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* Department of Microbiology, Molecular Genetics and Immunology, The University of Kansas Medical Center, Kansas City, KS 66160; and
The Affiliated Hospital of Ningxia Medical College, Yinchuan, Ningxia, People’s Republic of China
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.
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1 This research was supported by National Institutes of Health Grants 5K22AI060815 and 2P20RR016443-07 (to C.M.V.). The Flow Cytometry Core is supported by National Institutes of Health Grant P20 RR016443 from the National Center for Research Resources. Confocal imaging was conducted with staff support within an National Institute of Child Health and Human Development center supported facility, the Kansas Intellectual and Developmental Disabilities Research Center imaging core (National Institute of Child Health and Human Development HD 02528).
2 Address correspondence and reprint requests to Dr. Charlotte Vines, University of Kansas Medical Center, 3901 Rainbow Boulevard, Wahl Hall West 3025, Kansas City, KS 66160. E-mail address: cvines{at}kumc.edu
3 Abbreviations used in this paper: GPCR, G protein-coupled receptor; FPR, N-formyl peptide receptor; MEF, murine embryonic fibroblast; sf, serum free; siRNA, small interfering RNA.
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  D. A. Zidar, J. D. Violin, E. J. Whalen, and R. J. Lefkowitz Selective engagement of G protein coupled receptor kinases (GRKs) encodes distinct functions of biased ligands PNAS, June 16, 2009; 106(24): 9649 - 9654. [Abstract] [Full Text] [PDF]
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