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，用诺尼特P-40裂解。将细胞裂解液应用于明胶-琼脂糖亲和层析，并在SDS-PAGE上分析结合组分。除了纤维连接蛋白，还有一条47 kDa的条带与明胶结合。我们制备了抗47 kDa蛋白的多克隆和单抗，并对该蛋白在细胞内的定位进行了研究。47 kDa蛋白定位于成纤维细胞的内质网。在之前的一项研究中，Kurkinen等人。报道称，这种蛋白质，他们称之为“胶原素”，定位于细胞表面，但我们的观察表明，47 kDa的蛋白质并不是细胞表面的胶原受体。然而，这个4mkDa的蛋白质很有趣，因为它是由热休克诱导的，并通过Rous肉瘤病毒的恶性转化而减少。我们利用体外翻译系统在转录水平上调控47 kDa蛋白的表达，并鉴定出两种可能的47 kDa蛋白前体：一种是含有信号肽的42 kDa多肽，另一种是不含信号肽的41 kDa多肽。我们正在进行编码47 kDa蛋白的cDNA的克隆研究，另一方面，我们正在尝试利用表面标记的小鼠黑色素瘤细胞株，通过I型和IV型胶原亲和层析的方法来鉴定胶原受体(S)。

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.