Fuh G, Wells J A
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.
J Biol Chem. 1995 Jun 2;270(22):13133-7. doi: 10.1074/jbc.270.22.13133.
We investigated the mechanism of action of the human prolactin (hPRL) receptor on four different breast cancer cell lines, T-47D, MCF-7, BT-474, and SK-BR3, that express elevated levels of the receptor compared with normal cells. Cells treated with human growth hormone (hGH), which binds and activates the hPRL receptor, exhibited bell-shaped dose-response growth curves consistent with the sequential dimerization mechanism proposed for the hPRL receptor (Fuh, G., Colosi, P., Wood, W.I., and Wells, J.A. (1993) J. Biol. Chem. 268, 5376-5381). Growth stimulation was enhanced by Zn2+, which preferentially increases the affinity of hGH for the hPRL receptor. Furthermore, receptor-selective variants of hGH that bind the hPRL receptor but not the hGH receptor were agonistic, providing additional support that specific binding to the hPRL receptor can stimulate these breast cancer cells to grow. On this basis we produced variants of hGH and human placental lactogen (hPL) that were potential antagonists because they bind but do not dimerize the hPRL receptor. The hPL-based antagonist was less potent than the hGH-based antagonist toward the growth of MCF-7 cells, consistent with the lower affinity of hPL for hPRL receptor than for hGH. However, the hPL-based antagonist was more potent than the hGH antagonist for BT-474 cells. Antibodies to the hPRL receptor inhibited growth of FDC-P1 cells transfected with the hPRL receptor; these also inhibited MCF-7 cells and T47D cells but not BT-474 cells. A unique feature of BT-474 cells was found when screening its cDNA revealed the presence of a novel alternative splice of the hPRL receptor that codes for the soluble extracellular domain; this may explain these differential inhibitory effects. These studies provide further molecular insight into the potential role of the hPRL receptor in breast cancer and demonstrate that hPRL receptor antagonists can inhibit the growth of breast cancer cells.
我们研究了人催乳素（hPRL）受体对四种不同乳腺癌细胞系（T-47D、MCF-7、BT-474和SK-BR3）的作用机制，与正常细胞相比，这些细胞系中该受体表达水平升高。用人生长激素（hGH）处理细胞，hGH能结合并激活hPRL受体，处理后的细胞呈现钟形剂量反应生长曲线，这与为hPRL受体提出的顺序二聚化机制一致（Fuh, G., Colosi, P., Wood, W.I., and Wells, J.A. (1993) J. Biol. Chem. 268, 5376 - 5381）。Zn2+增强了生长刺激作用，它优先增加hGH对hPRL受体的亲和力。此外，能结合hPRL受体但不结合hGH受体的hGH受体选择性变体具有激动作用，这进一步证明与hPRL受体的特异性结合可刺激这些乳腺癌细胞生长。在此基础上，我们制备了hGH和人胎盘催乳素（hPL）的变体，它们可能是拮抗剂，因为它们能结合hPRL受体但不会使其二聚化。基于hPL的拮抗剂对MCF-7细胞生长的抑制作用比基于hGH的拮抗剂弱，这与hPL对hPRL受体的亲和力低于对hGH的亲和力一致。然而，基于hPL的拮抗剂对BT-474细胞的抑制作用比基于hGH的拮抗剂更强。抗hPRL受体抗体抑制了转染hPRL受体的FDC-P1细胞的生长；这些抗体也抑制MCF-7细胞和T47D细胞，但不抑制BT-474细胞。在筛选BT-474细胞的cDNA时发现了一个独特特征，即存在一种编码可溶性细胞外结构域的hPRL受体新型可变剪接；这可能解释了这些不同的抑制作用。这些研究为hPRL受体在乳腺癌中的潜在作用提供了进一步的分子见解，并证明hPRL受体拮抗剂可抑制乳腺癌细胞的生长。
