Department of Pathology, Emory University, Atlanta, GA 30322, USA.
Department of Neuroscience, Cell Biology and Anatomy, University of Texas Medical Branch, Galveston, TX 77555, USA.
G3 (Bethesda). 2022 Jul 6;12(7). doi: 10.1093/g3journal/jkac117.
Integrin plays a crucial role in the attachment of cells to the extracellular matrix. Integrin recruits many proteins intracellularly, including a 4-protein complex (kindlin, ILK, PINCH, and parvin). Caenorhabditis elegans muscle provides an excellent model to study integrin adhesion complexes. In Caenorhabditis elegans, UNC-112 (kindlin) binds to the cytoplasmic tail of PAT-3 (β-integrin) and to PAT-4 (ILK). We previously reported that PAT-4 binding to UNC-112 is essential for the binding of UNC-112 to PAT-3. Although there are crystal structures for ILK and a kindlin, there is no co-crystal structure available. To understand the molecular interaction between PAT-4 and UNC-112, we took a genetic approach. First, using a yeast 2-hybrid method, we isolated mutant PAT-4 proteins that cannot bind to UNC-112 and then isolated suppressor mutant UNC-112 proteins that restore interaction with mutant PAT-4 proteins. Second, we demonstrated that these mutant PAT-4 proteins cannot localize to attachment structures in nematode muscle, but upon co-expression of an UNC-112 suppressor mutant protein, mutant PAT-4 proteins could localize to attachment structures. Third, overexpression of a PAT-4 mutant results in the disorganization of adhesion plaques at muscle cell boundaries and co-expression of the UNC-112 suppressor mutant protein alleviates this defect. Thus, we demonstrate that UNC-112 binding to PAT-4 is required for the localization and function of PAT-4 in integrin adhesion complexes in vivo. The missense mutations were mapped onto homology models of PAT-4 and UNC-112, and taking into account previously isolated mutations, we suggest a surface of PAT-4 that binds to UNC-112.
整合素在细胞与细胞外基质的附着中起着至关重要的作用。整合素在细胞内招募许多蛋白质，包括一个 4 蛋白复合物（kindlin、ILK、PINCH 和 parvin）。秀丽隐杆线虫肌肉为研究整合素粘附复合物提供了一个极好的模型。在秀丽隐杆线虫中，UNC-112（kindlin）与 PAT-3（β-整合素）和 PAT-4（ILK）的细胞质尾巴结合。我们之前报道过，PAT-4 与 UNC-112 的结合对于 UNC-112 与 PAT-3 的结合是必不可少的。虽然有 ILK 和 kindlin 的晶体结构，但没有共晶体结构。为了理解 PAT-4 和 UNC-112 之间的分子相互作用，我们采用了遗传方法。首先，我们使用酵母双杂交方法分离了不能与 UNC-112 结合的突变 PAT-4 蛋白，然后分离了能够恢复与突变 PAT-4 蛋白相互作用的 UNC-112 突变蛋白。其次，我们证明了这些突变的 PAT-4 蛋白不能定位到线虫肌肉的附着结构中，但在共表达 UNC-112 突变体蛋白后，突变的 PAT-4 蛋白可以定位到附着结构中。第三，过表达 PAT-4 突变体导致粘附斑在肌肉细胞边界处的紊乱，而共表达 UNC-112 突变体蛋白可以减轻这种缺陷。因此，我们证明了 UNC-112 与 PAT-4 的结合对于 PAT-4 在整合素粘附复合物中的定位和功能是必需的。错义突变被映射到 PAT-4 和 UNC-112 的同源模型上，并考虑到之前分离的突变，我们提出了一个 PAT-4 与 UNC-112 结合的表面。
