Lee S J, Nathans D
Howard Hughes Medical Institute Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Biol Chem. 1988 Mar 5;263(7):3521-7.
Proliferin is a prolactin-related glycoprotein secreted by proliferating mouse cell lines and by mouse placenta. In an attempt to identify target sites for proliferin action, we looked for proliferin receptors in murine fetal and maternal tissues during pregnancy using proliferin purified from the conditioned medium of a constructed Chinese hamster ovary cell line carrying amplified copies of proliferin cDNA. Purified proliferin bound to membrane preparations from fetal or maternal liver and from placenta with a Kd of 1 to 2 nM. The amount of proliferin bound per microgram of membrane protein varied markedly during pregnancy; maximal binding to day 16 fetal liver membranes was approximately 25 times that to liver membranes from adult animals. Binding to fetal and maternal receptors was specifically and completely inhibited by mannose 6-phosphate, with half-maximal inhibition at 10 microM. Furthermore, non-glycosylated proliferin did not inhibit the binding of the glycosylated protein. A approximately 300 Kd proliferin receptor was purified from the liver of pregnant mice using a proliferin affinity column and elution with mannose 6-phosphate. This receptor reacted with antibodies directed against the rat cation-independent mannose 6-phosphate receptor. We conclude that 1) proliferin secreted by cultured cell binds to cation-independent mannose 6-phosphate receptors and therefore may be a lysosomal protein or targeted to lysosomes, and 2) the concentration or activity of mannose 6-phosphate receptors in murine fetal and maternal liver and in placenta is regulated during pregnancy.
增殖蛋白是一种与催乳素相关的糖蛋白，由增殖的小鼠细胞系和小鼠胎盘分泌。为了确定增殖蛋白的作用靶点，我们使用从携带增殖蛋白cDNA扩增拷贝的构建的中国仓鼠卵巢细胞系的条件培养基中纯化的增殖蛋白，在妊娠期间的小鼠胎儿和母体组织中寻找增殖蛋白受体。纯化的增殖蛋白与胎儿或母体肝脏以及胎盘的膜制剂结合，解离常数（Kd）为1至2 nM。每微克膜蛋白结合的增殖蛋白量在妊娠期间有显著变化；与成年动物肝脏膜相比，第16天胎儿肝脏膜的最大结合量约为其25倍。甘露糖6 - 磷酸可特异性且完全抑制与胎儿和母体受体的结合，半最大抑制浓度为10 microM。此外，非糖基化的增殖蛋白不抑制糖基化蛋白的结合。使用增殖蛋白亲和柱并用甘露糖6 - 磷酸洗脱，从怀孕小鼠的肝脏中纯化出一种约300 Kd的增殖蛋白受体。该受体与针对大鼠阳离子非依赖性甘露糖6 - 磷酸受体的抗体发生反应。我们得出结论：1）培养细胞分泌的增殖蛋白与阳离子非依赖性甘露糖6 - 磷酸受体结合，因此可能是一种溶酶体蛋白或靶向溶酶体；2）在妊娠期间，小鼠胎儿和母体肝脏以及胎盘中甘露糖6 - 磷酸受体的浓度或活性受到调节。
