Isolation and characterisation of a human calcitonin-gene-related-peptide receptor.
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.
Foord
S M
SM
Department of Biochemistry, University College and Middlesex School of Medicine, London, England.
Craig
R K
RK
D015740:Calcitonin Gene-Related Peptide; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009479:Neuropeptides / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism*; D018003:Receptors, Calcitonin; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*
D009479:Neuropeptides; D018003:Receptors, Calcitonin; D011956:Receptors, Cell Surface; D015740:Calcitonin Gene-Related Peptide