Complexin 2 Modulates Vesicle-associated Membrane Protein (VAMP) 2-regulated Zymogen Granule Exocytosis in Pancreatic Acini
Complexin 2 Modulates Vesicle-associated Membrane Protein (VAMP) 2-regulated Zymogen Granule Exocytosis in Pancreatic Acini
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DOI：
10.1074/jbc.m110.146597
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发表时间：
2010-11-12
影响因子：
4.8
通讯作者：
Groblewski, Guy E.
中科院分区：
文献类型：
--
作者：
Falkowski, Michelle A.;Thomas, Diana D. H.;Groblewski, Guy E.
复合蛋白是调节囊泡融合所必需的可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）复合物的活性的可溶性蛋白质。神经元特异性复合蛋白1通过夹持融合前状态的反式SNARE复合物并促进构象变化以促进细胞刺激后的膜融合，对胞吐具有抑制和刺激作用。复合蛋白不能结合单体SNARE蛋白，但以高亲和力结合三元SNARE复合物，并以较低亲和力结合靶SNARE复合物。对于复合蛋白在神经系统之外的功能，人们了解得少得多。RT-PCR和免疫印迹法检测胰腺腺泡表达复合蛋白2。免疫荧光显微镜显示复合蛋白2本地化沿着顶端质膜分泌的作用一致。因此，发现复合蛋白2与囊泡相关膜蛋白（VAMP）2、突触融合蛋白3和4相互作用，但不与VAMP 8或突触融合蛋白2相互作用。将重组复合蛋白2引入透性腺泡中以浓度依赖性方式抑制Ca 2+刺激的分泌，最大抑制率接近50%。中心α-螺旋结构域的突变减少了复合蛋白2 SNARE结合，同时消除了其抑制活性。令人惊讶的是，精氨酸59突变为组氨酸内的中央α-螺旋结构域并没有改变SNARE结合，而且，增加了130%的控制Ca 2+刺激分泌。与生化研究一致，胆囊收缩素-8刺激后，复合蛋白2与VAMP 2沿着顶端质膜共定位。这些数据表明复合蛋白2在神经系统外的功能作用，并表明它参与了酶原颗粒胞吐的Ca 2+敏感的调节途径。
Complexins are soluble proteins that regulate the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion. Neuronal specific complexin 1 has inhibitory and stimulatory effects on exocytosis by clamping trans-SNARE complexes in a prefusion state and promoting conformational changes to facilitate membrane fusion following cell stimulation. Complexins are unable to bind to monomeric SNARE proteins but bind with high affinity to ternary SNARE complexes and with lower affinity to target SNARE complexes. Far less is understood about complexin function outside the nervous system. Pancreatic acini express the complexin 2 isoform by RT-PCR and immunoblotting. Immunofluorescence microscopy revealed complexin 2 localized along the apical plasma membrane consistent with a role in secretion. Accordingly, complexin 2 was found to interact with vesicle-associated membrane protein (VAMP) 2, syntaxins 3 and 4, but not with VAMP 8 or syntaxin 2. Introduction of recombinant complexin 2 into permeabilized acini inhibited Ca2+-stimulated secretion in a concentration-dependent manner with a maximal inhibition of nearly 50%. Mutations of the central alpha-helical domain reduced complexin 2 SNARE binding and concurrently abolished its inhibitory activity. Surprisingly, mutation of arginine 59 to histidine within the central alpha-helical domain did not alter SNARE binding and moreover, augmented Ca2+-stimulated secretion by 130% of control. Consistent with biochemical studies, complexin 2 colocalized with VAMP 2 along the apical plasma membrane following cholecystokinin-8 stimulation. These data demonstrate a functional role for complexin 2 outside the nervous system and indicate that it participates in the Ca2+-sensitive regulatory pathway for zymogen granule exocytosis.