New insights into regulation of P-Rex1 autoinhibition.

Phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) is a guanine nucleotide exchange factor for Rho GTPases downstream of G protein coupled receptors (GPCRs) and tyrosine kinases. It is primarily expressed in hematopoietic cells and brain. Its ectopic expression is reported in different cancers including breast, prostrate, skin, and ovarian cancers, making it a potential therapeutic target. P-Rex1 is a multi-domain protein consisting of a catalytic Dbl-homology (DH) domain, followed by a PH, two DEP, two PDZ, and an inositolpolyphosphate-4 phosphatase-like (IP4P) domain. P-Rex1 is regulated by PIP3, Gbg binding and phosphorylation via PKC and PKA. In unstimulated cells, P-Rex1 is autoinhibited by intradomain interactions with its catalytic DH domain. Previously, our lab had shown that first DEP (DEP1) domain plays a key role in inhibition of DH/PH-DEP1 exchange activity. However, there is no information available as how these domains are engaged in regulation of full length P-Rex1. In our current study, we addressed this gap by employing single particle cryo-electron microscopy (cryo-EM) complemented by SAXS, HDX-MS, and functional enzymatic assays to understand P-Rex1 regulation. A structure with the head group of PIP3 (IP4) bound to the PH domain revealed interactions between DH and DEP1 domain that would block GTPase access to DH domain by coercing a novel configuration of the DH-PH module. In addition, we observed that the IP4P domain was anchored to PH domain via a previously unrecognized FAT domain. Mutagenesis of residues involved in the DH-DEP1 interface led to activation, suggestive of inhibitory role of this interface. IP4 is a soluble second messenger signaling molecule which is reported to compete with PIP3 binding site in other proteins. Therefore, we tested whether it could regulate P-Rex1 in a similar manner and found that IP4 competes with PIP3 and negatively regulates P-Rex1 activity. Based on these observations, we propose that in unstimulated cell, P-Rex1 is autoinhibited by intradomain interactions between the DEP1 and DH and between the FAT and PH domains, and that inositol phosphate levels may add an additional layer of desensitization by opposing P-Rex1 membrane translocation and favoring a closed autoinhibited state.