Kosugi S, Ban T, Akamizu T, Kohn L D
Cell Regulation Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1991 Oct 15;266(29):19413-8.
Residues 287 to 404 of the rat thyrotropin (TSH) receptor exhibit little homology to gonadotropin receptors. A large segment of this region, residues 303-382, has no determinants important for TSH to bind or elevate cAMP levels nor for the activity of thyroid-stimulating autoantibodies (TSAbs) from the sera of Graves' patients, i.e. deletions, substitutions, or mutations in this segment do not result in a loss of any of these activities in transfected Cos-7 cells. Critical residues for these activities do, however, flank both sides of this segment. Of particular interest, deletion or mutation of residues 299-301 and 387-395 results in a marked decrease in high affinity TSH binding but preserves the ability of a TSAb to increase cAMP levels. Tyrosine 385 is also of particular interest since its mutation to phenylalanine, alanine, threonine, or glutamine results in a receptor with a 20-fold decrease in the ability of TSH to bind or increase cAMP levels, but one whose TSAb activity is, once again, preserved. Because one activity is preserved, we can conclude that (a) the receptor must be fully integrated within the membrane of the cell without malfolding, (b) these sequences represent determinants involved in the high affinity TSH binding site, and (c) separate determinants exist for high affinity TSH binding and TSAb activity, consistent with the existence of autoantibodies in Graves' sera which inhibit TSH binding (TBIAbs) or which increase cAMP levels (TSAbs). Additionally, we show that a 16-mer peptide (residues 352-367), which reacts with the sera of greater than 80% of patients with Graves' disease, can induce the formation of antibodies to a peptide with no sequence homology, residues 377-397. This peptide flanks the region, residues 303-382, with no determinants important for TSH receptor binding or activity. As noted above, it contains residues involved in the high affinity TSH binding site but whose deletion or mutation has no effect on TSAb activity, i.e. residues which would appear to be required at an epitope important for TBIAb but not TSAb antibody activity.
大鼠促甲状腺激素（TSH）受体的287至404位残基与促性腺激素受体几乎没有同源性。该区域的一大段，即303 - 382位残基，对于TSH结合或提高cAMP水平以及格雷夫斯病患者血清中促甲状腺素刺激自身抗体（TSAbs）的活性都没有重要的决定因素，也就是说，该段的缺失、替换或突变不会导致转染的Cos - 7细胞中这些活性的任何丧失。然而，这些活性的关键残基位于该段的两侧。特别有趣的是，299 - 301位和387 - 395位残基的缺失或突变会导致高亲和力TSH结合显著降低，但保留了TSAb提高cAMP水平的能力。酪氨酸385也特别值得关注，因为将其突变为苯丙氨酸、丙氨酸、苏氨酸或谷氨酰胺会导致受体与TSH结合或提高cAMP水平的能力下降20倍，但再次保留了其TSAb活性。因为一种活性得以保留，我们可以得出结论：（a）受体必须在细胞膜内完全整合且无错误折叠；（b）这些序列代表参与高亲和力TSH结合位点的决定因素；（c）高亲和力TSH结合和TSAb活性存在单独的决定因素，这与格雷夫斯病血清中存在抑制TSH结合的自身抗体（TBIAbs）或提高cAMP水平的自身抗体（TSAbs）一致。此外，我们表明一种16肽（352 - 367位残基），它能与超过80%的格雷夫斯病患者血清发生反应，可诱导产生针对无序列同源性的肽（377 - 397位残基）的抗体。该肽位于303 - 382位残基区域的两侧，对于TSH受体结合或活性没有重要的决定因素。如上所述，它包含参与高亲和力TSH结合位点的残基，但其缺失或突变对TSAb活性没有影响，即这些残基似乎是对TBIAb但不对TSAb抗体活性重要的表位所必需的。
