Caveolin-1 functions as a novel Cdc42 guanine nucleotide dissociation inhibitor in pancreatic β-cells
Caveolin-1 functions as a novel Cdc42 guanine nucleotide dissociation inhibitor in pancreatic β-cells
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DOI：
10.1074/jbc.m603604200
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发表时间：
2006-07-14
影响因子：
4.8
通讯作者：
Thurmond, Debbie C.
中科院分区：
文献类型：
--
作者：
Nevins, Angela K.;Thurmond, Debbie C.
小Rho家族GTPase Cdc42的循环是胰岛素颗粒胞吐所必需的，尽管参与胰腺β细胞Cdc42循环的调节蛋白尚不清楚。在这里，我们证明了小泡蛋白caveolin-1 （Cav-1）是β细胞中的cdc42结合蛋白。Cav-1与cdc42 - vamp2结合颗粒在基础条件下存在于质膜附近。然而，葡萄糖刺激诱导Cav-1从Cdc42- VAMP2复合物中分离，与Cdc42激活的时间一致。对Cav-1支架结构域的分析揭示了鸟嘌呤核苷酸解离抑制剂（GDI）中保守的基序，这表明Cav-1在β细胞中作为Cdc42 GDI具有新的作用。1)体外结合分析表明Cav-1与Cdc42之间存在直接相互作用；2) GST-Cdc42相互作用实验显示，Cav-1与GTP-Cdc42的结合优于GTP-Cdc42；3) Cav-1耗竭研究导致在缺乏刺激的情况下不适当地诱导40%的活化Cdc42，并且MIN6细胞和胰岛的基础胰岛素释放增加40%。在Cav-1缺失的细胞中，野生型Cav-1的表达恢复了基础水平的正常分泌，而Cav-1的脚手架结构域突变体的表达未能使分泌正常化。综上所述，这些数据表明Cav-1在β细胞中作为Cdc42 GDI发挥作用，在缺乏刺激的情况下维持Cdc42处于非活性状态并调节基础分泌。通过与cdc42 - vamp2结合的胰岛素颗粒复合物的相互作用，Cav-1可能有助于颗粒特异性靶向由小泡组织的胞吐作用的“活性位点”。
The cycling of the small Rho family GTPase Cdc42 is required for insulin granule exocytosis, although the regulatory proteins involved in Cdc42 cycling in pancreatic beta-cells are unknown. Here we demonstrate that the caveolar protein caveolin-1 (Cav-1) is a Cdc42-binding protein in beta-cells. Cav-1 associated with Cdc42-VAMP2-bound granules present near the plasma membrane under basal conditions. However, stimulation with glucose induced the dissociation of Cav-1 from Cdc42- VAMP2 complexes, coordinate with the timing of Cdc42 activation. Analyses of the Cav-1 scaffolding domain revealed a motif conserved in guanine nucleotide dissociation inhibitors (GDIs), which suggested a novel role for Cav-1 as a Cdc42 GDI in beta-cells. The novel role was further supported by: 1) in vitro binding analyses that demonstrated a direct interaction between Cav-1 and Cdc42; 2) GST-Cdc42 interaction assays showing preferential Cav-1 binding to GDP-Cdc42 over that of GTP-Cdc42;3) Cav-1 depletion studies resulting in an inappropriate 40% induction of activated Cdc42 in the absence of stimuli and also a 40% increase in basal insulin release from both MIN6 cells and islets. Expression of wild-type Cav-1 in Cav-1-depleted cells restored basal level secretion to normal, whereas expression of a scaffolding domain mutant of Cav-1 failed to normalize secretion. Taken together, these data suggest that Cav-1 functions as a Cdc42 GDI in beta-cells, maintaining Cdc42 in an inactive state and regulating basal secretion in the absence of stimuli. Through its interaction with the Cdc42-VAMP2-bound insulin granule complex, Cav-1 may contribute to the specific targeting of granules to "active sites" of exocytosis organized by caveolae.