G protein Coupled Receptor Kinase 2 Levels Regulate Differential Internalization of C-C Chemokine Receptor 7 (CCR7) in Primary Mouse T Cells

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Abstract G protein-coupled receptor (GPCR) signaling can be regulated by phosphorylation via GPCR kinases (GRKs) and recruitment of b-arrestins. The C-C chemokine receptor 7 (CCR7) GPCR, has two ligands, CCL19 and CCL21 that promote chemotactic migration of CCR7 expressing immune cells, including naïve T cells. Distinct signaling pathways are activated in T cells depending upon which ligand is bound to the CCR7. It remains unclear to what extent GRKs contribute to differential internalization of ligand-bound CCR7 and subsequent signaling that controls T cell migration. We used naïve primary T cells from wild type C57 BL/6 mice (WT) or syngeneic mice heterozygous for the GRK2 gene that express 50% GRK2 protein levels compared to WT mice. T cells were incubated with 40 nM of CCL19 or CCL21 for up to 8 minutes. Incubation with CCL19, but not CCL21, led to CCR7 internalization; however, both ligands facilitated rapid, sustained CCR7 internalization in GRK2+/− T cells. The reduced GRK2 levels in the heterozygote T cells had no significant effect on migration to CCL19 or CCL21 compared to WT T cells. Titration transfection experiments in HEK293T cells or CRISPR-Cas9 knockdown of GRK2 in CEM T cells suggested that GRK2 is required for ligand-mediated CCR7 internalization, while higher GRK2 levels inhibit the internalization process. CCR7 internalization was blocked following transfection of a kinase dead GRK2 mutant indicating that GRK2 phosphorylation is required for CCR7 internalization. Immunomicroscopy suggested arrestin-3 formed clusters, independent of the ligand used. Overall, these data suggest that cellular concentration of GRK2 plays an important role in the internalization of ligand/CCR7.