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Structure-function studies of the C3a-receptor: C-terminal serine and threonine residues which influence receptor internalization and signaling

Settmacher, Britta; Rheinheimer, Claudia; Hamacher, Henning; Ames, Robert S; Wise, Alan; Jenkinson, Lesley; Bock, Daniel; Schaefer, Myriam; Kohl, Jorg and Klos, Andreas (2003) In European Journal of Immunology 33(4). p.920-927
Abstract
The anaphylatoxic peptide C3a is a pro-inflammatory mediator generated during complement activation, whose specific G protein coupled receptor is expressed on granulocytes, monocytes, mast cells, activated lymphocytes, and in the nervous tissue. We have generated RBL-2H3 cell clones stably expressing mutants of the human C3a-receptor (C3aR) with combined alanine (Ala) substitutions of ten C-terminal serine (Ser) or threonine (Thr) residues, which may represent putative phosphorylation sites to characterize their role in ligand-induced C3aR internalization and signaling. Ser475/479 and Thr480/481 as well as Ser449 seemed not to be involved in ligand-induced receptor internalization. Either directly or by a conformational change they even... (More)
The anaphylatoxic peptide C3a is a pro-inflammatory mediator generated during complement activation, whose specific G protein coupled receptor is expressed on granulocytes, monocytes, mast cells, activated lymphocytes, and in the nervous tissue. We have generated RBL-2H3 cell clones stably expressing mutants of the human C3a-receptor (C3aR) with combined alanine (Ala) substitutions of ten C-terminal serine (Ser) or threonine (Thr) residues, which may represent putative phosphorylation sites to characterize their role in ligand-induced C3aR internalization and signaling. Ser475/479 and Thr480/481 as well as Ser449 seemed not to be involved in ligand-induced receptor internalization. Either directly or by a conformational change they even "inhibit" C3aR internalization. In contrast, mutants with Ala substitutions at Ser465/470 and Thr463/466 were poorly internalized, and Thr463 seemed to be the most important C-terminal Thr or Ser residue directly effecting receptor internalization. However, it is likely that other C3aR regions additionally participate in this negative feed-back mechanism since even mutants with multiple Ala substitutions still internalized to a limited degree. Interestingly, in a mutant with a single exchange of Ser449 to Ala, the signal transduction assessed by a Ca(2+) assay and [(35)S]GTP gamma S-binding on HEK cells transiently co-transfected with G-alpha 16 or G-alpha O, respectively, was severely impaired, indicating that this residue of C3aR is involved in G protein coupling. (Less)
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author
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Complement, Receptor, Internalization, Inflammation, Regulation
in
European Journal of Immunology
volume
33
issue
4
pages
920 - 927
publisher
John Wiley & Sons
external identifiers
  • pmid:12672058
  • scopus:0038068127
ISSN
1521-4141
DOI
language
English
LU publication?
no
id
76defca9-7ab9-4ae4-8da6-af72805abeb9 (old id 1127670)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/12672058
http://onlinelibrary.wiley.com/doi/10.1002/eji.200323293/abstract;jsessionid=82F53A0AB2941B4D6AA2D40D552DE71F.f02t01
date added to LUP
2013-10-09 14:10:30
date last changed
2018-05-29 09:20:40
@article{76defca9-7ab9-4ae4-8da6-af72805abeb9,
  abstract     = {The anaphylatoxic peptide C3a is a pro-inflammatory mediator generated during complement activation, whose specific G protein coupled receptor is expressed on granulocytes, monocytes, mast cells, activated lymphocytes, and in the nervous tissue. We have generated RBL-2H3 cell clones stably expressing mutants of the human C3a-receptor (C3aR) with combined alanine (Ala) substitutions of ten C-terminal serine (Ser) or threonine (Thr) residues, which may represent putative phosphorylation sites to characterize their role in ligand-induced C3aR internalization and signaling. Ser475/479 and Thr480/481 as well as Ser449 seemed not to be involved in ligand-induced receptor internalization. Either directly or by a conformational change they even "inhibit" C3aR internalization. In contrast, mutants with Ala substitutions at Ser465/470 and Thr463/466 were poorly internalized, and Thr463 seemed to be the most important C-terminal Thr or Ser residue directly effecting receptor internalization. However, it is likely that other C3aR regions additionally participate in this negative feed-back mechanism since even mutants with multiple Ala substitutions still internalized to a limited degree. Interestingly, in a mutant with a single exchange of Ser449 to Ala, the signal transduction assessed by a Ca(2+) assay and [(35)S]GTP gamma S-binding on HEK cells transiently co-transfected with G-alpha 16 or G-alpha O, respectively, was severely impaired, indicating that this residue of C3aR is involved in G protein coupling.},
  author       = {Settmacher, Britta and Rheinheimer, Claudia and Hamacher, Henning and Ames, Robert S and Wise, Alan and Jenkinson, Lesley and Bock, Daniel and Schaefer, Myriam and Kohl, Jorg and Klos, Andreas},
  issn         = {1521-4141},
  keyword      = {Complement,Receptor,Internalization,Inflammation,Regulation},
  language     = {eng},
  number       = {4},
  pages        = {920--927},
  publisher    = {John Wiley & Sons},
  series       = {European Journal of Immunology},
  title        = {Structure-function studies of the C3a-receptor: C-terminal serine and threonine residues which influence receptor internalization and signaling},
  url          = {http://dx.doi.org/},
  volume       = {33},
  year         = {2003},
}