Hinkle P M, Tashjian A H
Biochemistry. 1975 Aug 26;14(17):3845-51. doi: 10.1021/bi00688a017.
Thyrotropin-releasing hormone (TRH), a hypothalamic tripeptide, binds rapidly and reversibly to specific membrane receptors on GH3 cells, a clonal strain of rat pituitary cells grown in culture. GH3 cells were incubated for 1-72 hr with unlabeled TRH, washed, and then incubated for 1 hr with [3H]TRH. Under these conditions 80% of any bound, unlabeled TRH exchanges with [3H]TRH in the medium, and the amount of radioactivity bound to the cells gives a measure of the number of TRH receptors. In GH3 cells, the number of available TRH receptors decreased from 92% of control after 1 hr to 35% after 48 or 72 hr of incubation with unlabeled TRH. Binding of [3H]TRH to both intact control and TRH-treated cells was half-maximal at 8 nM [3H]TRH, but the maximum amount of [3H]TRH bound was decreased by 75% in cells previously incubated for 48 hr with unlabeled TRH. Equilibrium binding studies were performed using membrane fractions prepared from control cells and cells previously exposed to TRH for various periods. The dissociation constant of the TRH-receptor complex was the same in all cases, but the maximum amount of TRH bound decreased progressively in membrane fractions from cells incubated with TRH for 1-51 hr. TRH receptors were not found in cytoplasmic fractions of control or TRH-treated cells. The loss of TRH receptors was reversible within 4 days. In the continued presence of the tripeptide the number of receptors remained low for 12 days. After incubation for 2 days with different concentrations of TRH, the number of receptors was decreased to 33% of control at 100-300 nM TRH, and half of this decrease occurred at about 1 nM TRH; half-maximal biological responses occur at 2 nM TRH. The biologically active Ntau-methylhistidyl derivative of TRH also effected a loss of receptors, while three inactive analogs of TRH did not cause reductions in the number of TRH receptors. In cultures incubated for 40 hr with cycloheximide, protein synthesis was inhibited by 85%, but the number of TRH receptors was 76% of control suggesting that the receptor has a long half-life. When GH3 cells were incubated with cycloheximide plus TRH, the number of TRH receptors decreased by only 23% as compared to a decrease of 73% in cells incubated with TRH alone, suggesting that receptor loss is partially dependent on active protein synthesis. We conclude that in GH3 cells TRH regulates the number of its own receptors.
促甲状腺激素释放激素（TRH）是一种下丘脑三肽，它能快速且可逆地与GH3细胞（一种在培养中生长的大鼠垂体细胞克隆株）上的特定膜受体结合。将GH3细胞与未标记的TRH孵育1至72小时，洗涤后，再与[3H]TRH孵育1小时。在这些条件下，任何结合的未标记TRH中有80%会与培养基中的[3H]TRH进行交换，细胞结合的放射性量可衡量TRH受体的数量。在GH3细胞中，与未标记的TRH孵育1小时后，可用TRH受体数量从对照的92%降至48或72小时后的35%。[3H]TRH与完整对照细胞和TRH处理细胞的结合在8 nM [3H]TRH时达到半数最大结合，但在先前用未标记的TRH孵育48小时的细胞中，[3H]TRH的最大结合量减少了75%。使用从对照细胞和先前在不同时间段暴露于TRH的细胞制备的膜组分进行平衡结合研究。TRH受体复合物的解离常数在所有情况下均相同，但在与TRH孵育1至51小时的细胞的膜组分中，TRH的最大结合量逐渐减少。在对照或TRH处理细胞的细胞质组分中未发现TRH受体。TRH受体的丧失在4天内是可逆的。在三肽持续存在的情况下，受体数量在12天内保持较低水平。用不同浓度的TRH孵育2天后，在100至300 nM TRH时，受体数量降至对照的33%，其中约一半的减少发生在约1 nM TRH时；半数最大生物学反应发生在2 nM TRH时。TRH具有生物活性的Nτ-甲基组氨酰衍生物也会导致受体丧失，而TRH的三种无活性类似物不会导致TRH受体数量减少。在用环己酰亚胺孵育40小时的培养物中，蛋白质合成受到85%的抑制，但TRH受体数量为对照的76%，这表明该受体具有较长的半衰期。当GH3细胞与环己酰亚胺加TRH一起孵育时，TRH受体数量仅减少23%，而单独用TRH孵育的细胞中减少了73%，这表明受体丧失部分依赖于活性蛋白质合成。我们得出结论，在GH3细胞中，TRH调节其自身受体的数量。
