Volume 28, Issue 4, Pages 569-583 (November 2007) Structural Basis and Mechanism of Autoregulation in 3-Phosphoinositide-Dependent Grp1 Family Arf GTPase Exchange Factors  Jonathan P. DiNitto, Anna Delprato, Meng-Tse Gabe Lee, Thomas C. Cronin, Shaohui Huang, Adilson Guilherme, Michael P. Czech, David G. Lambright  Molecular Cell  Volume 28, Issue 4, Pages 569-583 (November 2007) DOI: 10.1016/j.molcel.2007.09.017 Copyright © 2007 Elsevier Inc. Terms and Conditions

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 (kcat/Km) 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 ± deviation for n = 2. Molecular Cell 2007 28, 569-583DOI: (10.1016/j.molcel.2007.09.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

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. Molecular Cell 2007 28, 569-583DOI: (10.1016/j.molcel.2007.09.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

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. Molecular Cell 2007 28, 569-583DOI: (10.1016/j.molcel.2007.09.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

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 deviations were calculated for two independent measurements. Molecular Cell 2007 28, 569-583DOI: (10.1016/j.molcel.2007.09.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

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 deviations are plotted for two independent experiments. Approximately 100 cells were analyzed for each experiment. Molecular Cell 2007 28, 569-583DOI: (10.1016/j.molcel.2007.09.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Mechanisms for Partial Activation of Grp1 Family GEFs (A) Dependence of the observed rate constant for NΔ17Arf1 exchange catalyzed by Grp163–399 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 Grp163–399 in the presence and absence of 80 μM Arf6-GppNHp and 1 μM Ins(1,3,4,5)P4. (C) Catalytic efficiencies for Cytohesin-1 phosphoproteins compared with the R378C reference protein. (D) Dependence of the observed rate constant for NΔ17Arf1 exchange catalyzed by wild-type (WT) Cytohesin-153–398 or the 394DD395 double mutant on the concentration of 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 ± deviation for n = 2. Molecular Cell 2007 28, 569-583DOI: (10.1016/j.molcel.2007.09.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

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 toward the catalytically competent conformation. Other mechanisms, including phosphorylation of PKC sites in the polybasic motif of Cytohesin-1, may be required for full activation. Molecular Cell 2007 28, 569-583DOI: (10.1016/j.molcel.2007.09.017) Copyright © 2007 Elsevier Inc. Terms and Conditions