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 ␤-arrestins (␤-arr 1-and ␤-arr 2-EGFP) we show a persistent complex between C5aR and ␤-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. were not internalized even though observations by confocal microscopy indicated that ␤-arr 1-EGFP and/or ␤-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 ␤-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 ␤-arrestin 1 (␤-arr 1-(319 -418)-EGFP). Thus, the results suggest that the C5aR is internalized via a pathway dependent on ␤-arrestin, clathrin, and dynamin.

C5a Receptor Oligomerization

“…Similarly, 6 M urea treatment of CHO membranes containing C5aR receptor can uncouple receptor from G proteins. 2 The C5a receptor has been shown to interact with ␤-arrestins (38) and recently the Wiskott-Aldrich syndrome protein (39); interactions with these or other peripheral membrane proteins may aid in receptor oligomerization. The ability of C5aR to cross-link in the absence of peripheral membrane proteins argues that the oligomerization that we observe is a receptorautonomous process.…”

International Union of Basic and Clinical Pharmacology. LXXXVII. Complement Peptide C5a, C4a, and C3a Receptors

“…In some GPCR with cysteine residues in the C-terminal domain, such as bradykinin B2, helix 8 is critical for the association with b-arrestins, so the lack of a cysteine residue in this position in the C5a 1 receptor may be responsible for some of the differences in signaling observed for the complement peptide receptors. Unlike the C3a receptor, there is no evidence that the C5a 1 receptor signals through arrestins , although an association occurs (Braun et al, 2003;Kalant et al, 2005;van Lith et al, 2009). Kinase activation by the C5a 1 receptor is almost entirely Ga i dependent (Buhl et al, 1994;Gripentrog and Miettinen, 2008), with little evidence to date that arrestins are directly involved.…”

“…Arrestin signaling. Phosphorylation of the C5a 1 receptor leads to association with arrestins and subsequent targeting to clathrin-coated pits for internalization (Braun et al, 2003). PK-CbII and GRK2 interact with intracellular loop 3 and the C-terminal domain of the C5a 1 receptor and may be responsible for the serine/threonine phosphorylation of the receptor (Suvorova et al, 2008).…”

PMX-53 as a Dual CD88 Antagonist and an Agonist for Mas-Related Gene 2 (MrgX2) in Human Mast Cells

“…CD88 undergoes phosphorylation in response to agonist stimulation resulting in their desensitization (Christophe et al, 2000;Braun et al, 2003). Furthermore, phosphorylation-deficient mutants of CD88 are resistant to desensitization (Pollok-Kopp et al, 2007).…”