Abstract
Developmental angiogenesis and the maintenance of the blood-brain barrier involve endothelial cell adhesion, which is linked to cytoskeletal dynamics. GPR124 (also known as TEM5/ADGRA2) is an adhesion G protein-coupled receptor family member that plays a pivotal role in brain angiogenesis and in ensuring a tight blood-brain barrier. However, the signaling properties of GPR124 remain poorly defined. Here, we show that ectopic expression of GPR124 promotes cell adhesion, additive to extracellular matrix-dependent effect, coupled with filopodia and lamellipodia formation and an enrichment of a pool of the G protein-coupled receptor at actin-rich cellular protrusions containing VASP, a filopodial marker. Accordingly, GPR124-expressing cells also displayed increased activation of both Rac and Cdc42 GTPases. Mechanistically, we uncover novel direct interactions between endogenous GPR124 and the Rho guanine nucleotide exchange factors Elmo/Dock and intersectin (ITSN). Small fragments of either Elmo or ITSN1 that bind GPR124 blocked GPR124-induced cell adhesion. In addition, Gβγ interacts with the C-terminal tail of GPR124 and promotes the formation of a GPR124-Elmo complex. Furthermore, GPR124 also promotes the activation of the Elmo-Dock complex, as measured by Elmo phosphorylation on a conserved C-terminal tyrosine residue. Interestingly, Elmo and ITSN1 also interact with each other independently of their GPR124-recognition regions. Moreover, endogenous phospho-Elmo and ITSN1 co-localize with GPR124 at lamellipodia of adhering endothelial cells, where GPR124 expression contributes to polarity acquisition during wound healing. Collectively, our results indicate that GPR124 promotes cell adhesion via Elmo-Dock and ITSN. This constitutes a previously unrecognized complex formed of atypical and conventional Rho guanine nucleotide exchange factors for Rac and Cdc42 that is putatively involved in GPR124-dependent angiogenic responses.
Highlights
Developmental angiogenesis and the maintenance of the blood– brain barrier involve endothelial cell adhesion, which is linked to cytoskeletal dynamics
Accumulating evidence highlights the importance of GPR124 in developmental angiogenesis as well as tumor-induced angiogenesis, processes where this orphan adhesion GPCR modulates endothelial permeability and cell migration, indicating its contribution to adhesive properties and shape adjustments of endothelial cells [2,3,4, 7, 8, 28]
Using a pulldown assay to capture GTP-bound Cdc42 and Rac, based on their affinity for the CRIB region of PAK fused to GST, we revealed that GPR124 expression led to a significant activation of Cdc42 in cells left to adhere for 30 min (Fig. 1F, left)
Summary
GPR124 promotes cell adhesion, polarity, filopodia formation, and activation of Cdc and Rac (Ras-related C3 botulinum toxin substrate 1) GTPases. Pulldown assays revealed that the central region of Elmo contains the minimal binding site required to associate with the C-terminal tail of GPR124, which is different from the N-terminal region of Elmo binding to G␥ [16] (Fig. 3E) This minimal region, corresponding to the ELM domain, was tagged with Myc and used to test its effect on GPR124-dependent adhesion of transfected COS7 cells. We investigated the presence of Elmo and ITSN in the adhesion complex isolated from adhering COS7 cells previously shown to contain GPR124 and G␥. We found that GPR124 localizes with ITSN and Elmo at the extensions of adhering cells (Fig. 5H) These results are consistent with the idea that GPR124 promotes cell adhesion via direct interactions with a signaling complex composed of Elmo–Dock and ITSN
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