Roess D A, Rahman N A, Kenny N, Barisas B G
Department of Physiology, Colorado State University, Ft. Collins 80523.
Biochim Biophys Acta. 1992 Nov 17;1137(3):309-16. doi: 10.1016/0167-4889(92)90151-z.
To better understand the in situ organization of the luteinizing hormone receptor on rat luteal cells, we have examined the molecular motions of this receptor following binding of ovine luteinizing hormone (oLH) or human chorionic gonadotropin (hCG). Fluorescence photobleaching recovery (FPR) measurements of LH receptor lateral diffusion were performed using tetramethylrhodamine isothiocyanate (TRITC)-derivatized oLH or hCG as a probe. These experiments indicate that TRITC-oLH occupied LH receptors on luteal cells obtained from superovulated female rats have a lateral diffusion coefficient D of (1.7 +/- 0.6).10(-10) cm2s-1 at 27 degrees C with fluorescence recovery after photobleaching of 46 +/- 5%. In similar experiments, binding of TRITC-hCG caused a significant decrease in LH receptor lateral diffusion; fluorescence recovery after photobleaching was less than 20%. To determine whether hCG-occupied receptors might exist in large aggregates, we measured the rotational correlation times (RCT) of hCG and oLH bound to the LH receptor on intact cells using single cell polarized fluorescence depletion (PFD). At 4 degrees C, LH receptors occupied by eosin isothiocyanate (EITC)-hCG exhibited a slower RCT (64 microseconds) than did receptors occupied by EITC-oLH (43 microseconds). At this temperature both TRITC-oLH and TRITC-hCG occupied LH receptors were laterally immobile. These FPR and PFD results suggest that the molecular motions of the luteal cell LH receptor are significantly modulated by the subtle structural differences in various bound gonadotropins.
为了更好地理解大鼠黄体细胞上促黄体生成素受体的原位组织情况，我们检测了该受体在结合绵羊促黄体生成素（oLH）或人绒毛膜促性腺激素（hCG）后的分子运动。使用异硫氰酸四甲基罗丹明（TRITC）衍生的oLH或hCG作为探针，对促黄体生成素受体的侧向扩散进行荧光漂白恢复（FPR）测量。这些实验表明，在27℃时，TRITC - oLH占据的超排卵雌性大鼠黄体细胞上的促黄体生成素受体具有侧向扩散系数D为（1.7±0.6）×10⁻¹⁰ cm²s⁻¹，光漂白后的荧光恢复率为46±5%。在类似实验中，TRITC - hCG的结合导致促黄体生成素受体侧向扩散显著降低；光漂白后的荧光恢复率小于20%。为了确定hCG占据的受体是否可能以大聚集体形式存在，我们使用单细胞偏振荧光耗尽（PFD）测量完整细胞上与促黄体生成素受体结合的hCG和oLH的旋转相关时间（RCT）。在4℃时，异硫氰酸曙红（EITC） - hCG占据的促黄体生成素受体表现出比EITC - oLH占据的受体更慢的RCT（64微秒）（43微秒）。在此温度下，TRITC - oLH和TRITC - hCG占据的促黄体生成素受体在侧向均不移动。这些FPR和PFD结果表明，黄体细胞促黄体生成素受体的分子运动受到各种结合的促性腺激素细微结构差异的显著调节。
