Abstract

The Cys-Cys Chemokine Receptor 6 (CCR6) is a well established modulator of inflammation. Although several genetic associations have been identified between CCR6 polymorphisms and immune system disorders (e.g. rheumatoid arthritis, Crohn's disease), the pharmacological effects of naturally occurring missense mutations in this receptor have yet to be characterized. In this study, we initially assessed G protein-mediated signaling and observed that WT CCR6 exhibited ligand independent activity. In addition we found that the five most frequent CCR6 missense variants (A89T, A150V, R155W, G345S, and A369V) exhibited decreased basal and/or ligand induced Gαi protein signaling. To complement the study of these loss of function variants, we engineered a set of constitutively active CCR6 receptors. Selected mutations enhanced basal G protein-mediated signaling up to three fold relative to the WT value. Using a Bioluminescence Resonance Energy Transfer (BRET) assay we investigated the ability of each naturally occurring and engineered CCR6 receptor mutant to recruit beta arrestin. In contrast to G protein-mediated signaling, beta arrestin mobilization was largely unperturbed by the naturally occurring loss of function CCR6 variants. Elevated recruitment of beta arrestin was observed in one of the engineered constitutively active mutants (T98P). Our results demonstrate that point mutations in CCR6 can result in either a gain or loss of receptor function. These observations underscore the need to explore how CCR6 natural variants may influence immune cell physiology and human disease.