Chazenbalk G D, Kakinuma A, Jaume J C, McLachlan S M, Rapoport B
Thyroid Molecular Biology Unit, Veterans Administration Medical Center, San Francisco, California 94121, USA.
Endocrinology. 1996 Nov;137(11):4586-91. doi: 10.1210/endo.137.11.8895321.
The complementary DNA for the human TSH receptor (TSHR) translated region was amplified in the genome of stably transfected Chinese hamster ovary (CHO) cells using a dihydrofolate reductase minigene. Immunoprecipitation of TSHR in whole cells precursor-labeled with [35S]methionine and [35S]cysteine revealed an approximately 10-fold increase in TSHR expression in cells stabilized in 10,000 nM methotrexate (TSHR-10,000 cells) compared to cells with the same gene not subjected to amplification (TSHR-0 cells). Similarly, [125I]TSH cross-linking to the surface of intact CHO cells revealed a progressive increase in TSH-binding sites with dihydrofolate reductase minigene amplification, with a 12.8-fold increase in TSHR in TSHR-10,000 vs. TSHR-0 cells. Based on the known number of TSHR expressed by TSHR-0 cells, TSHR-10,000 express approximately 1.9 x 10(6) TSHR on their surface. Two ligand-TSHR complexes were evident under reducing conditions, representing the single chain holoreceptor of about 115 kDa and a dissociated A subunit of about 60 kDa. In the absence of TSH, basal cAMP levels in TSHR-10,000 cells were greater than those in TSHR-0 cells (6-fold in isotonic medium and 18.5-fold in hypotonic medium), indicating that the unliganded TSHR has significant constitutive activity. We assessed the kinetics of TSH binding to CHO cells overexpressing the TSHR using [125I]TSH in the presence of increasing concentrations of unlabeled TSH as well as by attempted saturation with labeled ligand. Surprisingly, in contrast to TSHR-0 cells (Kd = approximately 5 x 10(-10) M), we observed progressively lower affinities for TSH binding by TSHR-800 cells (Kd = approximately 10(-9) M) and TSHR-10,000 cells (Kd = approximately 2 x 10(-9) M). In summary, we report a high level of expression of TSHR in CHO cells and confirm the high constitutive activity of the TSHR in the absence of ligand as well as the binding of TSH to the single subunit, uncleaved TSHR. Moreover, we found that a high level of expression is associated with apparent negative cooperativity among the TSHR in terms of their affinity for ligand.
使用二氢叶酸还原酶小基因，在稳定转染的中国仓鼠卵巢（CHO）细胞基因组中扩增人促甲状腺激素受体（TSHR）翻译区的互补DNA。用[35S]甲硫氨酸和[35S]半胱氨酸对全细胞进行前体标记后，对TSHR进行免疫沉淀，结果显示，与未进行扩增的相同基因细胞（TSHR-0细胞）相比，在10000 nM甲氨蝶呤中稳定的细胞（TSHR-10000细胞）中TSHR表达增加了约10倍。同样，[125I]TSH与完整CHO细胞表面的交联显示，随着二氢叶酸还原酶小基因的扩增，TSH结合位点逐渐增加，TSHR-10000细胞中的TSHR比TSHR-0细胞增加了12.8倍。根据TSHR-0细胞表达的TSHR已知数量，TSHR-10000细胞表面表达约1.9×10(6)个TSHR。在还原条件下可明显看到两种配体-TSHR复合物，分别代表约115 kDa的单链全受体和约60 kDa的解离A亚基。在无TSH的情况下，TSHR-10000细胞中的基础cAMP水平高于TSHR-0细胞（等渗培养基中高6倍，低渗培养基中高18.5倍），表明未结合配体的TSHR具有显著的组成性活性。我们使用[125I]TSH，在未标记TSH浓度增加的情况下以及通过尝试用标记配体饱和，评估了TSH与过表达TSHR的CHO细胞结合的动力学。令人惊讶的是，与TSHR-0细胞（Kd =约5×10(-10) M）相比，我们观察到TSHR-800细胞（Kd =约10(-9) M）和TSHR-10000细胞（Kd =约2×10(-9) M）对TSH结合的亲和力逐渐降低。总之，我们报道了CHO细胞中TSHR的高表达水平，并证实了在无配体情况下TSHR的高组成性活性以及TSH与单亚基、未切割的TSHR的结合。此外，我们发现高表达水平与TSHR之间在配体亲和力方面存在明显的负协同性。
