Sphingosine-1-phosphate receptor S1P₁ is regulated by direct interactions with P-Rex1, a Rac guanine nucleotide exchange factor

Abstract

Sphingosine-1-phosphate (S1P) receptors S1P₁ are emerging molecular targets for the treatment of cancer, vascular and immune diseases, due to their pivotal role in cell migration and survival of immune and endothelial cells. A therapeutic strategy to control S1P₁ function is based on agonists that promote changes on S1P₁ expression at the plasma membrane. Here, we explored the hypothesis that cell surface expression and function of S1P₁ are influenced by direct interactions with P-Rex1, a guanine nucleotide exchange factor for Rac. We demonstrate that P-Rex1-PDZ domains interact with S1P₁-carboxyl terminal tail and full length receptor monomers and dimers. Endothelial cells transfected with P-Rex1-PDZ domains show an increased migratory response to S1P. S1P₁ trafficking to intracellular compartments is diminished by coexpression of P-Rex1. We conclude that S1P₁ signaling linked to cell migration is facilitated by a functional interaction with P-Rex1 via a mechanism that involves the maintenance of S1P₁ receptors at the cell membrane.

Introduction

Sphingosine-1-phosphate (S1P) is a physiological regulator of vascular, immune and central nervous systems during development and thereafter [1], [2], [3]. The functions of S1P are mediated by a group of five G protein coupled receptors named S1P₁–S1P₅[4], [5]. Gi-coupled S1P₁ controls lymphocyte trafficking and integrity of endothelial cell–cell adhesions, thus regulating immune and inflammatory responses. These events involve cytoskeletal changes downstream of Rac and kinase cascades including AKT and ERK [6], [7]. In addition, S1P₁ activation leads to angiogenic events sensitized by previous exposure to VEGF [8], [9]. This group of receptors and their downstream effectors are considered potential targets for pharmacological intervention in cancer and immune diseases [10], [11]. The carboxyl terminus of S1P₁ contributes to receptor desensitization via a mechanism dependent on phosphorylation and internalization [12], [13]. Recently, the carboxyl terminal region of different GPCRs has been recognized as the interacting site of novel GPCR-regulatory proteins; in particular, complex PDZ domain containing proteins that help to localize the receptors to restricted areas of the cell membrane, where signaling effectors are also concentrated [14], [15]. Proteins with more than one PDZ domain often interact with the carboxyl terminus of different receptors, helping to form functional aggregates.

P-Rex1 is a Rac guanine nucleotide exchange factor activated by Gβγ and phosphatidylinositol 3,4,5-trisphosphate (PIP3), a second messenger generated by different isoforms of PI3K, including those sensitive to Gi-coupled receptors acting via Gβγ[16]. In neutrophils, P-Rex1 leads to the production of reactive oxygen species [17], [18]. Whereas in the developing nervous system of mice, P-Rex1 is required for the radial migration of cortical neurons [19]. P-Rex1 is a structurally complex Rac guanine nucleotide exchange factor characterized by the presence of a catalytic Dbl-homology (DH) domain located in tandem with a pleckstrin-homology (PH) domain, this module is followed by two DEP and two PDZ domains and ends with a long carboxyl terminal tail. The family of P-Rex1 guanine nucleotide exchange factors also includes P-Rex2 with two splicing variants, P-Rex2a and P-Rex2b, both of them with a similar domain architecture but P-Rex2b having a shortest carboxyl terminal tail [20], [21]. Interestingly, P-Rex2b has been linked to S1P₁ signaling in endothelial cells [22]. The presence of multiple domains in P-Rex1 suggests its potential role as an integrator of GPCR signaling. A likely possibility is that once P-Rex1 is recruited to the plasma membrane via Gβγ and PIP3, which interact with the DH and PH domains respectively [23], the other domains of this RacGEF may establish different functional protein interactions. Recently, we described that P-Rex1 is a functional partner of the mammalian target of rapamycin (mTor), which interacts with P-Rex1 DEP domains, promoting Rac activation and cell migration [24]. The potential role of P-Rex1-PDZ domains in protein networks has not been reported. However, according to established properties of other PDZ domains as participants of protein complexes, in particular with the carboxyl terminus of integral plasma membrane proteins, it seems plausible that P-Rex1, mobilized to the plasma membrane in response to Gβγ and PIP3, interacts with plasma membrane proteins through its PDZ domains. In particular, the possibility that P-Rex1 establishes direct interactions with the carboxyl terminal region of G protein coupled receptors would be relevant for those Gi-coupled GPCRs that induce Rac activation via a Gβγ and PI3K-dependent mechanism, including S1P₁. Here, we tested the hypothesis that S1P₁ receptors interact with P-Rex1-PDZ domains and assessed the functional relevance of this interaction.