CELL-SURFACE PROTEIN DISULFIDE-ISOMERASE IS INVOLVED IN THE SHEDDING OF HUMAN THYROTROPIN RECEPTOR ECTODOMAIN

In human thyroid glands the TSH receptor undergoes a cleavage reaction which yields to an extracellular alpha subunit and a membrane spannin g beta subunit linked together by disulfide bridges. A similar reactio n is observed in transfected L cells although some uncleaved monomers persist in these cells. We have recently shown that the alpha subunit of the TSH receptor undergoes partial shedding in human thyroid cells and heterologous cells permanently transfected with an expression vect or encoding the receptor. This shedding is a two-step process. The fir st step consists in the cleavage of the proreceptor at the cell surfac e probably by a matrix metalloprotease and the second step in the redu ction of the disulfide bridge(s) (Couet. J., Sar, S., Jolivet, A., Vu Hai, M. T., Milgrom, E., & Misrahi. M. 1996, J. Biol. Chem. 271, 4545- 4552). We have used the transfected L cells to study the second step i nvolved in sTSHR shedding. The membrane impermeant sulfhydryl reagent DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) allowed us to confirm that the reduction of the TSH receptor disulfide bonds occurred at the cell surface. The antibiotic bacitracin even at low concentrations also el icited a marked inhibition of TSH receptor shedding. This led us to im plicate the enzyme protein disulfide isomerase (PDI, EC 5.3.4.1) in th is process. We thus tested the inhibitory activity of specific monoclo nal antibodies raised against PDI. All antibodies elicited a marked in hibition of sTSHR shedding. This confirmed that cell surface PDI is in volved in the shedding of the TSH receptor ectodomain. The shed alpha subunit may be at the origin of circulating TSH receptor ectodomain de tected in human blood.