Structural Dynamics in RhoGEF Signaling Investigated Using Model Cell Membranes

Abstract ID 129342 Poster Board 367 We are focused on understanding regulation of Rho guanine-nucleotide exchange factors (RhoGEFs) and how these proteins transition from autoinhibited to fully active states on a cell membrane. Phosphatidylinositol 3,4,5-trisphosphate (PIP₃)-dependent Rac exchanger (P-Rex) is a RhoGEF that regulates cell motility through its activation of small GTPases such as Rac1 and Cdc42. P-Rex is synergistically recruited to the cell membrane and activated by PIP₃ and Gβγ subunits, positioning the enzyme downstream of cell surface receptors including GPCRs. P-Rex1 functions as a critical regulator of neutrophil migration, but aberrant upregulation of P-Rex1 is strongly associated with cancer metastasis. The molecular mechanisms of P-Rex1 regulation are poorly understood. Here, we have taken a structural biology approach using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and cryo-EM to address this gap. We discovered that Ins(1,3,4,5)P₄ (IP₄) inhibits P-Rex1 activity and induces large decreases in backbone dynamics in diverse regions of the protein. The P-Rex1·IP₄ complex shows a conformation wherein the catalytic core makes unexpected contacts with the C-terminal domain, resulting in a condensed, "wrapped-up" conformation of P-Rex1 with the PH domain occluding the active site of the DH domain. This configuration is stabilized by interactions between the first DEP domain (DEP1) and the DH domain and between the PH domain and a 4-helix bundle that extends from the C-terminal domain of P-Rex1. Disruption of the DH–DEP1 interface enhanced activity, whereas mutations that constrain the occluded conformation led to decreased GEF activity. Variants of full-length P-Rex1 in which the DH–DEP1 and PH–4HB interfaces were disturbed exhibited enhanced activity during chemokine-induced cell migration. Interactions with PIP₃-containing liposomes led to disruption of these interfaces and increased dynamics protein-wide. Our results suggest that inositol phosphates such as IP₄ help to inhibit basal P-Rex1 activity in neutrophils. Research reported in this work was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R35GM146664 (J.N.C.) and by National Institutes of Health grants CA254402, CA221289, HL071818, P30CA023168 and by the Walther Cancer Foundation (J.J.G.T.).