Phosphorylation of key serine residues is required for internalization of the complement 5a (C5a) anaphylatoxin receptor via a β-arrestin, dynamin, and clathrin-dependent pathway

Abstract

The human complement 5a (C5a) anaphylatoxin receptor (CD88) is a G protein-coupled receptor involved in innate host defense and inflammation. Upon agonist binding, C5a receptor (C5aR) undergoes rapid phosphorylation on the six serine residues present in the C-terminal region followed by desensitization and internalization. Using confocal immunofluorescence microscopy and green fluorescent protein-tagged β-arrestins (β-arr 1- and β-arr 2-EGFP) we show a persistent complex between C5aR and β-arrestins to endosomal compartments. Serine residues in the C5aR C terminus were identified that control the intracellular trafficking of the C5aR-arrestin complex in response to C5a. Two phosphorylation mutants C5aR-A314,317,327,332 and C5aR-A314,317,332,334, which are phosphorylated only on Ser334/ Ser338 and Ser327/Ser338, respectively, recruited β-arr 1 and were internalized. In contrast, the phosphorylation-deficient receptors C5aR-A334,338 and C5aR A332,334,338 were not internalized even though observations by confocal microscopy indicated that β-arr 2-EGFP and/or β-arr 2-EGFP could be recruited to the plasma membrane. Altogether the results indicate that C5aR activation is able to promote a loose association with β-arrestins, but phosphorylation of either Ser334/Ser338 or Ser327/Ser338 is necessary and sufficient for the formation of a persistent complex. In addition, it was observed that C5aR endocytosis was inhibited by the expression of the dominant negative mutants of dynamin (K44E) and β-arrestin 1 (β-arr 1-(319-418)-EGFP). Thus, the results suggest that the C5aR is internalized via a pathway dependent on β-arrestin, clathrin, and dynamin.