Characterization of a fluorescent conjugate of the rabbit angiotensin AT(1) receptor.
1. The rabbit AT(1) receptor (AT(1)R) for angiotensin II (A(II)) has been conjugated to the yellow fluorescent protein (YFP) in order to establish the pharmacological profile of such a fusion protein and to facilitate the study of ligand-induced regulation. 2. A(II) bound AT(1)R-YFP (K(D) 8.1 nM in transiently transfected cells) and stimulated HEK 293 cells expressing the fusion protein at concentration ranges similar to the ones that stimulate the contraction of the isolated rabbit aorta. Antagonists found to be insurmountable in the latter assay (candesartan and EXP-3174 being the most extreme cases) were also insurmountable in the phospholipase A(2) assay applied to cells expressing AT(1)R-YFP, whereas losartan appeared to be surmountable in both assays. 3. Cells expressing AT(1)R-YFP exhibited a membrane-associated fluorescence that was partly and reversibly translocated into intracellular structures upon A(II) stimulation (confocal microscopy); the nonpeptide antagonists were not active in this respect, but prevented the effect of the agonist. 4. A(II) treatment increased the quantity of the fusion protein in cells, and phorbol 12-myristate 13-acetate (PMA) treatment even more so (immunoblot, confocal microscopy) but, unlike the agonist, the latter drug did not induce receptor endocytosis. A protein kinase C (PKC) inhibitor prevented the effect of either A(II) or PMA on AT(1)R-YFP abundance. 5. The conjugate AT(1)R-YFP retains the pharmacological properties of the parent rabbit AT(1)R. Agonist-induced downregulation was not documented using this system; to the contrary, we have observed a PKC-mediated increased expression AT(1)R-YFP likely to be the result of a decreased breakdown rate of the fusion protein.
Fortin
Jean-Philippe
JP
Centre de recherche en cancérologie, Centre Hospitalier Universitaire de Québec, Centre de recherche du Pavillon l'Hôtel-Dieu de Québec, 11, Côte-du-Palais, Quebec City, Québec, Canada G1R 2J6.
Bouthillier
Johanne
J
Bastien
Luc
L
Bachvarov
Dimcho R
DR
Marceau
François
F
D000818:Animals; D001011:Aorta / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000032:analysis / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011817:Rabbits; D044140:Receptor, Angiotensin, Type 1 / Q000032:analysis / Q000378:metabolism*
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D044140:Receptor, Angiotensin, Type 1; C054562:yellow fluorescent protein, Bacteria