Lane T A, Chen T T
Department of Zoology, University of Tennessee, Knoxville 37996.
Endocrinology. 1991 Apr;128(4):1833-40. doi: 10.1210/endo-128-4-1833.
Although the binding, internalization, and regulation of LH, PRL, and their respective receptors have been extensively studied, it is not known whether the receptors are coordinately regulated. Using double labeling experiments, we have previously shown that receptor-bound LH and PRL can be colocalized in identical endosomes of granulosa cells. We hypothesize that high levels of PRL may induce a heterologous down-modulation of LH receptors, consequently reducing ovarian responsiveness to further gonadotropin stimulation. In this study we used a novel procedure to enrich endosomes containing internalized PRL and to determine whether unoccupied LH receptors were cointernalized in granulosa cells. Porcine granulosa cells were obtained from medium-sized (3-5 mm) follicles and cultured for 4 days in the presence of FSH. Fluorescein isothiocyanate-labeled PRL (FITC-PRL) was used as a ligand to induce internalization of PRL receptors and as a marker to label endosomes. Granulosa cells were incubated with FITC-PRL at either 4 or 37 C for various times. At the end of the incubation, cells were trypsinized to remove surface receptors and then homogenized. The postnuclear fraction containing endosomes and other subcellular organelles was sorted using a FACStar Plus cell sorter. Results from 14 separate sorting experiments showed that 1) FITC-PRL-treated cells exhibited a sorting pattern distinct from that of FITC-BSA-treated or untreated cells; 2) excess unlabeled PRL partially shifted the sorting profile to one similar to that in controls; 3) the differences in sorting profiles were not due to free FITC; and 4) using this method, it was possible to isolate FITC-PRL-containing endosomes that were virtually devoid of other contaminating subcellular particles. Fluorescently positive (FITC-PRL-containing) organelles were collected and assayed for LH receptors using [125I]hCG as a tracer. When the cells were incubated with FITC-PRL at 37 C for 3 h, the number of available LH receptors (as determined by [125I]hCG binding) was 37% higher in particles containing FITC-PRL than in those devoid of FITC-PRL. If the cells were allowed to preincubate with FITC-PRL at 4 C for 10-16 h before raising the temperature to 37 C, the number of available LH receptors in FITC-PRL-containing endosomes was about 7-fold higher than that in FITC-negative endosomes. Results from this study suggest that PRL not only induces internalization of its own receptor, but also causes down-modulation of unoccupied LH receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
尽管对促黄体生成素（LH）、催乳素（PRL）及其各自受体的结合、内化和调节已进行了广泛研究，但尚不清楚这些受体是否受到协同调节。我们先前通过双重标记实验表明，与受体结合的LH和PRL可共定位于颗粒细胞的同一内体中。我们推测，高水平的PRL可能诱导LH受体的异源下调，从而降低卵巢对进一步促性腺激素刺激的反应性。在本研究中，我们采用一种新方法富集含有内化PRL的内体，并确定颗粒细胞中未被占据的LH受体是否会被共同内化。猪颗粒细胞取自中等大小（3 - 5毫米）的卵泡，并在促卵泡素（FSH）存在的情况下培养4天。异硫氰酸荧光素标记的PRL（FITC - PRL）用作诱导PRL受体内化的配体，并作为标记内体的标志物。将颗粒细胞与FITC - PRL在4℃或37℃孵育不同时间。孵育结束时，用胰蛋白酶处理细胞以去除表面受体，然后进行匀浆。使用FACStar Plus细胞分选仪对含有内体和其他亚细胞器的核后组分进行分选。14次独立分选实验的结果表明：1）FITC - PRL处理的细胞呈现出与FITC - 牛血清白蛋白（BSA）处理或未处理细胞不同的分选模式；2）过量未标记的PRL使分选图谱部分转变为与对照相似的图谱；3）分选图谱的差异不是由于游离的FITC所致；4）使用这种方法，可以分离出几乎不含其他污染性亚细胞颗粒的含FITC - PRL的内体。收集荧光阳性（含FITC - PRL）的细胞器，并用[125I]人绒毛膜促性腺激素（hCG）作为示踪剂检测LH受体。当细胞在37℃与FITC - PRL孵育3小时时，含FITC - PRL的颗粒中可用LH受体的数量（通过[125I]hCG结合测定）比不含FITC - PRL的颗粒高37%。如果在将温度升至37℃之前，先让细胞在4℃与FITC - PRL预孵育10 - 16小时，那么含FITC - PRL的内体中可用LH受体的数量比FITC阴性内体中的数量高约7倍。本研究结果表明，PRL不仅诱导其自身受体的内化，还导致未被占据的LH受体下调。（摘要截短于400字）
