Disruption of the first extracellular loop of thyrotropin receptor prevents ligand binding.
In order to understand the function of the first extracellular loop of the human thyrotropin receptor (hTSHR), each of two peptides of nine amino acids was inserted into the first extracellular loop of hTSHR. hTSHR cDNA was subcloned into the eukaryotic expression vector, pRc/CMV (hTSHR/pRc/CMV). B-hTSHR/pRc/CMV, a mutant hTSHR cDNA which encodes a hydrophilic peptide insert (AGTTRRVAI) and C-hTSHR/pRc/CMV which encodes a hydrophobic peptide insert (ATVLVVPMI) between +486 Ileu and +487 Asp of hTSHR were transfected into Chinese hamster ovary cells to generate the B-1 and C-6 cell lines, respectively. Neither thyrotropin (TSH) nor thyroid stimulating antibody (TSAb) stimulated cAMP production by B-1 or C-6 cells. An 125I-TSH binding assay showed that neither cell line bound TSH. Our data demonstrated that these mutations impaired both TSH binding and cAMP production. This evidence suggests that the first extracellular loop of hTSHR may have a crucial role in the TSH- and TSAb-dependent signal transduction.
Haraguchi
K
K
Third Department of Internal Medicine, University of Yamanashi Medical School, Japan.
Saito
T
T
Endo
T
T
Onaya
T
T
D000818:Animals; D000906:Antibodies / Q000378:metabolism / Q000494:pharmacology; D001323:Autoantibodies / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D016466:CHO Cells; D002999:Clone Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D005786:Gene Expression Regulation; D006801:Humans; D018828:Immunoglobulins, Thyroid-Stimulating; D008024:Ligands; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013961:Thyroid Gland / Q000276:immunology; D013972:Thyrotropin / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection
D000906:Antibodies; D001323:Autoantibodies; D017931:DNA Primers; D018828:Immunoglobulins, Thyroid-Stimulating; D008024:Ligands; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D004247:DNA; D000242:Cyclic AMP