Analysis of thyrotropin receptors by photoaffinity labelling. Orientation of receptor subunits in the cell membrane

Publisher Website
Google Scholar
Abstract
Porcine TSH receptors were purified by Sepharose-TSH affinity chromatography and crosslinked to a 125I-labeled photoactive derivative (N-hydroxysuccinimidyl-4-azidobenzoate; HSAB) of TSH (125I-HSAB-TSH). Purification of the crosslinked complexes on Sephacryl S-300 followed by polyacrylamide-gel electrophoresis in sodium dodecyl sulfate showed that the receptor contained 2 subunits. One subunit (A) with MW 45,000 was crosslinked to TSH and the other (B) subunit, MW 25,000, was linked to the A subunit by a disulfide bridge(s). Other, as yet unidentified, subunits may have been non-covalently associated with the A and B subunits. Analysis of reduced and non-reduced crosslinked TSH receptor-125I-HSAB-TSH on Sephacryl S-300 in the presence and absence of detergent indicated that the A subunit was a hydrophilic peptide. This was confirmed in studies of the release into aqueous solution by reducing agent treatment of 125I-HSAB-TSH crosslinked to the TSH receptor A subunit in thyroid membranes. Similar results were obtained with TSH receptors in human thyroid and guinea pig fat cell membranes. The hydrophilic A subunit of the receptor evidently forms a binding site for TSH on the outside surface of the cell membrane and is linked to the cell membrane by way of a disulfide bridge to the receptor B subunit.