Biochemical and molecular biological research for the function and regultion of a collagen receptor

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Chick embryo fibroblasts were labeled with ^<35>S-methionine, and lysed with Nonidet P-40. The cell lysate was applied to gelatin-Sepharose affinity chromatography, and the bound fraction was analysed on SDS-PAGE. In addition to fibronectin, a 47kDa band was shown to bind to gelatin. We prepared polyclonal and monoclonal antibodies against the 47kDa protein, and investigated the intracelular location of the protein. The 47kDa protein was shown to be localized in the endoplasmic reticulumn of the fibroblasts. In a previous study, Kurkinen et al. reported that this protein, which they termed "colligin", was localized on the cell surface, but our observation demonstrated the 47kDa protein was not the collagen receptor on the cell surface. However, this 4mkDa protein is interesting because the protein is induced by heat shock, and reduced by malignant transformation by Rous sarcoma virus. We demonstrated that the expression of the 47kDa protein is regulated at the level of transcription of mRNA by using in vitro translation system, and identified two possible precursors of 47kDa protein; one is 42kDa polypeptide with signal peptides and the other is 41kDa polypeptide without signal peptide. We are now performing the cloning study of the cDNAs which code the 47kDa protein.On the other hand, we are trying the identification of collagen receptor(s) using surface labeled mouse melanoma cell line by the methods of affinity chromtography of type I and type IV collagens.