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An Inactivating Mutation within the First Extracellular Loop of the Thyrotropin Receptor Impedes Normal Posttranslational Maturation of the Extracellular Domain

Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 690 (S.S.-T., C.A., J.L., N.d.R.), Hôpital Robert-Debré, 75935 Paris, France; Centre Hospitalier Sud-Réunion (S.D.), 97448 Saint Pierre, France; INSERM Unité 845 (S.S.-T., A.C., M.P.), Faculté de Médecine Necker-Enfants Malades, 75730 Paris, France; Service d’Endocrinologie Pédiatrique, Hôpital Necker-Enfants Malades, Assistance Publique-Hpitaux de Paris (AP-HP), 75743 Paris, France; University Paris Descartes-Paris 5 (M.P.), 75006 Paris, France; Service d’Endocrinologie Pédiatrique (J.L.) et Laboratoire de Biochimie Hormonale (N.d.R.), Hôpital Robert Debré, AP-HP, 75935 Paris, France; and University Paris Diderot-Paris 7 (N.d.R.), Institut Fédératif de Recherche 02, 75018 Paris, France

Address all correspondence and requests for reprints to: Nicolas de Roux, Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 690, Hôpital Robert-Debré, 48 Boulevard Serrurier, 75935 Paris Cedex 19, France. E-mail: nicolas.deroux{at}inserm.fr.

The TSH receptor (TSHR), a member of the large family of G protein-coupled receptors, controls both function and growth of thyroid cells; hence, mutations of this receptor result in thyroid dysfunction. Here, we took advantage of the description of a new inactivating TSHR mutation (Q489H) in two brothers with hypothyroidism, to precise maturation, intracellular trafficking, exporting pathways, and activation mechanisms of this receptor. Functional characterization of the Q489H-TSHR in transiently transfected HEK293 cells showed cell surface expression, normal TSH binding affinity, and its inability to generate intracellular cAMP in response to TSH stimulation. Western blot analysis of the whole membrane proteins or proteins expressed at the cell surface showed that Q489H-TSHR expressed in HEK293 transfected cells are restricted to mannose-rich uncleaved receptor. Analysis of the export pathway toward cell surface indicated that both Q489H and wild-type receptors followed the same intracellular route to cell surface throughout endoplasmic reticulum and Golgi apparatus. This study shows that Q489H substitution impedes complete glycosylation of TSHR extracellular domain within the Golgi apparatus and that Q489H-TSHR expressed at the cell surface is unable to undergo intramolecular cleavage as well as to switch toward an active conformation under TSH stimulation. Altogether, our results show that 1) Q489H substitution within the first extracellular loop induces a misfolding of TSHR, blocking it into an inactive conformation and impeding complete glycosylation and intramolecular cleavage, and 2) a misfolded G protein-coupled receptor can bypass endoplasmic reticulum or Golgi apparatus quality control and reach the cell surface as an immature receptor.