Cetani F, Tonacchera M, Vassart G
Institute de Recherche Interdisciplinaire (IRIBHN), Faculty of Medecine, Université Libre de Bruxelles, Belgium.
FEBS Lett. 1996 Jan 2;378(1):27-31. doi: 10.1016/0014-5793(95)01384-9.
The TSH receptor (TSHR) and the LH/CG receptor (LHR) are members of the family of G protein-coupled receptors. Recently, point mutations conferring constitutive activity to the TSHR and LHR have been observed as a cause of toxic adenoma and familial/sporadic male pseudo-precocious puberty, respectively. When evaluated by transfection in COS-7 cells the wild-type (wt) TSHR displays definite constitutive activity towards Gs-dependent adenylylcyclase stimulation, while available evidence shows that the LHR does not. In order to compare the constitutive activity of both receptors, we performed functional studies in COS-7 cells using different assay conditions. Human TSHR and LHR cDNAs subcloned in the expression vector pSVL were transiently expressed in COS-7 cells and cAMP production was determined following incubation in a medium containing physiological concentration of NaCl [isotonic (NaCl)] or in the same medium without NaCl [hypotonic (NaCl-)] or where NaCl was replaced by an isoosmolar concentration of sucrose [isotonic (sucrose)]. Cells transfected with the TSHR showed higher basal cAMP levels over cells transfected with pSVL in all conditions tested. The effect was stronger when cells were incubated in isotonic (sucrose) buffer. Cells expressing LHR exhibited a minimal increase of cAMP levels over cells transfected with pSVL in isotonic (NaCl) buffer; however, a marked increase in basal cAMP levels was observed when cells were assayed in hypotonic (NaCl-) or isotonic (sucrose) buffers. Varying the pH or incubation temperature was without effect on the results obtained with both receptors. Our data show that despite extensive sequence similarity, the LH and TSH receptors differ markedly in their basal activity. The differential sensitivity of both receptors to low NaCl concentrations, suggests that the unliganded TSH receptor is less constrained than its LH homolog and may be more susceptible to activation by a wide spectrum of mutations.
促甲状腺激素受体（TSHR）和促黄体生成素/绒毛膜促性腺激素受体（LHR）是G蛋白偶联受体家族的成员。最近，已观察到分别导致毒性腺瘤和家族性/散发性男性假性性早熟的TSHR和LHR的组成型活性点突变。当通过在COS-7细胞中进行转染评估时，野生型（wt）TSHR对Gs依赖性腺苷酸环化酶刺激表现出明确的组成型活性，而现有证据表明LHR没有。为了比较两种受体的组成型活性，我们在COS-7细胞中使用不同的测定条件进行了功能研究。亚克隆到表达载体pSVL中的人TSHR和LHR cDNA在COS-7细胞中瞬时表达，并在含有生理浓度NaCl的培养基[等渗（NaCl）]中孵育或在不含NaCl的相同培养基[低渗（NaCl-）]中或用等渗浓度的蔗糖代替NaCl [等渗（蔗糖）]后测定cAMP产生。在所有测试条件下，用TSHR转染的细胞比用pSVL转染的细胞显示出更高的基础cAMP水平。当细胞在等渗（蔗糖）缓冲液中孵育时，这种作用更强。在等渗（NaCl）缓冲液中，表达LHR的细胞与用pSVL转染的细胞相比，cAMP水平的增加最小；然而，当在低渗（NaCl-）或等渗（蔗糖）缓冲液中检测细胞时，观察到基础cAMP水平显著增加。改变pH或孵育温度对两种受体获得的结果没有影响。我们的数据表明，尽管序列有广泛的相似性，但LH和TSH受体的基础活性有显著差异。两种受体对低NaCl浓度的不同敏感性表明，未结合配体的TSH受体比其LH同源物受到的限制更少，可能更容易受到多种突变的激活。
