A single nucleotide polymorphism in the CCR3 gene ablates receptor export to the plasma membrane.
BACKGROUND
The chemokine receptor CCR3 orchestrates the migration of eosinophils, basophils, T(H)2 lymphocytes, and mast cells during the allergic response, with CCR3 blockade a potential means of therapeutic intervention. Non-synonymous single nucleotide polymorphisms (SNPs) within the ccr3 gene have previously been described, with little information regarding their effects on CCR3 function.


OBJECTIVE
To characterize the effects of nonsynonymous SNPs within the ccr3 gene.


METHODS
Site-directed mutagenesis was used to generate N-terminally tagged mutant CCR3 constructs corresponding to reported SNPs. Cell transfectants expressing either wild-type or mutant CCR3 were studied by flow cytometry, Western blotting, and confocal microscopy and examined for their ability to migrate to the CC chemokine ligand CCL11/eotaxin.


RESULTS
An L324P mutant CCR3 protein corresponding to the previously identified T971C SNP was not expressed at the cell surface, and cells remained unresponsive to CCL11 in chemotaxis assays. Confocal microscopy confirmed that L324P-CCR3 had a predominantly intracellular distribution compared with wild-type CCR3. A L324A variant of CCR3 had an identical phenotype to the L324P mutant, suggesting that L324 per se is critical for successful trafficking of nascent CCR3 to the cell membrane. The processes involved appear to be specific for CCR3, because an identical mutation in the homologous receptor CCR1 had minor effects.


CONCLUSIONS
Trafficking to the cell surface of nascent CCR3 is critically dependent on a C-terminal leucine residue, suggestive of specific mechanisms for CCR3 export. Manipulation of these mechanisms may suggest novel means of antagonizing CCR3 function in the treatment of allergy.
Wise
Emma L
EL
Leukocyte Biology Section, National Heart and Lung Institute, Imperial College London, London, United Kingdom.
Bonner
Kandace T
KT
Williams
Timothy J
TJ
Pease
James E
JE
D000818:Animals; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D020641:Polymorphism, Single Nucleotide; D021381:Protein Transport; D054397:Receptors, CCR3 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
C515351:Ccr3 protein, mouse; D054397:Receptors, CCR3