Characterization of a membrane glycoprotein having pharmacological and biochemical properties of an AT2 angiotensin II receptor from human myometrium

• Published in: European journal of biochemistry Vol. 220; no. 3; pp. 919 - 926
• Main Authors: LAZARD, Daniel, VILLAGEOIS, Phi, BRIEND‐SUTREN, Malène M., CAVAILLÉ, Françoise, BOTTARI, Serge, STROSBERG, A. Donny, NAHMIAS, Clara
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 15.03.1994; Blackwell
• Subjects: 1-Sarcosine-8-Isoleucine Angiotensin II - metabolism; Biological and medical sciences; Cell Membrane - metabolism; Cell receptors; Cell structures and functions; Cross-Linking Reagents; Female; Fundamental and applied biological sciences. Psychology; Hormone receptors. Growth factor receptors. Cytokine receptors. Prostaglandin receptors; Humans; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - metabolism; Molecular and cellular biology; Molecular Weight; Myometrium - chemistry; Oligopeptides - metabolism; Receptors, Angiotensin - chemistry; Receptors, Angiotensin - metabolism; Solubility; Human; Ligand binding; Molecular form; Membrane receptor; Purification; Peptide hormone; Glycoproteins; Selectivity; Angiotensin II; Physicochemical properties
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1994.tb18695.x

The angiotensin II receptors of human myometrial tissue were characterized using ligand binding, cross‐linking with radioactive label, detergent solubilization and partial purification by lectinaffinity chromatography. Human myometrial membrane preparations contained variable amounts (5–650 fmol/mg protein) of high affinity (Kd= 44–65 pM) binding sites for 125I‐CGP42112, a ligand specific for the AT2 subtype of angiotensin II receptors. Competition studies with AT1‐specific and AT2‐specific compounds indicated that angiotensin II receptors on these membranes were exclusively of the AT2 subtype. The binding sites for 125I‐CGP42112 were efficiently solubilized by the detergent Chaps, albeit with a marked decrease in affinity (Kd= 1.2 nM). The proteins in the myometrial membrane preparation were cross‐linked to 125I‐[Sar1, Ile8]angiotensin II (Sarile) with disuccinimidyl suberate. When low concentrations of cross‐linker were used, a single radiolabelled band of about 66–70 kDa was revealed on SDS/PAGE. At higher concentrations additional bands of about 105–120 kDa and 200 kDa were labelled. The 66–70‐kDa and 105–120‐kDa bands were separated by electroelution of SDS/PAGE gel slices and submitted to trypsin cleavage. The tryptic‐peptide maps were identical for both products, suggesting that the additional bands are homodimers and trimers of the labelled polypeptide. The Chaps‐solubilized receptor was retained on wheat‐germ‐agglutinin‐Sepharose and specifically eluted by the competing sugar triacetylchitotriose, leading to a fivefold purification factor. Treatment of the 125I‐Sarile‐labelled protein with N‐glycanase caused a shift in its apparent molecular mass on SDS/PAGE from 66–70 kDa to 33 kDa.