Alanine scanning mutagenesis of CCR3 reveals that the three intracellular loops are essential for functional receptor expression

Abstract

Intracellular signaling mediated by the eotaxin receptor, CCR3, has been implicated in allergic diseases involving the recruitment and activation of eosinophils. To investigate the structural requirements of the three intracellular loops (ICL) of CCR3, a panel of 15 alanine triplet mutants were generated and their effects on function assessed by assays of cell surface expression and chemotactic responsiveness. While the majority of constructs were efficiently expressed when compared with their wild-type counterpart, their abilities to migrate in response to eotaxin were relatively poor, suggesting that all three intracellular loops of CCR3 are involved to some degree in coupling to G proteins. - Another panel of 7 point mutants were then constructed to examine the DRY motif which resides in ICL2 and is highly conserved throughout the chemokine receptors identified to date. The conservative mutants D130E and R131K were well tolerated and gave chemotactic responses approaching 35 % of wild-type CCR3, but the less conserved substitutions D130A, D130N and R131L were non-functioning. Tyrosine 131 was particularly sensitive to mutation as both Y131F and Y131S mutants were poorly expressed and were chemotactically inactive. Together, this data suggest that the acidic / basic / polar nature of the DRY motif is a prerequisite for CCR3 function.