Noy Peter J, Yang Jing, Reyat Jasmeet S, Matthews Alexandra L, Charlton Alice E, Furmston Joanna, Rogers David A, Rainger G Ed, Tomlinson Michael G
From the School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom and.
School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom.
J Biol Chem. 2016 Feb 12;291(7):3145-57. doi: 10.1074/jbc.M115.703058. Epub 2015 Dec 14.
A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitously expressed transmembrane metalloprotease that cleaves the extracellular regions from its transmembrane substrates. ADAM10 is essential for embryonic development and is implicated in cancer, Alzheimer, and inflammatory diseases. The tetraspanins are a superfamily of 33 four-transmembrane proteins in mammals, of which the TspanC8 subgroup (Tspan5, 10, 14, 15, 17, and 33) promote ADAM10 intracellular trafficking and enzymatic maturation. However, the interaction between TspanC8s and ADAM10 has only been demonstrated in overexpression systems and the interaction mechanism remains undefined. To address these issues, an antibody was developed to Tspan14, which was used to show co-immunoprecipitation of Tspan14 with ADAM10 in primary human cells. Chimeric Tspan14 constructs demonstrated that the large extracellular loop of Tspan14 mediated its co-immunoprecipitation with ADAM10, and promoted ADAM10 maturation and trafficking to the cell surface. Chimeric ADAM10 constructs showed that membrane-proximal stalk, cysteine-rich, and disintegrin domains of ADAM10 mediated its co-immunoprecipitation with Tspan14 and other TspanC8s. This TspanC8-interacting region was required for ADAM10 exit from the endoplasmic reticulum. Truncated ADAM10 constructs revealed differential TspanC8 binding requirements for the stalk, cysteine-rich, and disintegrin domains. Moreover, Tspan15 was the only TspanC8 to promote cleavage of the ADAM10 substrate N-cadherin, whereas Tspan14 was unique in reducing cleavage of the platelet collagen receptor GPVI. These findings suggest that ADAM10 may adopt distinct conformations in complex with different TspanC8s, which could impact on substrate selectivity. Furthermore, this study identifies regions of TspanC8s and ADAM10 for potential interaction-disrupting therapeutic targeting.
解整合素金属蛋白酶10（ADAM10）是一种广泛表达的跨膜金属蛋白酶，可从其跨膜底物上切割细胞外区域。ADAM10对胚胎发育至关重要，并与癌症、阿尔茨海默病和炎症性疾病有关。四跨膜蛋白是哺乳动物中一个由33种四跨膜蛋白组成的超家族，其中TspanC8亚组（Tspan5、10、14、15、17和33）促进ADAM10的细胞内运输和酶促成熟。然而，TspanC8与ADAM10之间的相互作用仅在过表达系统中得到证实，其相互作用机制仍不明确。为了解决这些问题，研究人员开发了一种针对Tspan14的抗体，用于在原代人类细胞中显示Tspan14与ADAM10的共免疫沉淀。嵌合Tspan14构建体表明，Tspan14的大细胞外环介导了其与ADAM10的共免疫沉淀，并促进了ADAM10的成熟和向细胞表面的运输。嵌合ADAM10构建体表明，ADAM10的膜近端柄、富含半胱氨酸和去整合素结构域介导了其与Tspan14和其他TspanC8的共免疫沉淀。ADAM10从内质网中排出需要这个TspanC8相互作用区域。截短的ADAM10构建体揭示了柄、富含半胱氨酸和去整合素结构域对TspanC8结合的不同要求。此外，Tspan15是唯一能促进ADAM10底物N-钙黏蛋白切割的TspanC8，而Tspan14在减少血小板胶原受体GPVI切割方面具有独特性。这些发现表明，ADAM10与不同的TspanC8结合时可能会采取不同的构象，这可能会影响底物选择性。此外，本研究确定了TspanC8和ADAM10中可能用于破坏相互作用的治疗靶点区域。
