Mitogen-regulated protein (MRP) is secreted by certain immortal murine cell lines (Swiss 3T3, BNL) stimulated with serum or particular growth factors. We have identified a cDNA clone that encodes part of the protein and have confirmed that MRP is closely related to, if not identical to, the prolactin-related protein designated proliferin. MRP is not produced by primary mouse embryo fibroblasts to nearly the same extent as it is produced by many immortal or transformed lines. Control of expression of this protein by growth factors is achieved both by regulating the extent of transcription and by regulating the processing of the protein.Mitogen-regulated protein (MRP) was described by Nilsen-Hamilton et al. (12) (Mr, 30,000 to 38,000) released by Swiss mouse 3T3 cells some 18 to 28 h after stimulation with growth factors (serum, fibroblast growth factor, epidermal growth factor); platelet-derived growth factor and the tumor promoter 12-tetra-decanoylphorbol-13-acetate were shown subsequently to be effective at stimulating release also. Evidence for glycosylation includes the facts that treatment with endoglycosidase H reduces the apparent molecular weight substantially and that the protein synthesized in the presence of tunicamycin has a lower Mr of about 22,000. An antiserum raised against purified MRP has been used to identify the protein unambiguously.Not only mitogens, but also substances that increase intralysosomal pH (NH4CI, chloroquine, and the ionophores monensin and nigericin) enhance the production of MRP (14). This suggests that MRP levels are, in part, regulated at the level of protein degradation, since these latter substances inhibit degradation of lysosomal proteins. In agreement with this is the observation that the ionophore-stimulated release of MRP is not blocked by actinomycin D, in contrast to growth factor-induced release (M. Nilsen-Hamilton and R. T. Hamilton, unpublished data). Proteins known to be secreted by fibroblasts include plasminogen activator, collagenase, fibronectin, procollagen, and major excreted protein (13). Proteins for which there is evidence (in other cell types) that the secreted quantities are regulated by intracellular degradation include prolactin, parathormone, and procollagen (2).We have used an antiserum raised against MRP (R. T. Hamilton and M. Nilsen-Hamilton unpublished data) to screen a Xgtll expression library (18) for clones able to code for one or more epitopes found on MRP. Mouse BNL cells (15) were used as the source of poly(A) mRNA for cloning, since among the cell lines tested it produced the most MRP.
* Corresponding author.To provide initial enrichment for poly(A) mRNA molecules coding for MRP, the cytoplasmic poly(A) mRNA extracted from a growing population of mouse BNL cells was fractionated by velocity sedimentation in a sucrose gradient containing formamide. We recovered the RNA in separate pools and examined it by in vitro translation and immunoprecipitation to identify the fraction most enriched for MRP mRNA. The cDNA synthesized from that fra...