists; D005456:Fluorescent Dyes; D008024:Ligands; D011810:Quinoxalines; D018341:Receptors, Adrenergic, alpha-2; D011993:Recombinant Fusion Proteins; D000068438:Brimonidine Tartrate; C058525:2-methoxyidazoxan; D019204:GTP-Binding Proteins; D002118:Calcium; D019329:Idazoxan; D004837:Epinephrine