SPECIFIC ACTIVATION OF THE THYROTROPIN RECEPTOR BY TRYPSIN

The identification of 16 different activating mutations in the TSH rec eptor, found in patients suffering from toxic autonomous adenomas or c ongenital hyperthyroidism, leads to the concept that this receptor is in a constrained conformation in its wild-type form. We used mild tryp sin treatment of CHO-K1 cells or COS-7 cells, stably or transiently tr ansfected with the human TSH receptor, respectively, and measured its consequences on the TSH receptor coupled cascades, i.e. cyclic AMP and inositol-phosphates accumulation. A 2-min, 0.01% trypsin treatment in creased stably cyclic AMP but not inositol-phosphates formation. This was not observed after chymotrypsin, thrombin and endoproteinase glu C treatment. The TSH action on cyclic AMP was decreased by only 25%. Th e effect was also observed in cells expressing the dog TSH receptor. I t was not observed in MSH receptor, LH receptor expressing or mock tra nsfected cells (vector alone). It is therefore specific for the TSH re ceptor, for its action on the Gs/adenylate cyclase cascade, and for th e proteolytic cleavage caused by trypsin. Using monoclonal (A. Johnsto ne and P. Shepherd, personal communication) and polyclonal antibodies directed against the extracellular domain of the TSH receptor, it was shown that treatment by trypsin removes or destroys a VFFEEQ epitope ( residues 354-359) from the receptor. The effect mimics the action of T SH as it activates G(s alpha) and enhances the action of forskolin. It is not reversible in I h. The results support the concept that activa tion of the receptor (by hormone, autoantibodies, mutations or mild pr oteolysis) might involve the relief of a built-in negative constrain. They suggest that the C-terminal portion of the large extracellular do main plays a role in the maintenance of this constrain.