De Deyne P G, O'Neill A, Resneck W G, Dmytrenko G M, Pumplin D W, Bloch R J
Departments of Physiology, Surgery, Neurology and Anatomy & Neurobiology, School of Medicine, University of Maryland at Baltimore, Baltimore, MD 21201, USA.
J Cell Sci. 1998 Sep;111 ( Pt 18):2729-40. doi: 10.1242/jcs.111.18.2729.
Rat myotubes cultured in fetal calf serum adhere to vitronectin-coated substrates through two distinct structures, focal contacts and clathrin-coated membrane domains. We studied the integrins in myotubes to learn how they associate with these two domains. Double label immunofluorescence studies with antibodies specific for clathrin, vinculin and several forms of integrin showed that focal contacts and clathrin-coated membrane domains contain both vitronectin receptors (VnR, containing beta-3 and beta-5integrins) and fibronectin receptors (FnR, containing beta1-integrin). VnR but not FnR associates tightly with the substrate in both domains, as the VnR alone remains attached to the coverslip when the lipid bilayer and other membrane proteins are removed by detergent. Ultrastructural studies confirmed the localization of the beta5 subunit of the VnR at both domains. We used intracellular injection and affinity chromatography to test the possibility that clathrin at coated membrane domains associates with the cytoplasmic sequence of the beta5 subunit of the VnR. Injection of a synthetic peptide containing the NPXY motif from the cytoplasmic domain of the human beta5 subunit, SRARYEMASNPLYRKPIST, depleted clathrin from coated membrane domains without affecting clathrin in perinuclear structures or vinculin at focal contacts. Injection of the homologous beta1 peptide, MNAKWDTGENPIYKSAVITT, also containing an NPXY motif, had no significant effect on any of these structures. Affinity matrices containing the beta5 but not the beta1 peptide selectively retained clathrin from myotube extract, and bound clathrin could be selectively eluted by soluble forms of the beta5 but not the beta1 peptide. Thus, a sequence including the NPXY motif in the integrin beta5 subunit is involved in the specific anchoring of the VnR, but not the FnR, to clathrin-coated membrane.
在胎牛血清中培养的大鼠肌管通过两种不同结构，即粘着斑和网格蛋白包被膜区，附着于玻连蛋白包被的底物上。我们研究了肌管中的整合素，以了解它们如何与这两个区域相关联。用针对网格蛋白、纽蛋白和几种整合素形式的特异性抗体进行的双标记免疫荧光研究表明，粘着斑和网格蛋白包被膜区都含有玻连蛋白受体（VnR，含β-3和β-5整合素）和纤连蛋白受体（FnR，含β1整合素）。在这两个区域中，VnR而非FnR与底物紧密结合，因为当用去污剂去除脂质双层和其他膜蛋白时，仅VnR仍附着在盖玻片上。超微结构研究证实了VnR的β5亚基在这两个区域的定位。我们使用细胞内注射和亲和层析来测试网格蛋白包被膜区的网格蛋白与VnR的β5亚基的胞质序列相关联的可能性。注射包含人β5亚基胞质结构域的NPXY基序的合成肽SRARYEMASNPLYRKPIST，可使网格蛋白包被膜区的网格蛋白减少，而不影响核周结构中的网格蛋白或粘着斑处的纽蛋白。注射同源的β1肽MNAKWDTGENPIYKSAVITT（也含有NPXY基序）对这些结构均无显著影响。含有β5肽而非β1肽的亲和基质可从肌管提取物中选择性保留网格蛋白，并且结合的网格蛋白可用β5肽的可溶形式而非β1肽选择性洗脱。因此，整合素β5亚基中包含NPXY基序的序列参与了VnR而非FnR与网格蛋白包被膜的特异性锚定。
