A PH Domain in the Arf GTPase-activating Protein (GAP) ARAP1 Binds Phosphatidylinositol 3,4,5-Trisphosphate and Regulates Arf GAP Activity Independently of Recruitment to the Plasma Membranes

Abstract

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 (≈1.6 M), 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.

PTK6 Inhibits Down-regulation of EGF Receptor through Phosphorylation of ARAP1

“…Another possibility for the regulation of ARAP1 activation via Tyr 231 phosphorylation is through a change of localization of ARAP1. Campa et al (43) has shown that ARAP1 is recruited to the plasma membrane independent of phosphatidylinositol (3,4,5)-triphosphate (PIP 3 ) and that subsequent production of PIP 3 triggers Arf-GAP activity. Thus, we presume that ARAP1 might be phosphorylated by and recruited to PTK6 at the plasma membrane through its SH2 domain, prior to activation of Arf-GAP through the contact of its nearby PH domain with membrane PIP 3 .…”

“…ARAP1 is reported to be an Arf-GAP that preferentially uses Arf5, but can also use Arf1 and Arf6 (30,(43)(44). Arf6 has been implicated in multiple pathways of endocytosis (45).…”

Phosphatidylinositol-4-phosphate 5-Kinase and GEP100/Brag2 Protein Mediate Antiangiogenic Signaling by Semaphorin 3E-Plexin-D1 through Arf6 Protein

“…Large unilamellar vesicles (LUVs) containing various phospholipids were prepared by extrusion with lipids purchased from Avanti Polar Lipids and Echelon Biosciences as described previously (16). They contained molar ratios of 40% phosphatidylcholine, 25% phosphatidylethanolamine, 15% phosphatidylserine, 10% cholesterol, and 10% phosphatidylinositol (PI), or 9.5% PI plus 0.5% of various phosphatidylinositol phosphate (PIP), including PI 3-phosphate, PI 4-phosphate, PI 5-phosphate, PI 3,4-bisphosphate, PI 3,5-bisphosphate, PI(4,5)P 2 , and PI 3,4,5-trisphosphate.…”

Allosteric properties of PH domains in Arf regulatory proteins

“…76,77 All 4 subtypes of Arf GAPs may be allosterically regulated. 23,54,76,78 ASAP1 has been the most extensively examined Arf GAP and will be discussed here.…”

“…53 For the Arf GAPs examined, the PH domains do not have a role in membrane recruitment, but are critical for activity and for regulation of activity. 54,55 Cooperative binding of ligands controls activity of the Arf GAP ASAP1. 23 Figure 2.…”