ABSTRACT
Mitogen-regulated protein (MRP), a heterogeneously glycosylated mouse protein of Mr 34,000, is in the same protein family as prolactin, growth hormone, and placental lactogen. We show here that the level of translatable MRP mRNA is increased in response to fibroblast growth factor. Also, the amount of MRP secreted by 3T3 cells is modulated by the rate of degradation of newly synthesized MRP in the lysosomes. This is indicated by several results. First, agents that inhibit protein degradation by lysosomal proteases selectively increased by 2- to 6-fold the incorporation of [35S]methionine into MRP. These agents are ammonium chloride, the carboxylic ionophores, monensin and nigericin, and two thiol protease inhibitors, leupeptin and antipain. MRP that has already been secreted is not degraded by 3T3 cells. We examined the developmental appearance of MRP using immunofluorescence microscopy and found MRP localized in the mouse placenta between days 9 and 13 of development. The amount of MRP in the placenta drops suddenly after day 13. Whereas the appearance of MRP in the placenta follows the reported appearance of its mRNA, MRP disappears from the placenta more rapidly than its mRNA. On the basis of the results of our studies with cells in culture we propose that the production of MRP in the placenta is regulated similarly to prolactin. Thus we propose that the initial increase in MRP production in the placenta is due to pretranslational regulation by growth factors, and the later rapid decline is due to posttranslational regulation through degradation in the lysosomes.
Subject(s)
Epidermal Growth Factor/pharmacology , Fibroblast Growth Factors/pharmacology , Glycoproteins/biosynthesis , Placenta/metabolism , Ammonium Chloride/pharmacology , Animals , Antipain/pharmacology , Cell Line , Glycoproteins/genetics , Glycoproteins/metabolism , Glycosylation , Intercellular Signaling Peptides and Proteins , Kinetics , Leupeptins/pharmacology , Mice , Monensin/pharmacology , Placenta/drug effects , Prolactin , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Mitogen-regulated protein (MRP) is a glycoprotein secreted by Swiss murine 3T3 cells whose levels are increased 63-fold or more over the controls by growth factors. The sequence of a 226-bp MRP cDNA clone showed that a region close to the C terminus of MRP is identical to a sequence found in the cDNA-encoding proliferin (PLF). PLF, cloned from Balb/c 3T3 cells, is a member of the prolactin/growth-hormone family. Here we show that MRP and PLF are also antigenically identical. Antiserum raised against purified MRP specifically immunoprecipitated PLF secreted by CV-1 cells that had been transfected with PLF cDNA in an SV40 vector. Also, fibroblast growth factor (FGF) specifically increased the amount of PLF poly(A)+ RNA in Swiss 3T3 cells. We have previously shown that FGF increases the amount of MRP and MRP mRNA synthesized by the same cells. The anti-MRP antiserum recognized both unglycosylated and glycosylated forms of MRP and PLF. The unglycosylated and glycosylated forms of PLF had the same Mr values as those of the unglycosylated (21,500) and glycosylated (34,000) forms of MRP. However, the anti-MRP antiserum did not recognize mouse prolactin and anti-mouse prolactin antibody did not recognize MRP. Evidently, MRP/PLF is an immunologically distinct member of the prolactin/growth-hormone family of secreted, intercellular regulators.
