Mutation of alanine 623 in the third cytoplasmic loop of the rat thyrotropin (TSH) receptor results in a loss in the phosphoinositide but not cAMP signal induced by TSH and receptor autoantibodies.
Thyrotropin (TSH) and IgG preparations from patients with Graves' disease increase inositol phosphate as well as cAMP formation in Cos-7 cells transfected with rat TSH receptor cDNA. Mutation of alanine 623 in the carboxyl end of the third cytoplasmic loop of the TSH receptor, to lysine or glutamic acid, results in the loss of TSH- and Graves' IgG-stimulated inositol phosphate formation but not in stimulated cAMP formation. There is no effect of the mutations on basal or P2-purinergic receptor-mediated inositol phosphate formation. The mutations do not affect transfection efficiency or the synthesis, processing, or membrane integration of the receptor, as evidenced by the unchanged amount and composition of the TSH receptor forms on Western blots of membranes from transfected cells. The mutations increase the affinity of the TSH receptor for [125I]TSH and decrease Bmax; however, cells with an equivalently decreased Bmax as a result of transfection with lower levels of wild type receptor do not lose either TSH-induced inositol phosphate formation or cAMP signaling activity. Thus, in addition to discriminating between ligand-induced phosphatidylinositol bisphosphate and cAMP signals, the mutation appears to cause an altered receptor conformation which affects ligand binding to its large extracellular domain.
Kosugi
S
S
Cell Regulation Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Okajima
F
F
Ban
T
T
Hidaka
A
A
Shenker
A
A
Kohn
L D
LD
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001323:Autoantibodies / Q000494:pharmacology*; D001667:Binding, Competitive; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D006111:Graves Disease / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010716:Phosphatidylinositols / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013972:Thyrotropin / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection
D001323:Autoantibodies; D007074:Immunoglobulin G; D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D005576:Colforsin; D013972:Thyrotropin; D002772:Cholera Toxin; D000242:Cyclic AMP; D000409:Alanine