title: The middle portion in the second cytoplasmic loop of the thyrotropin receptor plays a crucial role in adenylate cyclase activation.
abstract: We have examined the role of the 2nd cytoplasmic loop of the TSH receptor (TSHR) in TSH- and TSHR autoantibody-stimulated cAMP and inositol phosphate formation using mutants created by substituting sequences from the alpha 1- or beta 2-adrenergic receptors (AR). Unlike similar substitution mutants involving the 3rd cytoplasmic loop that lose agonist-induced inositol phosphate but not cAMP increase after transfection into Cos-7 cells, mutants involving the 2nd loop showed significant change in generating both signals. Mutant B525, which substitutes residues 525-527 with a comparable beta 2-AR sequence, exhibited a complete loss in TSH- or Graves' immunoglobulin G-increased cAMP signaling and a lesser loss in phosphoinositide signaling. This is a unique mutant in which cAMP response was completely lost in all those involving the 2nd or 3rd cytoplasmic loop. On the other hand, mutant B528, in which residues 528-532 are substituted with a comparable beta 2-AR sequence, exhibited the most profound loss in phosphoinositide signaling. Mutants involving portions surrounding residues 528-532 in the 2nd cytoplasmic loop had milder losses in agonist-increased phosphoinositide signaling and much lesser losses in agonist-increased cAMP generation. The transfection efficiency of all transfectants was the same. All transfectants with mutant or wild type TSHR had a similar amount and identical profile of TSHR mRNA in Northern blots and TSHR forms on Western blots. Thus, the 2nd cytoplasmic loop is important for agonist-induced cAMP as well as for phosphoinositide signal generation, whereas the 3rd loop appears to be important only for the latter. The most important determinant for agonist-increased cAMP signal generation is in the middle of the 2nd loop, around residues 525-527. In contrast, the determinants most critical for agonist-induced phosphoinositide signaling are also located in the middle of the 2nd loop, around residues 528-532, and those with less importance are broadly distributed.
authors.affiliation: Department of Laboratory Medicine, Kyoto University School of Medicine, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Kosugi
authors.forename: L D
authors.initials: LD
authors.lastname: Kohn
authors.forename: T
authors.initials: T
authors.lastname: Akamizu
authors.forename: T
authors.initials: T
authors.lastname: Mori
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001323:Autoantibodies / Q000494:pharmacology; D002460:Cell Line; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000096:biosynthesis; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D006111:Graves Disease / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000494:pharmacology; D018828:Immunoglobulins, Thyroid-Stimulating; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000096:biosynthesis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011944:Receptors, Androgen / Q000737:chemistry* / Q000145:classification / Q000187:drug effects / Q000235:genetics; D011989:Receptors, Thyrotropin / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000187:drug effects* / Q000502:physiology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000187:drug effects*; D013972:Thyrotropin / Q000494:pharmacology*
chemical_list: D001323:Autoantibodies; D007074:Immunoglobulin G; D018828:Immunoglobulins, Thyroid-Stimulating; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D011944:Receptors, Androgen; D011989:Receptors, Thyrotropin; D011993:Recombinant Fusion Proteins; D013972:Thyrotropin; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases