Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.
J Biol Chem. 2011 Jul 8;286(27):24364-73. doi: 10.1074/jbc.M110.172445. Epub 2011 May 17.
The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31.
小分子 GTPase Rab5 循环结合 GDP 处于非活性状态和 GTP 结合的活性状态，在早期内吞途径中的膜出芽和运输中发挥重要作用。Rab5 通过各种液泡蛋白分选 9（VPS9）结构域包含的鸟嘌呤核苷酸交换因子激活。Rab21、Rab22 和 Rab31（Rab5 亚家族的成员）也参与早期内体的运输。控制 Rab5 亚家族成员激活的机制尚不清楚。RIN（Ras 和 Rab 相互作用）代表一个多功能蛋白家族，除了Src 同源 2（SH2）和 Ras 结合结构域外，还具有 VPS9 结构域。我们研究了 RIN 家族成员是否在生化和细胞形态水平上作为 Rab5 亚家族的鸟嘌呤核苷酸交换因子（GEF）起作用。RIN3 在无细胞和细胞 GEF 活性测定中刺激 GTP 结合的 Rab31 的形成。RIN3 还形成扩大的囊泡和管状结构，在 HeLa 细胞中与 Rab31 共定位。相比之下，RIN3 对 Rab21 没有表现出任何明显的影响。我们还发现，RIN3 中 SH2 和 RIN 家族同源结构域之间序列中的丝氨酸到丙氨酸取代特异性地消除了其对 Rab31 的 GEF 作用，但对 Rab5 没有影响。我们检查了 RIN3 是否影响阳离子依赖性甘露糖 6-磷酸受体（CD-MPR）的定位，该受体在高尔基网络和内吞隔室之间运输。我们发现 RIN3 将 CD-MPR 部分从高尔基网络转移到周围囊泡中，这依赖于其 Rab31-GEF 活性。这些结果表明 RIN3 特异性地作为 Rab31 的 GEF 起作用。
