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Does Thyrotropin Cleave Its Cognate Receptor?

Autoimmune Disease Unit, Cedars-Sinai Research Institute and School of Medicine, University of California, Los Angeles, Los Angeles, California 90048

Address all correspondence and requests for reprints to: Basil Rapoport, M.B., Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048. E-mail: rapoportb{at}cshs.org.

A recent report of major pathophysiological significance, and opposed to present concepts, is that TSH (but not MS-1, a hamster monoclonal thyroid-stimulating antibody), cleaves the single-chain TSH receptor (TSHR) on the cell surface into its two-subunit form. We reassessed the issue using two approaches. First we wished to confirm the flow-cytometric assay previously used to quantitate TSHR cleavage. We used CHO cell lines expressing large (TSHR-10,000 cells) or conventional (TSHR-0 cells) numbers of TSHR. Cells were preincubated (16 h) in either control medium or medium supplemented with TSH (5 x 10^-8 M) or MS-1 (10 µg/ml). After stringent washing to maximize removal of residual ligand, we performed flow cytometry with two antibodies, one recognizing only the single-chain TSHR, the other recognizing all (cleaved and uncleaved) TSHRs. TSH pretreatment did not appear to increase TSHR cleavage. Instead we observed ligand occupancy of the TSHR (with MS-1) or fewer receptors on the cell surface (down-regulation), particularly with the TSHR-0 cells. Second, we covalently cross-linked [¹²⁵I]TSH to monolayers of these cells, an unequivocal method to determine directly the proportion of single-chain and two-subunit TSHR forms. Pretreatment of TSHR-10,000 and TSHR-0 cells with TSH had no effect on the degree of TSHR cleavage. MS-1 slightly reduced spontaneous cleavage. In conclusion, in contrast to a recent report, we show that TSH does not alter the subunit structure of its cognate receptor, and we provide insight into the difficulties associated with the flow-cytometric assay for TSHR cleavage.

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