Human Molecular Genetics Advance Access published online on August 31, 2005
Human Molecular Genetics, doi:10.1093/hmg/ddi329
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1 Institute of Endocrine Sciences, University of Milan, Milan, 20122, Italy; Laboratory of Endocrinological Research, Istituto Auxologico Italiano, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Cusano Milanino (MI), 20095, Italy
* To whom correspondence should be addressed. TSH resistance is one of the causes of congenital hypothyroidism with thyroid gland in situ. We recently identified families with dominant transmission of partial TSH resistance due to heterozygous inactivating mutations in TSH receptor (TSHR) gene. Although we documented a poor routing of TSHR mutants to the cell membrane, the mechanism responsible for dominant inheritance of partial TSH resistance remained unexplained. We therefore co-transfected wild-type TSHR and mutant receptors found in these patients in Cos-7 cells. A variable impairment of cAMP response to bTSH stimulation was observed, suggesting that inactive TSHR mutants can exert a dominant negative effect on wild-type TSHR. We then generated chimeric constructs of wild-type or inactive TSHR mutants fused to different reporters. By fluorescence microscopy and immunoblotting, we documented an intracellular entrapment, mainly in the endoplasmic reticulum, and reduced maturation of wild-type TSHR in the presence of inactive TSHR mutants. Finally, fluorescence resonance energy transfer and co-immunoprecipitation experiments were performed to study the molecular interactions between wild-type and mutant TSHRs. The results are in agreement with the presence of oligomers formed by wild-type and mutant receptors in the endoplasmic reticulum. Such physical interaction represents the molecular basis for the dominant negative effect of inactive TSHR mutants. These findings provide an explanation for the dominant transmission of partial TSH resistance. This is the first report linking dominant negative mutations of a G protein-coupled receptor to an abnormal endocrine phenotype in heterozygous patients.
Received May 25, 2005
Revised July 11, 2005
Accepted August 16, 2005
Article
Intracellular entrapment of wild-type TSH receptor by oligomerization with mutants linked to dominant TSH resistance
2 Laboratory of Endocrinological Research, Istituto Auxologico Italiano, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Cusano Milanino (MI), 20095, Italy
3 Advanced Light and Electron Microscopy Bio-Imaging Center (ALEMBIC), Istituto Scientifico San Raffaele, Milan, 20132, Italy
4 Institute of Endocrine Sciences, University of Milan, Milan, 20122, Italy; Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milan, 20122, Italy
5 Institute of Endocrine Sciences, University of Milan, Milan, 20122, Italy; Laboratory of Endocrinological Research, Istituto Auxologico Italiano, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Via Zucchi 18, Cusano Milanino (MI), 20095, Italy
Luca Persani, E-mail: luca.persani{at}unimi.it
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