Abstract
CXCL12/CXCR4 axis relies on both heterotrimeric Gi protein and β-arrestin coupling to trigger downstream responses. G protein activation allows for calcium flux, chemotaxis and early extracellular-signal regulated kinases 1/2 (ERK1/2) phosphorylation, whereas β-arrestin recruitment leads to late signaling, receptor desensitization and internalization. Together they may regulate the balance between transactivation and transinhibition of epithelial growth factor receptor 1 (HER1). Since we have previously noted significant differences between CXCL12 and its structural variant [N33A]CXCL12 in CXCR4 signaling, we sought to better characterize them by performing cAMP inhibition and β-arrestin recruitment assays, as well as functional tests that separately investigate G protein and β-arrestin-induced responses. [N33A]CXCL12 showed reduced potency both in Gαi coupling and β-arrestin recruitment as compared to the wild type chemokine, acting as an unbiased ligand. While these findings translated into reduced potency within Gαi-dependent functions, β-arrestin-dependent modules were affected in a more peculiar way. Unlike CXCL12, the mutant analogue did not restore HB-EGF-stimulated HER1 from CXCR4-induced transinhibition, and did not trigger the late wave of ERK1/2 phosphorylation. Instead, CXCR4 internalization was not impaired upon [N33A]CXCL12 stimulation. These differences highlight the novel opportunity to dissect CXCL12 signaling within the β-arrestin layer, in which the mutant chemokine clearly favors the internalization module over the other pathways. Such functional selectivity has an impact on HER1 activation status and may play a relevant part in the crosstalk between tyrosine kinase and seven transmembrane receptors.
Highlights
CXCL12 is a CXC homeostatic chemokine, constitutively expressed by blood and stromal cells, which retains a central role in the regulation of embryogenesis [1], hematopoietic system [2], tissue repair mechanisms [3] and cancer biology [4]
We have demonstrated that, as compared to the wild type chemokine, the chemically synthetized mutant [N33A]CXCL12 (N33A) was not able to reverse G protein-dependent transinhibition of the heparin-binding EGF-like growth factor (HB-EGF)stimulated epithelial growth factor receptor 1 (HER1), amounting to an indirect proof that it might stabilize CXCR4 in a conformation favoring G protein signaling over β-arrestin recruitment [21]
By performing cAMP inhibition assay at 15 minutes in CXCR4-expressing chinese hamster ovary (CHO) cell line, we observed that EC50 was higher, whereas Emax was reduced for N33A as compared to CXCL12, demonstrating that N33A acts as a weak partial agonist in coupling to Gαi (Figure 1B)
Summary
CXCL12 is a CXC homeostatic chemokine, constitutively expressed by blood and stromal cells, which retains a central role in the regulation of embryogenesis [1], hematopoietic system [2], tissue repair mechanisms [3] and cancer biology [4]. It interacts with the seven transmembrane receptors (7TMRs) CXCR4 and CXCR7 to generate a complex network of intracellular biochemical signals. Apart from classical G protein signaling, CXCL12 induces G protein-coupled receptor kinases (GRK)mediated phosphorylation of CXCR4 cytoplasmic tail and subsequent recruitment of β-arrestin proteins [10, 11]. This mechanism accounts for the persistence of extracellularsignal regulated kinases 1/2 (ERK1/2) phosphorylation after the first, pertussin toxin-sensitive, spike, as well as the activation of AKT, PI3K and phosphodiesterase 4 (PDE4) [12,13,14]
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