Selective stimulation by mitogens of incorporation of 35S-methionine into a family of proteins released into the medium by 3T3 cells

Serum and three mitogens for mouse embryo 3T3 cells—fibroblast growth factor from brain, fibroblast growth factor from pituitary, and epidermal growth factor—specifically stimulate the synthesis and release into the medium by these cells of a group of proteins that travel together on SDS gel electrophoresis and that are detected by ³⁵S-methionine labeling. These proteins, designated mitogen-releasable proteins (MRPs), have a median, monomer molecular weight on SDS polyacrylamide gel electrophoresis of 34,000 daltons (30,000–38,000 daltons). Our evidence indicates that these proteins comprise a family of glycoproteins, probably with a common polypeptide backbone. The observations supporting this conclusion are that MRPs give a diffuse pattern of bands upon SDS gel electrophoresis; travel as a single, diffuse band when resolved by electrophoresis in the absence of SDS; adsorb to a pea-lectin-sepharose column and can be eluted with α-methyl mannose; and can be labeled metabolically with ³H-mannose. In addition, in the presence of tunicamycin, MRPs are not made—instead, a smaller molecular weight (22,000 dalton), and apparently homogeneous, protein appears. We believe this 22,000 dalton protein to be the unglycosylated form of MRP. Further support for this idea comes from our observation that treatment of MRPs with endoglycosidase H produces a protein with a molecular weight slightly greater than 22,000 daltons. The effect of mitogens on DNA synthesis and MRP release are correlated in the following ways. First, serum factors are required for both responses. Second, in 3T3 cells transformed by SV40, Moloney and Kirsten viruses that do not synthesize DNA in response to FGF, MRPs are not released in response to FGF. Third, in untransformed 3T3 cells, the dose-response curves for fibroblast growth factor on MRP release and thymidine incorporation are closely correlated. Fourth, insulin, a poor mitogen for 3T3 cells, does not enhance MRP release. Fifth, stimulation of MRP release by epidermal growth factor or fibroblast growth factor is inhibited by hydroxyurea and butyrate, both inhibitors of DNA synthesis in these cells. Sixth, if the mitogen is removed at any time during the 20 hr preincubation period, the effect on MRP release observed between 20 and 24 hr is severely diminished.