Foord S M, Craig R K
Department of Biochemistry, University College and Middlesex School of Medicine, London, England.
Eur J Biochem. 1987 Dec 30;170(1-2):373-9. doi: 10.1111/j.1432-1033.1987.tb13710.x.
We describe the identification and purification of a receptor for calcitonin-gene-related peptide (CGRP) from human term placenta, using lectin and beta-CGRP-Affigel affinity chromatography. The membrane-bound receptor has an estimated Mr of 240,000, as determined by cross-linking 125I-labelled alpha-CGRP (125I-alpha-CGRP) using discuccinimidyl suberate and SDS/polyacrylamide gel electrophoresis, or of 263,000, as judged by sucrose gradient centrifugation of the soluble partially purified native receptor preparation. Cross-linking studies with disuccinimidyl suberate and N-hydroxysuccinimidyl-4-azidobenzoate using membrane-solubilised, partially purified and CGRP-affinity-purified preparations, show a number of 125I-alpha-CGRP binding subunit(s) of Mr 62,000-68,000. Silver staining of the purified CGRP receptor preparation showed two distinct doublets in this plus a number of minor doublets of lower Mr. The receptor binds human beta-CGRP with greater affinity than alpha-CGRP, and showed little affinity for human calcitonin. Adsorption isotherms and Scatchard analysis of 125I-alpha-CGRP binding to the membrane-bound or soluble purified receptor are consistent, under the conditions used, with a single binding site of high affinity. Molecular cloning at present in progress should define the amino acid sequence and subunit composition of the human placental CGRP receptor, since at present the observed heterogeneity of CGRP-binding proteins may be interpreted in a number of ways, for instance: receptor heterogeneity, variable glycosylation of one of two subunits, or limited proteolysis of a single subunit during purification.
我们描述了利用凝集素和β -降钙素基因相关肽（CGRP）-Affigel亲和层析从人足月胎盘中鉴定和纯化CGRP受体的过程。通过使用辛二酸二琥珀酰亚胺酯和SDS/聚丙烯酰胺凝胶电泳对125I标记的α - CGRP（125I-α - CGRP）进行交联，确定膜结合受体的估计分子量为240,000；或者通过对可溶性部分纯化的天然受体制剂进行蔗糖梯度离心判断，其分子量为263,000。使用膜溶解、部分纯化和CGRP亲和纯化的制剂，用辛二酸二琥珀酰亚胺酯和N -羟基琥珀酰亚胺-4-叠氮苯甲酸进行交联研究，显示出一些分子量为62,000 - 68,000的125I-α - CGRP结合亚基。纯化的CGRP受体制剂的银染显示在此处有两个不同的双峰以及一些分子量较低的小双峰。该受体与人β - CGRP的结合亲和力高于α - CGRP，对人降钙素的亲和力很小。在所用条件下，125I-α - CGRP与膜结合或可溶性纯化受体结合的吸附等温线和Scatchard分析与单个高亲和力结合位点一致。目前正在进行的分子克隆应能确定人胎盘CGRP受体的氨基酸序列和亚基组成，因为目前观察到的CGRP结合蛋白的异质性可能有多种解释方式，例如：受体异质性、两个亚基之一的可变糖基化，或纯化过程中单个亚基的有限蛋白水解。
