Molecular basis of dual signal transduction via the human TSH receptor

Background: Growth and differentiation are regulated in the thyroid mainly by thyroid-stimulating hormone (TSH) and the G-protein-coupled TSH receptor (TSHR). Mutations of TSH are characterized as defined clinical pictures: a ctivated mutations in autonomous adenomas and carcinomas, inactivated mutat ions in congenital hypothyroidism. The activated wild-type TSHR influences thyrocyte functions via two effector systems: Gs/adenylate cyclase and Gq/1 1/phospholipase C. In a congenital hypothyroidism, a Y601H exchange was det ected at the conserved position 601, classified as a silent polymorphism. T his amino-acid exchange (TSHR-Y601H) ih also found in the first submitted h uman TSHR sequence, which functions as reference sequence in most databases . We examined the prevalence and signal transduction properties of TSHR-Y60 1H. Methods: The prevalence of TSHR-Y601 was examined by PCR and restriction an alysis of the TSHR gene. To investigate the functional importance of Y601, seven TSHR mu rants were developed by PCR using TSHR-pcDPS as the expressio n-plasmid. The TSH-induced accumulation of cAMP and inositol phosphate was examined in COS-7 cells that transiently expressed the TSHR mutants. The su rface expression of TSHR was measured by ELISA. Results: The Y601H exchange could not be diagnosed in 503 patients with var ious thyroid disorders and healthy test subjects. The functional expression of TSHR-Y601H in COS-7 cells revealed altered signal transduction properti es of the receptor: a complete loss of endogenous constitutive cAMP formati on and TSH-induced phospholipase C activation. TSH-induced stimulation of a denylate cyclase activity and surface expression of TSHR were unaltered. Sy stematic exchange of amino-acid residues at position 601 (Y601A, Y601D, Y60 1F,Y601K, Y601P,Y601S, Y601W) did not lead to restoration of wild type sign al transduction properties in any of the mutants. Conclusion: These findings indicate that a precisely situated hydroxyl grou p at position 601 has a functional impact on dual signal transduction and e ndogenous constitutive cAMP formation. Our findings indicate, that the prim ary sequence of human TSHR must be corrected in most databases. TSHR-Y601H is neither a wild-type receptor nor a silent polymorphism but a rare partia lly inactivated isoform of TSHR.