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Endocrinology Vol. 138, No. 7 2893-2899
Copyright © 1997 by The Endocrine Society

ARTICLES

Evidence That the Thyrotropin Receptor Ectodomain Contains Not One, But Two, Cleavage Sites1

Gregorio D. Chazenbalk, Kunihiko Tanaka, Yuji Nagayama2, Ayumu Kakinuma, Juan Carlos Jaume3, Sandra M. McLachlan and Basil Rapoport

Thyroid Molecular Biology Unit (G.D.C., Y.N., A.K., J.C.J., S.M.M., B.R.), Veterans’ Administration Medical Center, and the University of California, San Francisco, California 94121; and Department of Pharmacology 1 (K.T., Y.N.), Nagasaki University School of Medicine, Nagasaki 852, Japan

Address all correspondence and requests for reprints to: Basil Rapoport, M.B. or Gregorio Chazenbalk, Ph.D., Veterans’ Administration Medical Center, Thyroid Molecular Biology Unit (111T), 4150 Clement Street, San Francisco, California 94121.

TSH receptor (TSHR) cleavage into two subunits (A and B) was explored using two new mammalian cell lines expressing the recombinant receptor; 1) TSHR-10,000 CHO cells overexpressing the TSHR; 2) TSHRmyc cells with a c-myc epitope inserted at residues 338–349. Immunoprecipitation or immunoblotting of TSHR-10,000 cells with mAb to either the A subunit or the B subunit revealed multiple forms of the TSHR: 1) uncleaved receptors of approximately 115 kDa and approximately 100 kDa with complex carbohydrate and high mannose carbohydrate, respectively; 2) two subunit TSHR with an approximately 62 kDa A subunit containing complex carbohydrate. The A subunit was approximately 35 kDa after enzymatic deglycosylation (predicted C-terminus near residue 330). The nonglycosylated B subunit was evident primarily as an approximately 42 kDa band (predicted N terminus near residue 380). The sum of the A and B subunit polypeptide backbones was smaller than the predicted size of the TSHR, a polypeptide backbone (84.5 kDa), raising the possibility that an approximately 5-kDa polypeptide fragment was excised during intramolecular cleavage. This hypothesis was supported by data obtained with the TSHRmyc cells. Thus, mAb to the c-myc epitope and to amino acid residues 22–35 (mAb A10) were equally effective in detecting the single chain forms of the TSHR in these cells. However, the 35 kDa, deglycosylated A subunit was clearly visible on immunoprecipitation with mAb A10 to the TSHR amino terminus, but not with the anti-myc mAb, indicating loss of the c-myc epitope at residues 338–349. Further, even though the A subunit was not detected in TSHRmyc cells with anti-myc mAb, 125I-TSH cross-linking to the cell surface showed similar A subunit expression in TSHRmyc and wild-type TSHR expressing cells.

In summary, our study provides a surprising and novel finding for G protein-coupled receptors. Contrary to the prevailing concept of one cleavage site in the TSHR, we present evidence that there are, in fact, two such sites. The TSHR, like insulin, may release a C peptide during intramolecular cleavage into two subunits.




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