The study investigates the regulation of P-Rex1, a key guanine nucleotide exchange factor (GEF) for Rac that plays central roles in neutrophil chemotaxis and cancer metastasis. P-Rex1 is synergistically activated by PIP3 and the heterotrimeric Gβγ subunits, but the mechanistic details of its regulation remain poorly understood.
The researchers 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. Cryo-electron microscopy analysis of the P-Rex1·IP4 complex revealed a conformation where the pleckstrin homology (PH) domain occludes the active site of the Dbl homology (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 (4HB) subdomain that extends from the C-terminal domain of P-Rex1.
Disruption of the DH-DEP1 interface in a DH/PH-DEP1 fragment enhanced activity and led to a more extended conformation in solution, 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, confirming that the observed structure represents the autoinhibited state in living cells.
Interactions with PIP3-containing liposomes led to disruption of these interfaces and increased dynamics protein-wide. The results suggest that inositol phosphates such as IP4 help to inhibit basal P-Rex1 activity in neutrophils, similar to their inhibitory effects on phosphatidylinositol-3-kinase.
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by Ravala,S. K.... at www.biorxiv.org 09-16-2023
https://www.biorxiv.org/content/10.1101/2023.09.15.557836v3Deeper Inquiries