Analysis of thyrotropin receptors by photoaffinity labelling. Orientation of receptor subunits in the cell membrane.
Porcine thyrotropin (TSH) receptors have been purified by Sepharose-TSH affinity chromatography and crosslinked to a 125I-labelled 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 sulphate showed that the receptor contained two subunits. One subunit (A) with Mr 45 000 was crosslinked to TSH and the other (B) subunit, Mr 25 000, was linked to the A subunit by a disulphide 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. These studies suggest that the hydrophilic A subunit of the receptor forms a binding site for TSH on the outside surface of the cell membrane and that the A subunit is linked to the cell membrane by way of a disulphide bridge to the receptor B subunit.
Kajita
Y
Y
Rickards
C R
CR
Buckland
P R
PR
Howells
R D
RD
Rees Smith
B
B
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006801:Humans; D046911:Macromolecular Substances; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011989:Receptors, Thyrotropin; D013552:Swine; D013961:Thyroid Gland / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D011956:Receptors, Cell Surface; D011989:Receptors, Thyrotropin; C029534:hydroxysuccinimidyl-4-azidobenzoate; D004229:Dithiothreitol