Cheresh D A, Spiro R C
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037.
J Biol Chem. 1987 Dec 25;262(36):17703-11.
M21 human melanoma cells express an Arg-Gly-Asp-directed adhesion receptor composed of noncovalently associated alpha and beta chains. To establish the structural and functional properties of this receptor on M21 human melanoma cells, stable variant cell lines were selected that express altered alpha chain levels. One of these variants, M21-L, fails to synthesize alpha chain protein or its mRNA, yet does produce normal levels of the beta chain. In these cells the beta chain does not reach the cell surface but rather accumulates within the cell. M21-L cells lacking the alpha chain are incapable of attaching to vitronectin, von Willebrand factor, fibrinogen, or an Arg-Gly-Asp-containing heptapeptide yet attach normally to fibronectin, whereas the unselected M21 cells attach to all of these adhesive proteins. In addition, a monoclonal antibody, LM609 generated to a functional site on the intact receptor, is capable of preventing M21 cell attachment to vitronectin, von Willebrand factor, fibrinogen, and the Arg-Gly-Asp peptide but not to fibronectin. Following a 2-min biosynthetic pulse-label, the newly synthesized alpha chain remains in free form for 5 min and then associates with previously synthesized beta chain present in an intracellular pool. Once oligomerization takes place, the receptor gains the capacity to recognize Arg-Gly-Asp, and at this time the epitope recognized by monoclonal antibody LM609 is formed.
M21人黑素瘤细胞表达一种由非共价结合的α链和β链组成的、以精氨酸 - 甘氨酸 - 天冬氨酸(Arg - Gly - Asp)为导向的黏附受体。为了确定该受体在M21人黑素瘤细胞上的结构和功能特性,我们筛选了表达水平改变的α链的稳定变异细胞系。其中一个变异体M21 - L无法合成α链蛋白或其mRNA,但能产生正常水平的β链。在这些细胞中,β链无法到达细胞表面,而是在细胞内积累。缺乏α链的M21 - L细胞无法附着于玻连蛋白、血管性血友病因子、纤维蛋白原或含Arg - Gly - Asp的七肽,但能正常附着于纤连蛋白,而未筛选的M21细胞能附着于所有这些黏附蛋白。此外,针对完整受体上一个功能位点产生的单克隆抗体LM609能够阻止M21细胞附着于玻连蛋白、血管性血友病因子、纤维蛋白原和Arg - Gly - Asp肽,但不能阻止其附着于纤连蛋白。经过2分钟的生物合成脉冲标记后,新合成的α链以游离形式存在5分钟,然后与细胞内池中先前合成的β链结合。一旦发生寡聚化,受体就获得了识别Arg - Gly - Asp的能力,此时单克隆抗体LM609识别的表位形成。