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) controls lymphocyte migration to secondary lymphoid organs. Although CCR7 has been implicated in targeting the metastasis of cancers to lymph nodes, the role of CCR7 in the metastasis of breast cancer, along with the molecular mechanisms that are controlled by CCR7 that target breast cancer metastasis to the lymph nodes, has yet to be defined. To explore the cellular and molecular mechanisms of breast cancer cell migration to the lymph nodes, we used the mouse MMTV-PyVmT mammary tumor cells (PyVmT) transfected with CCR7 and the human CCR7-expressing MCF10A and MCF7 mammary cell lines. We found that the CCR7 ligands CCL19 and CCL21, controlled cell migration using the β(1)-integrin heterodimeric adhesion molecules. To define a physiological significance for CCR7 regulation of migration, we used the FVB syngeneic mouse model of metastatic breast cancer. When CCR7-negative PyVmT cells transfected with control vector were orthotopically transferred to the mammary fat pad of FVB mice, tumors metastasized to the lungs (10/10 mice) but not to the lymph nodes (0/10). In contrast, CCR7-expressing PyVmT (CCR7-PyVmT) cells metastasized to the lymph nodes (6/10 mice) and had a reduced rate of metastasis to the lungs (4/10 mice). CCR7-PyVmT tumors grew significantly faster than PyVmT tumors, which mirrored the growth in vitro, of CCR7-PyVmT, MCF7, and MCF10A mammospheres. This model provides tools for studying lymph node metastasis, CCR7 regulation of tumor cell growth, and targeting of breast cancer cells to the lymph nodes.

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.