Importance of the extracellular domain of the human thyrotrophin receptor for activation of cyclic AMP production.
The mechanism by which the TSH receptor is activated is unknown. Current knowledge leads us to consider that G protein-coupled receptors are activated by positioning of their ligand in the pocket formed by the hydrophobic transmembrane segments. Furthermore, activation of an N-terminally truncated LH receptor lacking most of the extracellular domain has been described, suggesting the existence of a mechanism involving a direct interaction between LH and the transmembrane segments. The high conservation of the transmembrane segments among G protein-coupled receptors is a strong indication for a common mechanism of receptor activation. To test this hypothesis for the TSH receptor we have constructed four N-terminally truncated TSH receptor mutants with 5 or 69 amino acids of the extracellular domain joined to signal peptide regions consisting of the first 23 or 33 amino acids. The four fragments were amplified by PCR and subcloned into pBSK+. Sequences were confirmed after subcloning in M13. After joining the four fragments in pBSK+, the four TSH receptor constructs were subcloned in pSVL and transiently or stably expressed in COS and Chinese hamster ovary (CHO) cells respectively. In contrast to results obtained for the LH receptor, stimulation of the transfectants with 10 microM human chorionic gonadotrophin or 350 mU TSH/ml did not increase cyclic AMP (cAMP) concentrations in cultures of transiently transfected COS cells or stably transfected CHO cells. However, mRNA for the TSH receptor could be detected by RNase protection assay in all stable transfectants used for stimulation of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)
Service de Génétique Médicale, Université Libre de Bruxelles, Belgium.
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R
Paschke
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M
Parmentier
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G
Vassart
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D003001:Cloning, Molecular; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis*; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011989:Receptors, Thyrotropin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013972:Thyrotropin / Q000494:pharmacology; D014162:Transfection
D006063:Chorionic Gonadotropin; D017931:DNA Primers; D010446:Peptide Fragments; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D000242:Cyclic AMP