Kim Doo Yeon, Ingano Laura A MacKenzie, Kovacs Dora M
Neurobiology of Disease Laboratory, Genetics and Aging Research Unit, Center for Aging, Genetics and Neurodegeneration, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA.
J Biol Chem. 2002 Dec 20;277(51):49976-81. doi: 10.1074/jbc.M210179200. Epub 2002 Oct 9.
Nectin-1 is a member of the immunoglobulin superfamily and a Ca(2+)-independent adherens junction protein involved in synapse formation. Here we show that nectin-1alpha undergoes intramembrane proteolytic processing analogous to that of the Alzheimer's disease amyloid precursor protein, mediated by a presenilin (PS)-dependent gamma-secretase-like activity. 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of Chinese hamster ovary cells activated a first proteolytic event, resulting in ectodomain shedding of nectin-1alpha. Subsequent cleavage of the remaining 26-kDa membrane-anchored C-terminal fragment (CTF) was inhibited independently by three specific gamma-secretase inhibitors and by expression of the dominant negative form of PS1. The PS/gamma-secretase-like cleavage product was detected in vivo following proteasome inhibitor treatment of cells. An in vitro gamma-secretase assay confirmed the generation of a 24-kDa nectin-1alpha intracellular domain, peripherally associated with the membrane fraction. We also found nectin-1alpha to interact with the N-terminal fragment of PS1. Finally, gamma-secretase inhibition resulted in beta-catenin release from cell junctions, concomitantly with the accumulation of the 26-kDa nectin-1alpha CTF, suggesting that high levels of nectin-1alpha CTF interfere with TPA-induced remodeling of cell-cell junctions. Our results are consistent with a previously reported role for PS/gamma-secretase in adherens junction function involving cleavage of cadherins. Similar to nectin-1, other members of the immunoglobulin superfamily involved in synapse formation may also serve as substrates for PS/gamma-secretase-like intramembrane proteolytic activity.
Nectin-1是免疫球蛋白超家族的成员,是一种参与突触形成的不依赖Ca(2+)的黏附连接蛋白。在此我们表明,nectin-1α经历了类似于阿尔茨海默病淀粉样前体蛋白的膜内蛋白水解过程,这一过程由早老素(PS)依赖的γ-分泌酶样活性介导。用12-O-十四烷酰佛波醇-13-乙酸酯(TPA)处理中国仓鼠卵巢细胞激活了首个蛋白水解事件,导致nectin-1α的胞外域脱落。剩余26 kDa膜锚定C端片段(CTF)的后续切割被三种特异性γ-分泌酶抑制剂以及PS1显性负性形式的表达独立抑制。在蛋白酶体抑制剂处理细胞后,在体内检测到了PS/γ-分泌酶样切割产物。体外γ-分泌酶分析证实生成了一个24 kDa的nectin-1α细胞内结构域,其与膜组分外周相关。我们还发现nectin-1α与PS1的N端片段相互作用。最后,γ-分泌酶抑制导致β-连环蛋白从细胞连接处释放,同时26 kDa的nectin-1α CTF积累,这表明高水平的nectin-1α CTF会干扰TPA诱导的细胞间连接重塑。我们的结果与之前报道的PS/γ-分泌酶在涉及钙黏蛋白切割的黏附连接功能中的作用一致。与nectin-1类似,参与突触形成的免疫球蛋白超家族的其他成员也可能作为PS/γ-分泌酶样膜内蛋白水解活性的底物。