tailieunhanh - Báo cáo khoa học: Proper targeting and activity of a nonfunctioning thyroid-stimulating hormone receptor (TSHr) combining an inactivating and activating TSHr mutation in one receptor

Activating mutations of the thyroid-stimulating hormone receptor (TSHr) have been identified as a cause of toxic adenomas. Germline-inactivating TSHr mutations have beendescribedas a cause of congenital hypothyroidism. The effects of combining activating and inactivating mutations within a single receptor was studied. The double mutant T477I/P639S contained an activating TSHr mutation (P639S) togetherwithan inactivatingone (T477I). Theother one (I486M/P639S) contained two activating mutations. | Eur. J. Biochem. 270 3839-3847 2003 FEBS 2003 doi Proper targeting and activity of a nonfunctioning thyroid-stimulating hormone receptor TSHr combining an inactivating and activating TSHr mutation in one receptor Patrizia Aaretti1 GiuseDDina De Marco1 Paola Collecchi2 Luca Chiovato3 Paolo Vitti Aldo Pinchera1 and Massimo Tonacchera1 1 Dipartimento di Endocrinologia e Metabolismo Ortopedia e Traumatologia Medicina del Lavoro Universita di Pisa Pisa Italy 2Dipartimento di Oncologia Divisione di Anatomia Patologica Universita di Pisa Pisa Italy Cattedra di Endocrinologia Fondazione S Maugeri IRCCS Pavia Italy Activating mutations of the thyroid-stimulating hormone receptor TSHr have been identified as a cause of toxic adenomas. Germline-inactivating TSHr mutations have been described as a cause of congenital hypothyroidism. The effects of combining activating and inactivating mutations within a single receptor was studied. The double mutant T477I P639S contained an activating TSHr mutation P639S together with an inactivating one T477I . The other one I486M P639S contained two activating mutations. Constructs were expressed in COS-7 cells and basal and TSH-stimulated cyclic AMP cAMP accumulation and inositol phosphate IP production were determined. The expression at the cell surface was studied both with binding and fluorescence-activated cell scanning analysis. Our results show that the effect of combining the two activating muta tions is an increase in the constitutive activity only for the cAMP pathway and not for the IP pathway suggesting that different mutations result in receptor conformations with different relative abilities to couple to Gs-alpha or Gq-alpha. Surprisingly the double mutant containing the T477I behaves as an activating receptor with constitutive activity both for the cAMP and IP pathways. These data show that an inactive form of the TSHr which is trapped inside a cell after transfection is able to gain the .