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.

Figure 1. Grp1 family Arf GEFs are autoinhibited by the polybasic motif

(A) Representative time courses for NΔ17Arf1 nucleotide exchange catalyzed by Grp1 constructs including (green) or lacking (blue) the polybasic motif. (B) Concentration dependence of the observed rate constant for NΔ17Arf1 nucleotide exchange catalyzed by Grp1 constructs including (green) or lacking (blue) the polybasic motif. (C) Summary of catalytic efficiencies (k_cat/K_m) and oligomeric state for Grp1, ARNO, and Cytohesin-1 constructs and Grp1-Grsp1 complexes. Oligomeric state was determined by sedimentation equilibrium at concentrations of 4.5–37 μM. Values are mean ± s.d. for n = 2.

Figure 2. Structural organization of an autoinhibited Grp1 construct

(A) Ribbon rendering showing the Sec7 domain (blue), linker (red), PH domain (green), and C-terminal helix (orange). Ins(1,3,4,5)P₄ is represented by red spheres (oxygen atoms) and yellow spheres (carbon and phosphate atoms). (B) Annotated sequence alignment of Grp1 family paralogs/homologs.

Figure 3. Pseudosubstrate autoinhibition by the Sec7-PH linker and C-terminal helix

(A) Intramolecular interactions at the interface between the linker and Sec7 domain. (B) Intramolecular interactions at the interface between the C-terminal helix and Sec7 domain. (C and D) Comparison of the linker and C-terminal helix of Grp1 with the switch I and II regions of Arf1-GDP from the complex with the E156K mutant of the ARNO Sec7 domain (PDB ID code 1R8S) after superposition of Cα atoms.

Figure 4. Structure-based mutational analysis of the determinants of autoinhibition

Catalytic efficiencies are plotted on a log scale in units of the catalytic efficiency for the corresponding wild type Sec7-PH-polybasic construct. Mean values and standard deviations were calculated for two independent measurements.

Figure 5. Determinants of plasma membrane targeting in 3T3 L1 adipocytes

(A) Representative examples of the localization of GFP-fusion constructs in serum starved (−) and insulin stimulated (+) cells. (B) Percentage of cells for which plasma membrane targeting was observed. Mean values and standard deviations are plotted for 2 independent experiments. Approximately 100 cells were analyzed for each experiment.

Figure 6. Mechanisms for partial activation of Grp1 family GEFs

(A) Dependence of the observed rate constant for NΔ17Arf1 exchange catalyzed by Grp1_63-399 on the concentration of GppNHp-loaded or GDP-loaded Arf GTPases. Solid lines represent fitted model functions for a 1:1 binding/activation isotherm. (B) Dependence of the observed rate constant for NΔ17Arf1 exchange on the concentration of Grp1_63-399 in the presence and absence of 80 μM Arf6-GppNHp and 1 μM Ins(1,3,4,5)P₄. (C) Catalytic efficiencies for Cyothesin-1 phosphoproteins compared with the R378C reference protein. (D) Dependence of the observed rate constant for NΔ17Arf1 exchange catalyzed by wild type (WT) Cytohesin-1_53-398 or the ³⁹⁴DD³⁹⁵ double mutant on the concentration Arf6 loaded with GppNHp or GDP in the presence of 10 μM Ins(1,3,4,5)P₄. Values and error bars for panels A, C, and D are mean ± s.d. for n = 2.

Figure 7. Model for autoregulation of Grp1 family GEFs

After PtdIns(3,4,5)P₃-dependent plasma membrane recruitment of Grp1 family GEFs, lateral association with Arf6-GTP simultaneously enhances membrane partitioning and shifts the equilibrium towards the catalytically competent conformation. Other mechanisms, including phosphorylation of PKC sites in the polybasic motif of Cytohesin-1, may be required for full activation.