β-Arrestin recruitment and G protein signaling by the atypical human chemokine decoy receptor CCX-CKR

J Biol Chem. 2013 Mar 8;288(10):7169-81. doi: 10.1074/jbc.M112.406108. Epub 2013 Jan 22.

Abstract

Chemokine receptors form a large subfamily of G protein-coupled receptors that predominantly activate heterotrimeric Gi proteins and are involved in immune cell migration. CCX-CKR is an atypical chemokine receptor with high affinity for CCL19, CCL21, and CCL25 chemokines, but is not known to activate intracellular signaling pathways. However, CCX-CKR acts as decoy receptor and efficiently internalizes these chemokines, thereby preventing their interaction with other chemokine receptors, like CCR7 and CCR9. Internalization of fluorescently labeled CCL19 correlated with β-arrestin2-GFP translocation. Moreover, recruitment of β-arrestins to CCX-CKR in response to CCL19, CCL21, and CCL25 was demonstrated using enzyme-fragment complementation and bioluminescence resonance energy transfer methods. To unravel why CCX-CKR is unable to activate Gi signaling, CCX-CKR chimeras were constructed by substituting its intracellular loops with the corresponding CCR7 or CCR9 domains. The signaling properties of chimeric CCX-CKR receptors were characterized using a cAMP-responsive element (CRE)-driven reporter gene assay. Unexpectedly, wild type CCX-CKR and a subset of the chimeras induced an increase in CRE activity in response to CCL19, CCL21, and CCL25 in the presence of the Gi inhibitor pertussis toxin. CCX-CKR signaling to CRE required an intact DRY motif. These data suggest that inactive Gi proteins impair CCX-CKR signaling most likely by hindering the interaction of this receptor with pertussis toxin-insensitive G proteins that transduce signaling to CRE. On the other hand, recruitment of the putative signaling scaffold β-arrestin to CCX-CKR in response to chemokines might allow activation of yet to be identified signal transduction pathways.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arrestins / genetics
  • Arrestins / metabolism*
  • Binding, Competitive / drug effects
  • Blotting, Western
  • CHO Cells
  • Cell Line, Tumor
  • Chemokine CCL19 / metabolism
  • Chemokine CCL19 / pharmacology
  • Chemokine CCL21 / metabolism
  • Chemokine CCL21 / pharmacology
  • Chemokines, CC / metabolism
  • Chemokines, CC / pharmacology
  • Cricetinae
  • Cricetulus
  • GTP-Binding Protein alpha Subunits, Gi-Go / metabolism*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Microscopy, Fluorescence
  • Models, Biological
  • Pertussis Toxin / pharmacology
  • Protein Binding / drug effects
  • Protein Transport / drug effects
  • Receptors, CCR / genetics
  • Receptors, CCR / metabolism*
  • Signal Transduction*
  • beta-Arrestins

Substances

  • ACKR4 protein, human
  • Arrestins
  • CCL25 protein, human
  • Chemokine CCL19
  • Chemokine CCL21
  • Chemokines, CC
  • Receptors, CCR
  • beta-Arrestins
  • Green Fluorescent Proteins
  • Pertussis Toxin
  • GTP-Binding Protein alpha Subunits, Gi-Go