Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada.
J Biol Chem. 2010 Apr 2;285(14):10627-37. doi: 10.1074/jbc.M109.050930. Epub 2010 Feb 12.
Rabs constitute the largest family of monomeric GTPases, yet for the majority of Rabs relatively little is known about their activation and recruitment to vesicle-trafficking pathways. We recently identified connecdenn (DENND1A), which contains an N-terminal DENN (differentially expressed in neoplastic versus normal cells) domain, a common and evolutionarily ancient protein module. Through its DENN domain, connecdenn functions enzymatically as a guanine-nucleotide exchange factor (GEF) for Rab35. Here we identify two additional connecdenn family members and demonstrate that all connecdenns function as Rab35 GEFs, albeit with different levels of activity. The DENN domain of connecdenn 1 and 2 binds Rab35, whereas connecdenn 3 does not, indicating that Rab35 binding and activation are separable functions. Through their highly divergent C termini, each of the connecdenns binds to clathrin and to the clathrin adaptor AP-2. Interestingly, all three connecdenns use different mechanisms to bind AP-2. Characterization of connecdenn 2 reveals binding to the beta2-ear of AP-2 on a site that overlaps with that used by the autosomal recessive hypercholesterolemia protein and betaarrestin, although the sequence used by connecdenn 2 is unique. Loss of connecdenn 2 function through small interference RNA knockdown results in an enlargement of early endosomes, similar to what is observed upon loss of Rab35 activity. Our studies reveal connecdenn DENN domains as generalized GEFs for Rab35 and identify a new AP-2-binding motif, demonstrating a complex link between the clathrin machinery and Rab35 activation.
Rabs 构成了单体 GTPases 家族中最大的家族，然而对于大多数 Rabs，其激活和招募到囊泡运输途径的机制相对知之甚少。我们最近鉴定了 connecdenn（DENND1A），它包含一个 N 端 DENN（在肿瘤与正常细胞中差异表达）结构域，这是一个常见且古老的蛋白质模块。通过其 DENN 结构域，connecdenn 作为 Rab35 的鸟嘌呤核苷酸交换因子（GEF）发挥酶促作用。在这里，我们鉴定了另外两个 connecdenn 家族成员，并证明所有 connecdenn 都作为 Rab35 GEF 发挥作用，尽管活性水平不同。connecdenn 1 和 2 的 DENN 结构域与 Rab35 结合，而 connecdenn 3 则不结合，这表明 Rab35 的结合和激活是可分离的功能。通过其高度分化的 C 末端，每个 connecdenn 都与网格蛋白和网格蛋白衔接蛋白 AP-2 结合。有趣的是，所有三种 connecdenn 都使用不同的机制与 AP-2 结合。connecdenn 2 的特性揭示了在与常染色体隐性高胆固醇血症蛋白和 betaarrestin 重叠的位点与 AP-2 的 beta2 耳结合，尽管 connecdenn 2 使用的序列是独特的。通过小干扰 RNA 敲低丧失 connecdenn 2 功能会导致早期内体增大，类似于 Rab35 活性丧失时观察到的情况。我们的研究揭示了 connecdenn 的 DENN 结构域作为 Rab35 的通用 GEF，并确定了一个新的 AP-2 结合基序，证明了网格蛋白机制与 Rab35 激活之间的复杂联系。
