Anouk Allgeier

SPECIFIC ACTIVATION OF THE THYROTROPIN RECEPTOR BY TRYPSIN

The identification of 16 different activating mutations in the TSH receptor, found in patients suffering from toxic autonomous adenomas or congenital hyperthyroidism, leads to the concept that this receptor is in a constrained conformation in its wild-type form. We used mild trypsin treatment of CHO-K1 cells or COS-7 cells, stably or transiently transfected 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 increased 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%. The effect was also observed in cells expressing the dog TSH receptor. It was not observed in MSH receptor, LH receptor expressing or mock transfected cells (vector alone). It is therefore specific for the TSH receptor, for its action on the Gs/adenylate cyclase cascade, and for the proteolytic cleavage caused by trypsin. Using monoclonal (A. Johnstone 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 TSH as it activates Gs alpha and enhances the action of forskolin. It is not reversible in 1 h. The results support the concept that activation of the receptor (by hormone, autoantibodies, mutations or mild proteolysis) might involve the relief of a built-in negative constrain. They suggest that the C-terminal portion of the large extracellular domain plays a role in the maintenance of this constrain.

Multiple G-protein coupling of the dog thyrotropin receptor.

We investigated, in dog thyroid membranes, the ability of the dog thyrotropin (TSH) receptor to interact with the endogenous G proteins expressed in this tissue. Activation of the receptor led to increased incorporation of the photoreactive GTP analog [alpha-(32)P]GTP azidoanilide into immunoprecipitated alpha subunits of three G protein families: G(s), G(q/11), G(i/o). This effect was not due to a general loss of receptor G protein specificity since carbamylcholine, in the same membrane preparations, only stimulated the binding of the GTP analog to the alpha subunits of G(q/11) proteins. To investigate the multiple coupling of the dog TSH receptor in intact cells, cyclic AMP accumulation, IP(3) formation and (45)Ca2+ efflux experiments were performed. When thyrocytes were pretreated with pertussis toxin (PTX), the TSH receptor-mediated accumulation of cAMP increased by approximately 45% with TSH at 1 mU/ml, suggesting that the TSH receptor coupled to both G(s) and G(i) in vivo. On the other hand, no increase in IP(3) accumulation nor Ca2+ efflux was observed in the presence of thyrotropin. These data in intact cells are thus in contradiction with those obtained in membranes, suggesting that receptor-mediated transmembrane signalling may implicate a specificity which itself may reflect a localization and organization of the different components (receptors, G proteins, ...) in the plasma membrane of intact cells. As in some cells, G(i) activates mitogenesis by hormone activated G-protein-coupled receptors, we tested its role in the stimulation by TSH of the proliferation of thyrocytes. This was not affected by PTX, suggesting that the mitogenic effect of TSH does not involve G(i)-proteins.

Expression of multiple adenylyl cyclase isoforms in human and dog thyroid.

Although the TSH receptor and Galpha(s), which activate the cAMP cascade in the thyroid gland have been much studied, nothing is known about the adenylyl cyclase (AC) isoforms which are actually involved in this pathway. To characterize the cAMP generation in the dog and human thyroid gland, resulting from the presence of distinct adenylyl cyclase families, the responses to various agents (Ca2+, calmodulin (CaM), phorbol esters (TPA) and thapsigargin (Tg)) were studied. These experiments suggest a role of at least two families of cyclases: cyclases negatively modulated by Ca2+ (ACV or ACVI) and cyclases positively modulated by PKC (ACII, ACIII or ACVII). To further analyze by other experimental procedures the expression pattern of the cyclase isoforms in the thyroid gland, Northern blotting, Western blotting and RT-PCR experiments were performed. The results clearly suggest that in both species, three different adenylyl cyclases ACIII, ACVI and ACIX are mainly expressed in thyrocytes.