Genty N, Paly J, Edery M, Kelly P A, Djiane J, Salesse R
Unité d'Ingéniérie des Protéines, INRA-Biotechnologies, Jouy-en-Josas, France.
Mol Cell Endocrinol. 1994 Mar;99(2):221-8. doi: 10.1016/0303-7207(94)90011-6.
Molecular cloning of the prolactin (PRL) receptor cDNA has revealed different forms of the receptor: among them, the longest form encodes a transmembrane protein of 592-598 amino acids and was originally found in rabbit mammary gland as well as in human and rat tissues. It contains a cytoplasmic domain of 358 amino acids. In CHO cells transfected with the PRL receptor cDNA, PRL is able to induce the specific expression of a reporter gene provided with the promoter of the milk protein gene beta-lactoglobulin. The cDNA encoding this long receptor form has been expressed permanently after stable transfection of Chinese hamster ovary (CHO) cells. In these cells, we have determined the fate of the bound hormone and of the receptor. At 37 degrees C, transfected cells were able to endocytose 125I-labeled human growth hormone (hGH) or ovine prolactin (oPRL) at an initial rate of about 1 fmol/h at 100 pM labeled hormone and 10(6) cells/well. Lowering the temperature to 15 degrees C slowed the endocytosis of [125I]hGH by a factor of 5. These results were confirmed by electron microscopy with oPRL labeled with colloidal gold. At 37 degrees C, the receptor underwent rapid insertion to the cell surface and constitutive endocytosis (half-life 80 min). This rate of endocytosis was enhanced in the presence of 10 nM oPRL (half-life 8 min), leading to down-regulation of the receptor by exhaustion of the intracellular receptor pool. After down-regulation, the cell surface was replenished with newly synthesized PRL receptor with a half-time of 8-10 min. If cycloheximide was added, almost no receptors could be found on the cell surface. These results indicate that in transfected cells the PRL receptor behaved largely as in classical target cells. A "conveyor belt" endocytosis behavior was found, with degradation of the endocytosed receptors, and occupation by the hormone enhancing this process. Moreover, since the PRL receptor belongs to a family of receptors in which companion protein(s) seem to play important roles, transfected CHO cells appear to provide the expressed receptors with the necessary element(s) to function as in normal PRL target cells.
催乳素（PRL）受体cDNA的分子克隆揭示了该受体的不同形式：其中，最长的形式编码一种由592 - 598个氨基酸组成的跨膜蛋白，最初在兔乳腺以及人类和大鼠组织中发现。它包含一个由358个氨基酸组成的胞质结构域。在用PRL受体cDNA转染的CHO细胞中，PRL能够诱导由乳蛋白基因β-乳球蛋白启动子驱动的报告基因的特异性表达。编码这种长受体形式的cDNA在稳定转染中国仓鼠卵巢（CHO）细胞后实现了永久表达。在这些细胞中，我们确定了结合激素和受体的命运。在37℃时，转染细胞能够以100 pM标记激素和每孔10⁶个细胞的初始速率内吞¹²⁵I标记的人生长激素（hGH）或绵羊催乳素（oPRL），速率约为1 fmol/h。将温度降至15℃会使[¹²⁵I]hGH的内吞作用减慢5倍。用胶体金标记的oPRL进行电子显微镜观察证实了这些结果。在37℃时，受体迅速插入细胞表面并进行组成型内吞作用（半衰期80分钟）。在10 nM oPRL存在的情况下，这种内吞速率会加快（半衰期8分钟），导致细胞内受体池耗尽，从而使受体下调。下调后，细胞表面会以8 - 10分钟的半衰期被新合成的PRL受体补充。如果加入放线菌酮，细胞表面几乎找不到受体。这些结果表明，在转染细胞中，PRL受体的行为在很大程度上与经典靶细胞中的行为相同。发现了一种“传送带”式的内吞行为，内吞的受体发生降解，激素的占据会增强这一过程。此外，由于PRL受体属于一个伴侣蛋白似乎起重要作用的受体家族，转染的CHO细胞似乎为表达的受体提供了在正常PRL靶细胞中发挥功能所需的要素。
