Phosphorylation of key serine residues is required for internalization of the complement 5a (C5a) anaphylatoxin receptor via a beta-arrestin, dynamin, and clathrin-dependent pathway.
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 beta-arrestins (beta-arr 1- and beta-arr 2-EGFP) we show a persistent complex between C5aR and beta-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-A(314,317,327,332) and C5aR-A(314,317,332,334), which are phosphorylated only on Ser(334)/Ser(338) and Ser(327)/Ser(338), respectively, recruited beta-arr 1 and were internalized. In contrast, the phosphorylation-deficient receptors C5aR-A(334,338) and C5aR-A(332,334,338) were not internalized even though observations by confocal microscopy indicated that beta-arr 1-EGFP and/or beta-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 beta-arrestins, but phosphorylation of either Ser(334)/Ser(338) or Ser(327)/Ser(338) 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 beta-arrestin 1 (beta-arr 1-(319-418)-EGFP). Thus, the results suggest that the C5aR is internalized via a pathway dependent on beta-arrestin, clathrin, and dynamin.
Braun
Laurence
L
Département de Réponse et Dynamique Cellulaires/Biochimie et Biophysique des Systèmes Intégrés, (UMR 5092, Commissariat à l'Energie Atomique (CEA)/CNRS/Université Joseph Fourier), CEA/Grenoble, 17 rue des Martyrs, France.
Christophe
Thierry
T
Boulay
François
F
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D019390:Arrestins / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism*; D017931:DNA Primers; D034281:Dynamins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism*; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins
C000605253:ARRB1 protein, human; D015703:Antigens, CD; D019390:Arrestins; D002966:Clathrin; D017931:DNA Primers; D008164:Luminescent Proteins; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D011993:Recombinant Fusion Proteins; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D012694:Serine; D034281:Dynamins