The Phosphatidylinositol (3,4,5)-Trisphosphate-dependent Rac Exchanger 1·Ras-related C3 Botulinum Toxin Substrate 1 (P-Rex1·Rac1) Complex Reveals the Basis of Rac1 Activation in Breast Cancer Cells

Abstract

The P-Rex (phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of the Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion, and metastasis in several human cancers. The family is unique among Rho GEFs, as their activity is regulated by the synergistic binding of PIP3 and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1·Rac1 interface revealed a critical role for this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. The structural data indicated that the PIP3/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP3 and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site.

Keywords: G protein-coupled receptor (GPCR); P-Rex1; PREX1; PREX2; Ras-related C3 botulinum toxin substrate 1 (Rac1); Rho (Rho GTPase); breast cancer; crystallography; guanine nucleotide exchange factor (GEF); receptor tyrosine kinase.

FIGURE 1.

Structure of the P-Rex1·Rac1 complex. A, schematic representation of the domain layout of P-Rex1, highlighting the position of the DH (yellow) and PH (purple) domains. Rac1 is shown in teal. B, the structure of P-Rex1-(34–399) bound to Rac1-(1–176) shown schematically. The position of the missing β3-β4 loop within the P-Rex1 PH domain is indicated.

FIGURE 2.

The P-Rex1·Rac1 interface. A, the P-Rex1 DH domain helices α1, α5, and α6 interact with the Rac1 switch 1 and 2 regions. Rac1 has been rotated 180° about the y axis relative to P-Rex1 to show the interface between each protein. Interacting residues involved in switch 1 binding (blue) and switch 2 binding (orange) are highlighted as spheres. B, 2F_o − F_c electron density map contoured at 1.0 σ, highlighting interactions between P-Rex1 (gray) and the Rac1 switch 1 region (blue). C, structural alignment of P-Rex1-bound Rac1 with Rac1 bound to GDP and Mg²⁺ (gray; PDB code 1I4D (64), with an r.m.s. deviation of 1.008 Å over 169 residues). Conformational changes in the Rac1 switch 1 (blue) and switch 2 (orange) regions occur upon P-Rex1 binding, whereas the conformation of the P-loop (green) is unchanged. Residues within the Rac1 switch regions that undergo large conformational changes upon P-Rex1 binding are displayed as sticks.

FIGURE 3.

Structural basis of Rac1 activation by P-Rex1. A, location of the Rac1 switch 1 and switch 2 interaction interfaces within the P-Rex1·Rac1 structure. B, P-Rex1 DH domain residues Glu-56 and Gln-197 interact with the switch 1 region of Rac1. C, P-Rex1 DH domain residues Lys-201, Asn-238, Arg-242, and Glu-245 mediate interactions with the switch 2 region of Rac1. D, multiple sequence alignment of P-Rex1 family members with related GEFs. The P-Rex1 secondary structure is illustrated above the alignment with residues targeted for mutational analysis indicated. E, the rate of in vitro GEF activity of the P-Rex1 DH-PH domain was significantly decreased for E56R, E56A, and Q197A mutants. F, the rate of in vitro GEF activity of the P-Rex1 DH-PH domain was significantly decreased for R242A, K201A, and N238A, but not E245R, mutants. Curves show the average of at least three independent experiments. G, GEF activity at 60 min following P-Rex1 DH-PH addition. H, impaired interactions between wild-type His₆-Rac1 and untagged P-Rex1-(1–404) mutants compared with the wild-type control. Error bars indicate means ± S.E. ***, p < 0.001, one-way ANOVA with Sidak's multiple comparison test. The activity of the mutants is expressed relative to the wild-type P-Rex1 DH-PH domain. Binding was assessed by pulldown assays using His₆-Rac1 bound to Ni-NTA-agarose beads. Purified untagged wild-type P-Rex1 and the indicated P-Rex1 mutants were added to the pulldown assay at a concentration of 1 μm.

FIGURE 4.

EGFR, CXCR4, and βAR require P-Rex1 for Rac1 activation in breast cancer cells. A, Rac1 activity in MCF7 cells following the addition of 100 nm isoprenaline, 30 nm SDF1α, 10 ng/ml EGF, or vehicle (0.13% (w/v) BSA in PBS) (406–526 cells). The FRET response for each cell is expressed relative to the maximal FRET change (F/F_max). B, representative ratiometric pseudocolor images of MCF7 cells expressing RaichuEV-Rac1 at baseline (0 min) following stimulation with ligands and the maximal FRET response to the positive control (Positive). C, effect of endogenous P-Rex1 knockdown on the Rac1 response in MCF7 cells (26–101 cells). AUC, area under the curve. D, expression of endogenous P-Rex1 in MCF7 cells is knocked down by P-Rex1 siRNA but not by a scrambled siRNA control. E, effect of P-Rex1 overexpression on the Rac1 response in MDA-MB-231 cells (307–484 cells). F, P-Rex1 is not endogenously expressed in MDA-MB-231 cells but can be expressed following transfection. Error bars indicate means ± S.E. ***, p < 0.001 versus vehicle control; ^∧∧, p < 0.01; and ^∧∧∧, p < 0.001 versus scrambled siRNA controls, two-way ANOVA with Dunnett's multiple comparison test.

FIGURE 5.

The P-Rex1 DH domain activates Rac1 in human breast cancer cells. Transfection with HA-tagged full-length P-Rex1 wild-type (WT) or point mutants induces P-Rex1 overexpression in MCF7 (A) and MDA-MB-231 (B) cells. P-Rex1-dependent Rac1 activity is shown following the addition of vehicle (0.13% (w/v) BSA in PBS), 100 nm isoprenaline, 30 nm SDF1α, or 10 ng/ml EGF in MCF7 cells transfected with wild-type P-Rex1 (347–446 cells) (C), P-Rex1 Q197A (142–206 cells) (D), P-Rex1 R242A (144–287 cells) (E), P-Rex1 K201A (131–155 cells) (F), P-Rex1 R242A/Q197A (94–114 cells) (G), or P-Rex1 E56A/N238A (51–98 cells) (H). I, effect of overexpression of P-Rex1 mutants on Rac1 activity in MCF7 cells (51–556 cells). J, effect of overexpression of P-Rex1 mutants on Rac1 activity in MDA-MB-231 cells (30–484 cells). AUC, area under the curve. Bars/symbols represent means, and error bars = S.E. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 versus vehicle control; ^, p < 0.05; ^^, p < 0.01; and ^^^, p < 0.001 versus wild-type P-Rex1, two-way ANOVA with Dunnett's multiple comparison test.

FIGURE 6.

Structural insights into P-Rex1·Gβγ and P-Rex1·PIP₃ binding. A, alignment of the PH domain of P-Rex1 with the GRK2 PH·Gβγ complex (green; PDB code 1OMW (34), with an r.m.s. deviation of 2.046 Å across 93 residues) positions the Gβγ heterodimer on the opposite side of P-Rex1 from Rac1. In the alignment, Gβγ contacts both the P-Rex1 DH and PH domains. Inset, shows PIP₃ modeled by structural alignment of the P-Rex1 PH domain with the CENTA1 PH·PIP₃ complex (purple; PDB code 3LJU (65), with an r.m.s. deviation of 1.815 Å over 86 residues). B, electrostatic surface of P-Rex1 in the same orientation as shown in A, highlighting a highly negatively charged surface patch on P-Rex1 at the site of Gβγ binding and a highly positively charged pocket, where modeling places the negatively charged PIP₃ head group. C and D, the membrane-interacting regions of each component of the P-Rex1·Rac1·Gβγ tetramer are located on the same face of the complex (C), including the C terminus of Rac1 extending from Val-176 (orange), the PIP₃-binding pocket of the P-Rex1 PH domain, and the C-terminal Cys-68 of Gγ (blue) (D). PIP₃ (green) shown as modeled in A. Val-176 of Rac1 and Cys-68 of Gγ are highlighted as spheres.

FIGURE 7.

Model of Gβγ and PIP₃-dependent P-Rex1 GEF activity. Low basal P-Rex1 activity is maintained through inhibitory interactions between the DH-PH tandem domain and C-terminal domains (gray) that block the GTPase binding site (DH domain, orange). At the plasma membrane, PIP₃ binds via a positively charged pocket within the PH domain (purple), and Gβγ subunits bind to a negatively charged patch that spans the DH and PH domains. The binding of PIP₃ and Gβγ subunits releases the C-terminal domains and exposes the Rac1 binding interface on the opposite side of P-Rex1. This allows Rac1 nucleotide exchange via the P-Rex1 DH domain.