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.
Mesh Terms:
Amino Acid Sequence, Antigens, CD, Arrestins, Base Sequence, Blotting, Western, Cell Line, Clathrin, DNA Primers, Dynamins, Green Fluorescent Proteins, Humans, Luminescent Proteins, Microscopy, Confocal, Molecular Sequence Data, Phosphorylation, Precipitin Tests, Protein Binding, Receptor, Anaphylatoxin C5a, Receptors, Complement, Recombinant Fusion Proteins, Sequence Homology, Amino Acid, Serine
Amino Acid Sequence, Antigens, CD, Arrestins, Base Sequence, Blotting, Western, Cell Line, Clathrin, DNA Primers, Dynamins, Green Fluorescent Proteins, Humans, Luminescent Proteins, Microscopy, Confocal, Molecular Sequence Data, Phosphorylation, Precipitin Tests, Protein Binding, Receptor, Anaphylatoxin C5a, Receptors, Complement, Recombinant Fusion Proteins, Sequence Homology, Amino Acid, Serine
J. Biol. Chem.
Date: Feb. 07, 2003
PubMed ID: 12464600
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