Arrestin 3 Mediates Endocytosis of CCR7 following Ligation of CCL19 but Not CCL21

Abstract

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.

Dendritic cell chemotaxis in 3D under defined chemokine gradients reveals differential response to ligands CCL21 and CCL19

“…1, indicating that ligand-receptor binding kinetics play important roles in DC chemotaxis in gradients of CCR7 ligands. At the highest ∇C tested (220 nM∕mm), the chemotactic response appeared to decrease for CCL19 but not CCL21; this may reflect differences in receptor availability due to CCR7 recycling only after binding CCL21 but not CCL19 (8,9,40). Such receptor recycling differences would lead to a higher apparent K D of CCL21 than CCL19 for binding CCR7, which is supported by the effective K D values estimated from fitting the data in Fig.…”

“…Both ligands have similar affinity to CCR7 (4,5), but drive different fates: CCL19 signaling leads to receptor internalization and temporal desensitization while CCL21 can signal from the cell membrane (6,9,40,44,45), a well described liganddependent process for G-protein-coupled receptors. This differential fate of activated CCR7 could lead to increased receptor density and signaling on the cell side dominated by CCL21 (46), consistent with our observation of increased DC response to CCL21 gradients above 50 nM (Fig.…”

“…Interestingly, CCL19 and CCL21 have similar binding affinities for CCR7 (4)(5)(6) and similar chemotactic potential for DCs and T cells under 2D conditions (7,8), but differ in their internalization (8,9) as well as their binding affinity to extracellular matrix (ECM) proteoglycans. Specifically, the positively charged C terminus of CCL21 gives it strong binding affinity to a wide range of proteoglycans [as well as type IV collagen (10)], unlike CCL19 (11)(12)(13).…”

Dendritic Cells Distinguish Individual Chemokine Signals through CCR7 and CXCR4

“…Despite CCL19 and CCL21 binding the same receptor, our studies and several others (7,50,51) showed that the cell perceives them as unique entities. In cross-desensitization studies, CCL19 inhibited CCL21 signaling but not vice versa (45,51), and despite similar G protein activation and calcium mobilization, differences were shown in receptor desensitization, internalization, and recycling (50)(51)(52)(53). In our direct competition assays between CCL19 and CCL21, opposing gradients of equal concentrations resulted in a biased response toward CCL19.…”

CXCR7/CXCR4 Heterodimer Constitutively Recruits β-Arrestin to Enhance Cell Migration

“…These results and others suggest that biased ligands or conditions that lead to selective activation of one pathway over the other will result in distinct physiological effects, possibly by stabilizing different "active" receptor conformations (33,34). There are indeed such examples of selective ligands; for example, CCL19 and CCL21 are two CCR7-specific endogenous chemokine ligands that are equally effective in their ability to activate G proteins, but only CCL19 can induce ␤-arrestin recruitment and ␤-arrestin-dependent ERK activation (44). Similarly, both ␤ 2 -adrenergic and parathyroid hormone receptors have been shown to have specific ligands that are biased toward either G protein-dependent or ␤-arrestin-dependent signaling (45,46).…”