Signal termination of the chemokine receptor CCR9 is governed by an arrestin-independent phosphorylation mechanism.

The C-C chemokine receptor type 9 (CCR9) coordinates immune cell migration from the thymus to the small intestine along gradients of the chemokine CCL25. Receptor dysregulation is associated with a variety of inflammatory bowel diseases such as Crohn's and ulcerative colitis, whereas aberrant CCR9 overexpression correlates with tumor metastasis. Despite being an attractive therapeutic target, attempts to clinically antagonize CCR9 have been unsuccessful. This highlights the need for a deeper understanding of its specific regulatory mechanisms and signaling pathways. CCR9 is a G protein-coupled receptor (GPCR) and activates G(i) and G(q) pathways. Unexpectedly, live-cell bioluminescence resonance energy transfer assays reveal only limited G protein activation, and signaling is rapidly terminated. Truncating the receptor C terminus significantly enhanced G protein coupling, highlighting a regulatory role of this domain. Signal suppression was not because of canonical arrestin-coordinated desensitization. Rather, removal of GPCR kinase phosphorylation led to sustained and robust G protein activation by CCR9. Using site-directed mutagenesis, we identified specific phosphorylation motifs that attenuate G protein coupling. Receptor internalization did not correlate with G protein activation capabilities. Instead, CCR9 phosphorylation disrupted the interaction of G protein heterotrimers with the receptor. This interference may lead to rapid loss of productive coupling and downstream signaling as phosphorylation would effectively render the receptor incapable of G protein coupling. An arrestin-independent, phosphorylation-driven deactivation mechanism could complement arrestin-dependent regulation of other GPCRs and have consequences for therapeutically targeting these receptors.