Characterization of a fluorescent conjugate of the rabbit angiotensin AT₁ receptor

Abstract

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.

Discovery of a Dual-Function Peptide That Combines Aminopeptidase N Inhibition and Kinin B1Receptor Antagonism

“…The reaction that cleaves angiotensin III into angiotensin IV is also reported to be catalyzed by aminopeptidase N (Robertson et al, 1992); to modelize this in the rabbit aorta contractility assay, cumulative concentration-effect curves were constructed for angiotensin II, III (des-Asp 1 -angiotensin II), and IV [des-(Asp 1 , Arg 2 )-angiotensin II; Sigma-Aldrich] in separate tissues pre-equilibrated for 1 h as described previously (Fortin et al, 2003a; the first peptide was included for comparison). The same tissues were exposed to the AT 1 receptor antagonist losartan (100 nM, gift of Merck Research Laboratories, Rahway, NJ) in the period 2 to 3 h postmounting, and the curves were constructed again at time 3 h, a time point where control tissues show a great constancy of response relative to the time 1 h (Fortin et al, 2003a).…”

“…The same tissues were exposed to the AT 1 receptor antagonist losartan (100 nM, gift of Merck Research Laboratories, Rahway, NJ) in the period 2 to 3 h postmounting, and the curves were constructed again at time 3 h, a time point where control tissues show a great constancy of response relative to the time 1 h (Fortin et al, 2003a). The aims of these preliminary experiments were to clarify that the metabolism of angiotensin III into angiotensin IV is practically a functional inactivation and to confirm that all forms of angiotensin contract the rabbit aorta via the AT 1 receptor, as reported for the rat aorta (Li et al, 1995).…”

A Novel Nonpeptide Antagonist of the Kinin B1Receptor: Effects at the Rabbit Receptor

“…Contractility studies in the aortic preparation were based upon the construction of cumulative concentration-responses curves for des-Arg 9 -BK (a B 1 R agonist on this tissue) and an additional one for Ang II (an AT 1 R agonist in the aorta; Fortin et al, 2003a). These studies aimed to investigate the potency, surmountability, and specificity of the B 1 receptor antagonist in the vascular smooth muscle preparation.…”

“…These drugs were present during the 60-min period allowed for radioligand binding equilibration in the binding buffer specific for each assay. Competition of [ 3 H]Ang II binding to HEK 293 cells expressing the fusion protein AT 1 R-YFP by unlabeled drug was performed as described previously (Fortin et al, 2003a).…”

“…HEK 293 cells stably expressing AT 1 R-YFP (Fortin et al, 2003a) or transiently expressing B 1 R-YFP (Sabourin et al, 2002a) were used in confocal microscopy (35-mm petri dishes), precisely as described previously (Sabourin et al, 2002a). COS-1 or HEK 293 cells transiently transfected with myc-B 1 R coding or control vectors were fixed and submitted to indirect immunofluorescence using an anti-myc monoclonal antibody (clone 9e10, dilution 1:1000; Convance, Richmond, CA) revealed using an Alexa-Fluor 488-labeled anti-mouse goat antibody (dilution 1:1000; Molecular Probes, Eugene, OR).…”