A single amino acid substitution in CXCL12 confers functional selectivity at the beta-arrestin level

Abstract

CXCL12/CXCR4 axis relies on both heterotrimeric Gi protein and ß-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 ß-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 ß-arrestin recruitment assays, as well as functional tests that separately investigate G protein and ß-arrestin-induced responses. [N33A] CXCL12 showed reduced potency both in Gai coupling and ß-arrestin recruitment as compared to the wild type chemokine, acting as an unbiased ligand. While these findings translated into reduced potency within Gai-dependent functions, ß-arrestindependent 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 ß-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.