Abstract
The chemokine receptor CXCR4, a G protein-coupled receptor (GPCR) capable of heteromerizing with other GPCRs, is involved in many processes, including immune responses, hematopoiesis, and organogenesis. Evidence suggests that CXCR4 activation reduces thrombin/protease-activated receptor 1 (PAR1)-induced impairment of endothelial barrier function. However, the mechanisms underlying cross-talk between CXCR4 and PAR1 are not well-understood. Using intermolecular bioluminescence resonance energy transfer and proximity ligation assays, we found that CXCR4 heteromerizes with PAR1 in the HEK293T expression system and in human primary pulmonary endothelial cells (hPPECs). A peptide analog of transmembrane domain 2 (TM2) of CXCR4 interfered with PAR1:CXCR4 heteromerization. In HTLA cells, the presence of CXCR4 reduced the efficacy of thrombin to induce β-arrestin-2 recruitment to recombinant PAR1 and enhanced thrombin-induced Ca2+ mobilization. Whereas thrombin-induced extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation occurred more transiently in the presence of CXCR4, peak ERK1/2 phosphorylation was increased when compared with HTLA cells expressing PAR1 alone. CXCR4-associated effects on thrombin-induced β-arrestin-2 recruitment to and signaling of PAR1 could be reversed by TM2. In hPPECs, TM2 inhibited thrombin-induced ERK1/2 phosphorylation and activation of Ras homolog gene family member A. CXCR4 siRNA knockdown inhibited thrombin-induced ERK1/2 phosphorylation. Whereas thrombin stimulation reduced surface expression of PAR1, CXCR4, and PAR1:CXCR4 heteromers, chemokine (CXC motif) ligand 12 stimulation reduced surface expression of CXCR4 and PAR1:CXCR4 heteromers, but not of PAR1. Finally, TM2 dose-dependently inhibited thrombin-induced impairment of hPPEC monolayer permeability. Our findings suggest that CXCR4:PAR1 heteromerization enhances thrombin-induced G protein signaling of PAR1 and PAR1-mediated endothelial barrier disruption.
Highlights
Already reached the clinical arena as a drug approved by the United States Food and Drug Administration for mobilization of stem cells in cancer patients [5,6,7]
We utilized intermolecular bioluminescence resonance energy transfer (BRET) titration assays to test whether CXCR4 heteromerizes with protease-activated receptor 1 (PAR1) (Fig. 1A)
Consistent with nonspecific bystander BRET signals, BRET signals in cells transfected with CXCR4-RLuc and yellow fluorescent protein (YFP) were low and increased linearly with increasing energy acceptor/donor ratios
Summary
Chemokine (CXC motif) receptor 4 (CXCR4) is essential during embryonic development and plays multifaceted roles in biology after birth [1,2,3,4]. As an alternative explanation for the effects of CXCL12 on thrombin-induced RhoA activation and endothelial cell barrier function, depletion of the CXCR4:PAR1 heteromer from the cell surface upon binding of CXCL12 to CXCR4 could contribute to these effects To test this possibility, we pretreated hPPECs with the TM2 or the TM control peptide, followed by stimulation with thrombin or CXCL12. The apparent paradox that the CXCR4 agonist CXCL12 as well as the TM2 peptide, which inhibits G protein–mediated signaling and function of CXCR4, inhibit thrombin-induced G protein–mediated signaling of PAR1 and endothelial barrier function impairment can be explained by the similarity of their effects on CXCR4:PAR1 heteromerization Both molecules reduce expression of CXCR4:PAR1 heteromers on the cell surface without affecting PAR1 expression levels. Our observations further support the concept that the development of drugs targeting GPCR heteromers could provide new therapeutic opportunities in the future
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