Evidence for a Two-Step Model for Activation of GPR25 by the Chemoattractant CXCL17

CXCL17 was recently reported to activate GPR25, a receptor expressed by T-regulatory cells. Although classified as a chemokine, the activity of CXCL17 is ablated by minor C-terminal truncation, suggesting a novel mode of receptor activation. We set out to test this hypothesis by mutagenesis. GPR25 was expressed in the murine pre-B cell line L1.2 and mediated robust migration of transfectants to nanomolar concentrations of recombinant CXCL17 (24-119). The N-terminally truncated form of CXCL17 (64-119) was also chemotactic for GPR25 transfectants, albeit with severely reduced potency. Modelling of CXCL17:GPR25 implied multiple interactions between the N- and C-termini of CXCL17 with GPR25, which was validated by mutagenesis. Cells expressing a chimeric FPR1:GPR25 construct responded chemotactically to CXCL17 but with significantly reduced potency compared with wild-type GPR25 transfectants, implicating the GPR25 N-terminus in CXCL17 recognition. Mutagensis of the GPR25 residues W95, R178 and R264 resulted in a complete loss of chemotactic responsiveness to CXCL17, consistent with the residues interacting with the C-terminal CXCL17 motif. In conclusion, we verify GPR25 as a bona fide CXCL17 receptor and suggest a two-step model of GPR25 activation, in which the receptor N-terminus orients CXCL17 for activation of GPR25 via its C-terminus. We also advocate the reclassification of CXCL17 as a chemoattractant distinct from the chemokine family.