Characterization of AT4 Receptor from Bovine Aortic Endothelium with Photosensitive Analogues of Angiotensin IV

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• 作者：Sylvie G. Bernier ; Julie M. L. Bellemare ; Emanuel Escher ; and Gaé ; tan Guillemette
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：March 24, 1998
• 年：1998
• 卷：37
• 期：12
• 页码：4280 - 4287
• 全文大小：144K
• 年卷期：v.37,no.12(March 24, 1998)
• ISSN：1520-4995

文摘

Newly developed photosensitive analogues of AngIV were used tocharacterize the AT₄ receptorof bovine aortic endothelial cells. The photoactivatable AngIVanalogues [N₃-Phe⁶]AngIV and[Bpa⁶]AngIV displayed high affinities for AT₄ receptor, withIC₅₀'s of 3.7 ± 0.3 and 19.1 ± 3.5 nM,respectively.The radioiodinated ligands showed a good efficiency ofphotoaffinity labeling demonstrated by highproportions (60-75%) of acid-resistant binding. Covalentlylabeled receptor was solubilized under reducingor nonreducing conditions and subjected to SDS-PAGE. Undernonreducing conditions, autoradiographiesrevealed a major band of M_r 186 ± 2 kDa and aminor band of M_r 241 ± 6 kDa. Thelabeling of thesebands was completely abolished in the presence of 10 ges/entities/mgr.gif">M AngIV.Under reducing conditions, only thelow M_r 186 kDa band was revealed. Afterendoglycosidase digestion with an enzyme that cleavesN-linkedsaccharides, the M_r of the denaturedAT₄ receptor was decreased by 31% to a value of 129 ± 10kDa.Kinetic studies revealed a stepwise process of AT₄receptor deglycosylation by endoglycosidase F,suggesting at least two different sites of N-linked saccharides.Mild trypsin treatment of photolabeledendothelial cell membranes released a large fragment ofM_r 177 ± 3 kDa which accounts for about95%of the whole receptor molecular mass. These results demonstratethat [N₃-Phe⁶]AngIV and[Bpa⁶]AngIVare very efficient tools for selective photoaffinity labeling ofAT₄ receptor. We have shown thatAT₄receptor is a 186 kDa integral membrane glycoprotein with a very largeextracellular domain. Theseproperties are consistent with those of a growth factor or cytokinereceptor.