Campa Fanny, Yoon Hye-Young, Ha Vi Luan, Szentpetery Zsofia, Balla Tamas, Randazzo Paul A
Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892.
Program for Developmental Neuroscience, NICHD, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 2009 Oct 9;284(41):28069-28083. doi: 10.1074/jbc.M109.028266. Epub 2009 Aug 7.
ARAP1 is a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3))-dependent Arf GTPase-activating protein (GAP) with five PH domains that regulates endocytic trafficking of the epidermal growth factor receptor (EGFR). Two tandem PH domains are immediately N-terminal of the Arf GAP domain, and one of these fits the consensus sequence for PtdIns(3,4,5)P(3) binding. Here, we tested the hypothesis that PtdIns(3,4,5)P(3)-dependent recruitment mediated by the first PH domain of ARAP1 regulates the in vivo and in vitro function of ARAP1. We found that PH1 of ARAP1 specifically bound to PtdIns(3,4,5)P(3), but with relatively low affinity (approximately 1.6 microm), and the PH domains did not mediate PtdIns(3,4,5)P(3)-dependent recruitment to membranes in cells. However, PtdIns(3,4,5)P(3) binding to the PH domain stimulated GAP activity and was required for in vivo function of ARAP1 as a regulator of endocytic trafficking of the EGFR. Based on these results, we propose a variation on the model for the function of phosphoinositide-binding PH domains. In our model, ARAP1 is recruited to membranes independently of PtdIns(3,4,5)P(3), the subsequent production of which triggers enzymatic activity.
ARAP1是一种依赖磷脂酰肌醇3,4,5-三磷酸（PtdIns(3,4,5)P(3)）的Arf GTP酶激活蛋白（GAP），具有五个PH结构域，可调节表皮生长因子受体（EGFR）的内吞运输。两个串联的PH结构域位于Arf GAP结构域的紧邻N端，其中一个符合PtdIns(3,4,5)P(3)结合的共有序列。在此，我们检验了一个假设，即由ARAP1的第一个PH结构域介导的PtdIns(3,4,5)P(3)依赖性募集调节ARAP1在体内和体外的功能。我们发现ARAP1的PH1特异性结合PtdIns(3,4,5)P(3)，但亲和力相对较低（约1.6微摩尔），并且这些PH结构域不介导细胞中PtdIns(3,4,5)P(3)依赖性向膜的募集。然而，PtdIns(3,4,5)P(3)与PH结构域的结合刺激了GAP活性，并且是ARAP1作为EGFR内吞运输调节剂在体内发挥功能所必需的。基于这些结果，我们提出了一种关于磷酸肌醇结合PH结构域功能模型的变体。在我们的模型中，ARAP1独立于PtdIns(3,4,5)P(3)被募集到膜上，随后PtdIns(3,4,5)P(3)的产生触发酶活性。
