The chemokine receptor CCX‐CKR mediates effective scavenging of CCL19 in vitro

Abstract

The chemokines CCL19, CCL21 and CCL25, by signalling through the receptors CCR7 or CCR9, play critical roles in leukocyte homing. They also bind another heptahelical surface protein, CCX-CKR. CCX-CKR cannot couple to typical chemokine receptor signalling pathways or mediate chemotaxis, and its function remains unclear. We have proposed that it controls chemokine bioavailability. Here, using transfected HEK293 cells, we have shown that both CCX-CKR and CCR7 mediate rapid CCL19 internalisation upon initial chemokine exposure. However, internalised CCL19 was more efficiently retained and degraded after uptake via CCX-CKR. More importantly, CCR7 rapidly became refractory for CCL19 uptake, but the sequestration activity of CCX-CKR was enhanced. These properties endowed CCX-CKR with an impressive ability to mediate progressive sequestration and degradation of large quantities of CCL19, and conversely, prevented CCR7-expressing cells from extensively altering their chemokine environment. These differences may be linked to the routes of endocytosis used by these receptors. CCX-CKR, unlike CCR7, was not critically dependent on b-arrestins or clathrin-coated pits. However, over-expression of caveolin-1, which stabilises caveolae, blocked CCL19 uptake by CCX-CKR while having no impact on other chemokine receptors, including CCR7. These data predict that CCX-CKR scavenges extracellular chemokines in vivo to modify responses through CCR7.See accompanying commentary: http://dx

β-Arrestin Recruitment and G Protein Signaling by the Atypical Human Chemokine Decoy Receptor CCX-CKR

“…HEK293T cells transiently transfected with CCX-CKR internalized 125 I-CCL19 substantially faster than cells expressing CCR7 (Fig. 1A), which confirms previous observations (10). Next, CCX-CKR was stably expressed in U2OS human osteosarcoma cells that already express GFP-tagged ␤-arrestin2.…”

“…This rank order of binding affinities is in line with the previously reported 50% inhibitory concentration (IC 50 ) values (4). Hitherto, the only functional read-out for CCX-CKR has been the measurement of chemokine internalization (10). HEK293T cells transiently transfected with CCX-CKR internalized 125 I-CCL19 substantially faster than cells expressing CCR7 (Fig.…”

“…Moreover, CCR7 and CCR9 have been shown to play a role in various cancers (9). CCX-CKR scavenges CCL19 and CCL21 both in vitro and in vivo, thereby decreasing the free concentration of these chemokines (7,8,10). Indeed, low CCX-CKR expression levels in breast cancer tumor cells were correlated with an increase in lymph node metastasis and consequently poor survival rate of patients (11).…”

“…However, a more recent study provided evidence that ␤-arrestin was not necessarily involved in D6 internalization but increased receptor stability (21). CCX-CKR has been previously suggested to internalize CCL19 in a ␤-arrestin-independent manner (10). In the present study, however, we demonstrate for the first time the concentrationdependent recruitment of ␤-arrestins to the atypical chemokine receptor CCX-CKR upon stimulation with CCL19, CCL21, or CCL25 using three different methodologies in various transfected cell lines.…”

Multiple Roles for the C-terminal Tail of the Chemokine Scavenger D6

“…Cell Culture and Transfection-HEK293 cells were maintained and transfected as described previously (12), as were WT and ␤-arrestin null MEFs (provided by R. Lefkowitz, Howard Hughes Medical Institute, Durham, NC) (28,32,33). Stably transfected HEK293 pools were used throughout because transiently expressed D6, or D6-GFP, accumulates in cytoplasmic deposits in these cells causing considerable cell death.…”