Characterization of the subunit structure of the thyrotropin receptor in the FRTL-5 rat thyroid cell line.

Abstract

Radioiodinated TSH was covalently cross-linked to monolayers of FRTL-5 rat thyroid cells using the homobifunctional cross-linking agent disuccinimidyl suberate. Analysis of the cross-linked samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions demonstrated the presence of three specifically labeled complexes with apparent mol wt of 68,000, 85,000, and 145,000, in addition to the TSH alpha-beta dimer and its alpha- and beta-subunits. When bound [125I]iodo-TSH was cross-linked with increasing concentrations of disuccinimidyl suberate, the formation of the 68,000 and 85,000 mol wt complexes was sequential, with the 68,000 complex appearing first. These two complexes were also observed after labeling with radioiodinated TSH hybrid molecules (alpha-beta or alpha-beta), in which the label is in only one subunit, or immuno-precipitation with antibodies against either the alpha- or beta-subunit of TSH. Similar complexes (65,000, 82,000, and 145,000 mol wt) were also formed after cross-linking with the alkaline-cleavable cross-linker (bis-[2-(succinimidooxycarbonyloxy)ethyl]sulfone. Again, the appearance of these three complexes was sequential and dependent on the cross-linker concentration. At low concentrations and under reducing conditions, the 65,000 mol wt complex was the major band. However, at high concentrations, especially under nonreducing conditions, most of the radioactivity was present in the 145,000 mol wt complex. Alkaline cleavage of these three complexes followed by electrophoresis in a second dimension resulted in the release of three components with approximate mol wt of 31,000, 17,000, and 63,000 in addition to the TSH alpha-beta dimer and its alpha- and beta-subunits. Reduction by dithiothreitol followed by electrophoresis in a second dimension resulted in the release of only the 17,000 and 63,000 components. Taken together, these results suggest that 1) both the 65,000 and 82,000 complexes are formed after incremental cross-linking of TSH alpha-beta dimer to receptor subunits; and 2) the TSH receptor may be an oligomer composed of three different subunits, 31,000, 17,000, and 63,000, of which only the 31,000 subunit binds TSH.