Photoaffinity labeling of subtype 2 angiotensin receptor of human myometrium.
Angiotensin II (AII) binding sites were characterized in human myometrium membrane preparations. The sites were saturable and of high affinity (Kd of 0.09 nM and Bmax of about 200 fmol/mg of protein). PD 123319 completely inhibited 125I-AII binding, with an IC50 of 30 nM, whereas L-158,809 (1 microM) had no significant effect on 125I-AII binding. These results indicate that human myometrium contains almost exclusively the AT2 receptor subtype. Association and dissociation studies performed with 125I-AII on human myometrium membranes revealed that AII had a very high affinity for AT2 receptors, with a Kd of 0.01 nM (association rate constant K1 = 1.056 x 10(12) mol-1 min-1; dissociation rate constant K2 = 0.003 min-1). The photoactivable AII analogue [Sar1, Val5, D-Phe8(N3)]AII displayed a high affinity for AT2 receptors (IC50 of 0.18 nM), but its radioiodinated form showed poor efficiency in photoaffinity labeling experiments. A newly synthesized photoactivatable analogue of AII, [Sar1, p-benzoyl-Phe8]AII, (AII-Bpa), also displayed a high affinity for AT2 receptors of human myometrium (IC50 of 0.3 nM). Photoaffinity labeling experiments were performed with 125I-AII-Bpa, and a high yield (70%) of covalent incorporation to human myometrium membranes was obtained upon photolysis. Covalently labeled receptors were solubilized, denatured, and subjected to polyacrylamide gel electrophoresis. Autoradiography of the polyacrylamide gel revealed a single band, of 68 kDa, and the labeling of this band was completely abolished in the presence of 1 microM PD 123319, indicating selective labeling of the AT2 receptor subtype. These results demonstrate that AII-Bpa is a very efficient tool for selective photoaffinity labeling of the AT2 receptor.
Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Québec, Canada.
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Servant
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Boulay
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Bossé
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Escher
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Guillemette
D000302:Adrenal Cortex / Q000378:metabolism; D000328:Adult; D000345:Affinity Labels; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008875:Middle Aged; D009215:Myometrium / Q000378:metabolism*; D010777:Photochemistry; D011869:Radioligand Assay; D011945:Receptors, Angiotensin / Q000032:analysis* / Q000145:classification
D000345:Affinity Labels; D011945:Receptors, Angiotensin; D000804:Angiotensin II