Biosynthesis of a novel transformation-sensitive heat-shock protein that binds to collagen. Regulation by mRNA levels and in vitro synthesis of a functional precursor.
The synthesis of a major collagen-binding glycoprotein of molecular weight 47,000 was previously shown to be altered by malignant transformation as well as by heat shock in chick embryo fibroblasts (Nagata, K., and Yamada, K.M. (1986) J. Biol. Chem. 261, 7531-7536 and Nagata, K., Saga, S., and Yamada, K.M. (1986) J. Cell Biol. 103, 223-229). In this paper, we examined the synthesis of this heat shock protein (hsp47) in terms of possible functional precursors and its regulation after heat shock and transformation by Rous sarcoma virus. Actinomycin D inhibited the induction of hsp47 after heat shock. Messenger RNAs purified from chick embryo fibroblasts (CEF), heat-treated CEF, and transformed CEF were analyzed in an in vitro translation system. In vitro translated products readily bound to gelatin-Sepharose, and levels were increased after heat shock and decreased after transformation. The increase in mRNA after heat shock was shown more directly by Northern assay using a synthetic oligonucleotide probe. We identified two putative precursors of hsp47 using an in vitro translation/processing system and tunicamycin: one is a 42-kDa primary translation product and the second is a 41-kDa polypeptide lacking signal peptide and carbohydrate moieties. Both of these precursors are biologically active as determined by gelatin-binding activity, in contrast to the lack of binding activity of precursors in several other membrane-associated receptor systems.
Nagata
K
K
Membrane Biochemistry Section, National Cancer Institute, Bethesda, Maryland 20892.
Hirayoshi
K
K
Obara
M
M
Saga
S
S
Yamada
K M
KM
D000818:Animals; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D002642:Chick Embryo; D003094:Collagen / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D011498:Protein Precursors / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
D006360:Heat-Shock Proteins; D011498:Protein Precursors; D012333:RNA, Messenger; D014415:Tunicamycin; D003609:Dactinomycin; D003094:Collagen