Internalization of ligand bound G protein-coupled receptors, an important cellular function that mediates receptor desensitization, takes place via distinct pathways, which are often unique for each receptor. The C-C chemokine receptor (CCR7) G protein-coupled receptor is expressed on naive T cells, dendritic cells, and NK cells and has two endogenous ligands, CCL19 and CCL21. Following binding of CCL21, 21 ± 4% of CCR7 is internalized in the HuT 78 human T cell lymphoma line, while 76 ± 8% of CCR7 is internalized upon binding to CCL19. To determine whether arrestins mediated differential internalization of CCR7/CCL19 vs CCR7/CCL21, we used small interfering RNA (siRNA) to knock down expression of arrestin 2 or arrestin 3 in HuT 78 cells. Independent of arrestin 2 or arrestin 3 expression, CCR7/CCL21 internalized. In contrast, following depletion of arrestin 3, CCR7/CCL19 failed to internalize. To examine the consequence of complete loss of both arrestin 2 and arrestin 3 on CCL19/CCR7 internalization, we examined CCR7 internalization in arrestin 2−/−/arrestin 3−/− murine embryonic fibroblasts. Only reconstitution with arrestin 3-GFP but not arrestin 2-GFP rescued internalization of CCR7/CCL19. Loss of arrestin 2 or arrestin 3 blocked migration to CCL19 but had no effect on migration to CCL21. Using immunofluorescence microscopy, we found that arrestins do not cluster at the membrane with CCR7 following ligand binding but cap with CCR7 during receptor internalization. These are the first studies that define a role for arrestin 3 in the internalization of a chemokine receptor following binding of one but not both endogenous ligands.

C-C Chemokine Receptor 7 (CCR7) regulates migration of naïve T cells to lymph nodes, via chemotaxis to the ligand, CCL21. Approximately 72 hours after lymph node entry, Endothelial Differentiation Gene 1 (EDG-1) is up-regulated on T cells and mediates lymph node egress. It is unclear how EDG-1 expression is up-regulated. We hypothesized that lymph node egress occurs in response to CCR7 receptor interaction with dendritic cells presenting the second CCR7 ligand, CCL19. To test this hypothesis, we used murine or human T cells, stimulated with CCL19 and found that EDG-1 mRNA was up-regulated at 24, 48, and 72 hours. The cells displayed increased migration to EDG-1 ligand S1P over the same time period confirming surface expression of EDG-1. Extracellular Regulated Kinase 5 (ERK5) can mediate transcription of Kruppel-Like Factor 2 (KLF-2). KLF-2 directly mediates transcription of EDG-1. To determine if the hypothesized ERK5-KLF-2-EDG-1 pathway was active in our system, we assayed murine and human T cells for changes in ERK5 and KLF-2. We found that stimulation with CCL19 led to increased phosphorylation and expression of ERK5 at 24, 48, and 72 hours. Similarly, KLF-2 levels were increased over the same interval. Taken together, our data suggests that after lymph node entry of T cells via CCR7/CCL21, T cells exit the lymph nodes via EDG-1 in response to activation of T cell CCR7 by CCL19 on activated dendritic cells.