Structural Basis and Mechanism of Autoregulation in 3-Phosphoinositide-Dependent Grp1 Family Arf GTPase Exchange Factors

Abstract

Arf GTPases regulate membrane trafficking and actin dynamics. Grp1, ARNO, and Cytohesin-1 comprise a family of phosphoinositide-dependent Arf GTPase exchange factors with a Sec7-pleckstrin homology (PH) domain tandem. Here, we report that the exchange activity of the Sec7 domain is potently autoinhibited by conserved elements proximal to the PH domain. The crystal structure of the Grp1 Sec7-PH tandem reveals a pseudosubstrate mechanism of autoinhibition in which the linker region between domains and a C-terminal amphipathic helix physically block the docking sites for the switch regions of Arf GTPases. Mutations within either element result in partial or complete activation. Critical determinants of autoinhibition also contribute to insulin-stimulated plasma membrane recruitment. Autoinhibition can be largely reversed by binding of active Arf6 to Grp1 and by phosphorylation of tandem PKC sites in Cytohesin-1. These observations suggest that Grp1 family GEFs are autoregulated by mechanisms that depend on plasma membrane recruitment for activation.

Allosteric regulation of Arf GTPases and their GEFs at the membrane interface

“…63 Arf-GTP did not modify the activity of BRAG2, indicating that the PH domain is not involved in a feedback mechanism. 29 This major difference between BRAG and cytohesins is consistent with the observation that the side of the PH domain that binds Arf-GTP in cytohesins 33 is masked by the linker in BRAG2. 29 The origin of the allosteric effect of membranes on BRAG efficiency is best explained by multiple discrete intramolecular, protein-protein and protein-lipid interactions that result in an optimized conformation and orientation of the Arf/BRAG complex at the membrane interface.…”

“…k cat /K m toward Arf1 in solution and on membranes determined using a range of GEF concentrations are given in Table 1 for Sec-PH constructs of representative members of the cytohesin (ref. 33, this study), BRAG 29 and EFA6 49 subfamilies. In contrast to the similarities of these 3 subfamilies in their lipid and Arf specificities, their regulation is unexpectedly different.…”

“…Communication between the membrane and the GEF domain involves little direct contact, if any, and can thus be described in the framework of allostery. It is well established that Sec7 domains have little conformational flexibility, 26,27,[29][30][31][32][33] which makes it unlikely that the GEF activity is regulated by conformational changes at the level of the Sec7 domain. In the following sections, we discuss our current knowledge on the regulatory roles of non-catalytic domains of eukaryotic and bacterial ArfGEFs at the membrane interface.…”

“…Elements proximal to the PH domain (Sec7-PH linker and C-terminal helix/polybasic region) shut down the GEF activity by obstructing the active site of the Sec7 domain. 33 Autoinhibition is released by Arf1-GTP and Arf6-GTP, which bind to the PH domain as effectors 33,59,60 and enable a positive feedback mechanism whereby the activated Arfs modulate the GEF activity of cytohesins 58,61 and enhance their recruitment to membranes. 58 Another activated small GTPase, Arl4-GTP, binds to the PH domain of cytohesins and recruits them to the plasma membrane, although whether this is associated with autoinhibition release is not known.…”

“…58 Another activated small GTPase, Arl4-GTP, binds to the PH domain of cytohesins and recruits them to the plasma membrane, although whether this is associated with autoinhibition release is not known. 62 The phosphoinositide-binding site of cytohesins is fully accessible in their autoinhibited conformation, 33 which suggests that phosphoinositides can recruit cytohesins to membranes regardless of whether or not they are autoinhibited or bound to Arf-GTP. Thus, the PH domain of cytohesins has a dual regulatory role on membranes: it responds to PIP 2 or PIP 3 phosphoinositides signals by recruiting cytohesins to membranes through its canonical phosphoinositidebinding site; and it modulates the shape of the Arf-GTP signal by autoinhibition of the Arf-binding site and its release by membrane-bound Arf-GTP.…”