Structure-function studies of the C3a-receptor: C-terminal serine and threonine residues which influence receptor internalization and signaling.
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.
Settmacher
Britta
B
Department of Medical Microbiology, Medical School Hannover, Hannover, Germany.
Rheinheimer
Claudia
C
Hamacher
Henning
H
Ames
Robert S
RS
Wise
Alan
A
Jenkinson
Lesley
L
Bock
Daniel
D
Schaefer
Myriam
M
Köhl
Jörg
J
Klos
Andreas
A
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004705:Endocytosis; D044385:GTP-Binding Protein alpha Subunits; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins; D009154:Mutation; D051381:Rats; D011951:Receptors, Complement / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D012694:Serine / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013912:Threonine / Q000235:genetics / Q000502:physiology*
D044385:GTP-Binding Protein alpha Subunits; D008565:Membrane Proteins; D011951:Receptors, Complement; C085567:complement C3a receptor; C084618:olfactory G protein subunit alpha olf; D013912:Threonine; D012694:Serine; C112631:G protein alpha 16; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium