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

鸡胚成纤维细胞用s -蛋氨酸标记，用Nonidet P-40裂解。细胞裂解液采用明胶- sepharose亲和层析，结合部分采用SDS-PAGE进行分析。除了纤维连接蛋白外，一个47kDa的条带被证明与明胶结合。我们制备了47kDa蛋白的多克隆和单克隆抗体，并研究了该蛋白在细胞内的定位。47kDa蛋白被证明定位于成纤维细胞的内质网。在之前的研究中，Kurkinen等报道了这种被他们称为“胶原蛋白”的蛋白定位于细胞表面，但我们的观察表明47kDa蛋白并不是细胞表面的胶原受体。然而，这个4mkDa蛋白是有趣的，因为该蛋白由热休克诱导，并通过劳斯肉瘤病毒的恶性转化而减少。我们利用体外翻译系统证实了47kDa蛋白的表达在mRNA转录水平上受到调控，并鉴定了两种可能的47kDa蛋白前体；一种是42kDa多肽，含信号肽，另一种是41kDa多肽，不含信号肽。我们现在正在进行编码47kDa蛋白的cdna的克隆研究。另一方面，我们正在尝试用I型和IV型胶原亲和层析的方法，利用表面标记的小鼠黑色素瘤细胞系鉴定胶原受体。

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.