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G Servant, DT Dudley, E Escher and G Guillemette
Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Canada.
We recently described the photoaffinity labeling and partial characterization of the angiotensin type 2 (AT2) receptor from human myometrium. In the present study, specific receptors for angiotensin II (AII) were also analyzed in a murine fibroblast cell line (R3T3) and a rat pheochromocytoma cell line (PC-12). Dose-displacement experiments with PD 123319 (an AT2-selective antagonist) completely inhibited 125I- AII binding, whereas L-158,809 (an AT1-selective antagonist) had no significant effect on 125I-AII binding, thus revealing that these two cell lines express exclusively AT2 sites. High yields of covalent and selective labelling of AT2 receptors from human myometrium, R3T3 cells, and PC-12 cells were obtained with the photosensitive analogue 125I- [Sar1,Val5,p-benzoyl-Phe8]AII. Gel permeation chromatography of Triton X-100-solubilized AT2 receptors from the three different sources revealed similar Stokes' radii of about 65 A. Interestingly, upon electrophoresis under reducing conditions, marked disparities were observed between the apparent molecular masses of AT2 receptors from the three different sources. As observed previously, AT2 receptors from human myometrium showed a molecular mass of 68 +/- 4.6 kDa. AT2 receptors from PC-12 cells showed a larger molecular mass of 113 +/- 12 kDa, whereas AT2 receptors from R3T3 cells showed a molecular mass of 91 +/- 7.8 kDa. After endoglycosidase digestion with an enzyme that cleaves N-linked saccharides, the molecular masses of the denatured AT2 receptors of human myometrium, R3T3 cells, and PC-12 cells were decreased by 54%, 66%, and 73%, to 31.3 +/- 1.6 kDa, 30.9 +/- 0.7 kDa, and 30.6 +/- 0.8 kDa, respectively. Kinetic studies with AT2 receptors from human myometrium revealed a complex, multiple-step process of deglycosylation involving at least three different sites of N-linked saccharides. These results suggest that the disparity in the sizes of AT2 receptors from different sources is mostly related to different degrees of N-glycosylation. They also imply that the AT2 receptor contains at least three asparagine-linked sites of glycosylation.
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