Beta-arrestin- but not G protein-mediated signaling by the "decoy" receptor CXCR7

Abstract

Ubiquitously expressed seven-transmembrane receptors (7TMRs) classically signal through heterotrimeric G proteins and are commonly referred to as G protein-coupled receptors. It is now recognized that 7TMRs also signal through beta-arrestins, which act as versatile adapters controlling receptor signaling, desensitization, and trafficking. Most endogenous receptors appear to signal in a balanced fashion using both beta-arrestin and G protein-mediated pathways. Some 7TMRs are thought to be nonsignaling "decoys" because of their inability to activate typical G protein signaling pathways; it has been proposed that these receptors act to scavenge ligands or function as coreceptors. Here we demonstrate that ligand binding to the decoy receptor CXCR7 does not result in activation of signaling pathways typical of G proteins but does activate MAP kinases through beta-arrestins in transiently transfected cells. Furthermore, we observe that vascular smooth muscle cells that endogenously express CXCR7 migrate to its ligand interferon-inducible T-cell alpha chemoattractant (ITAC), an effect that is significantly attenuated by treatment with either a CXCR7 antagonist or beta-arrestin depletion by siRNA. This example of an endogenous "beta-arrestin-biased" 7TMR that signals through beta-arrestin in the absence of G protein activation demonstrates that some 7TMRs encoded in the genome have evolved to signal through beta-arrestin exclusively and suggests that other receptors that are currently thought to be orphans or decoys may also signal through such nonclassical pathways.

Fig. 1.

CXCR7 recruits β-arrestin resulting in MAP kinase activation in transiently transfected HEK293 cells. (A) β-arrestin recruitment in live HEK 293 cells transiently transfected with CXCR7 and GFP-labeled β-arrestin2 before and 30 min after treatment with SDF-1α and ITAC. (B) Thirty minutes after treatment with either ligand, there is a pattern of pERK formed in CXCR7-transfected cells. Fixed cells were labeled for CXCR7 (blue) and pERK (red) after transfection with CXCR7 and β-arrestin 2-GFP (green). In cells treated with SDF-1α, there is clear localization of all of these components in cytoplasmic vesicles on merged images (white), whereas this change is not as pronounced in those cells treated with ITAC.

Fig. 2.

Expression of chemokine receptors for SDF-1α and ITAC in rVSMCs. (A) CXCR7 shares ligands with the typical chemokine receptors CXCR4 and CXCR3. Other ligands for CXCR3 that are not shown are IP10 and Mig. (B) mRNA expression levels of CXCR7 (R7), CXCR4 (R4) and CXCR3 (R3) in passage 0 rVSMCs by semiquantitative RT-PCR at 30 and 40 cycles. (C) Immunoblot analysis of receptor expression in rVSMCs using antibodies against CXCR7, 4, and 3.

Fig. 3.

Chemokine receptor expression and lack of G protein activation by ITAC in rVSMCs. (A) Treatment with unlabeled ITAC or SDF competes off radiolabeled SDF-1α from rVSMCs. (B) Activation of G_αi in SDF-1α-treated rVSMCs (SDF) but not in untreated (UT) or ITAC-treated (ITAC) samples. The negative control was incubated with GDP (GDP) and the positive control incubated with GTP (GTP) for 90 min prior to immunoprecipitation. Data shown are mean ± SEM from three independent experiments. (C) Absence of calcium influx as assessed by change in Fura-2 fluorescence emission ratio upon stimulation of rVSMCs with SDF-1α (blue) or ITAC (red) compared to Angiotensin II (black) from a representative experiment from three independent experiments.

Fig. 4.

rVSMC migration in response to ITAC is a CXCR7- and β-arrestin-dependent process. (A) Migration of rVSMCs to 1–100 nM of SDF-1α and ITAC compared to PDGF positive control. Shown is representative data from at least three independent experiments. (B) Effects of CCX704 (inactive control), CCX733 (CXCR7 antagonist), T497 (CXCR3 antagonist), and AMD3100 (CXCR4 antagonist) on ITAC-stimulated migration. Only treatment with CCX733 resulted in a significant inhibitory effect (∗ p < 0.05). (C) Significant inhibition of ITAC-stimulated rVSMC migration by β-arrestin2 depletion (∗ p < 0.05) but not β-arrestin1 depletion. Data shown are mean ± SEM from at least four independent experiments.

Fig. 5.

Balanced and biased signaling by 7TMRs. (A) In a balanced system, signaling is mediated by G proteins and β-arrestins, while β-arrestins also control desensitization and receptor trafficking. (B) Treatment of an unbiased receptor with a biased agonist results in a biased response, in this case, β-arrestin-mediated signaling only. (C) In the case of a β-arrestin-biased receptor, treatment of the receptor with an unbiased ligand results in β-arrestin-mediated signaling only.