The C terminus of the human C5a receptor (CD88) is required for normal ligand-dependent receptor internalization.
The biological effects of the potent inflammatory mediator C5a, a complement split product, on human neutrophils and monocytes are limited by the rapid internalization of its specific receptor (C5aR, CD88). The C terminus of the C5aR is phosphorylated after stimulation with C5a of phorbol ester, and this phosphorylation might lead to receptor internalization. In this context, we have studied the effects on C5aR internalization of C5a, phorbol 12-myristate 13-acetate (PMA), the protein kinase inhibitor staurosporine, and pertussis toxin on rat basophilic RBL.2H3 cells stably transfected with the human wild-type or mutant C5aR. C5aR mutants lacked either part of the cytosolic C terminus, including suggested major phosphorylation sites, or a putative phosphorylation motif for protein kinase C in the third cytosolic loop. Additionally, agonist-induced internalization was analyzed on HEK293 cells co-transfected with C5aR and the pertussis toxin-resistant G protein alpha subunit, G alpha 16. Staurosporine-sensitive agonist-dependent C5aR internalization could be detected, suggesting that C5aR phosphorylation, most likely of the C terminus, participates in this type of internalization. In contrast, PMA-induced C5aR internalization seems to be independent of putative phosphorylation sites in either the truncated section of the C terminus or the third cytosolic loop. The phorbol ester-induced C5aR internalization may, therefore, be caused by an indirect and less specific effect of protein kinase C on the internalization machinery. Manipulation of the pertussis toxin-sensitive or -resistant G protein-dependent signal transduction had no effect on ligand-induced internalization.
Bock
D
D
Institut für Medizinische Mikrobiologie der MHH, Hannover, Germany.
Martin
U
U
Gärtner
S
S
Rheinheimer
C
C
Raffetseder
U
U
Arseniev
L
L
Barker
M D
MD
Monk
P N
PN
Bautsch
W
W
Köhl
J
J
Klos
A
A
D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D002460:Cell Line; D015936:Complement C5a / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000235:genetics; D006801:Humans; D007457:Iodine Radioisotopes; D007668:Kidney / Q000166:cytology / Q000196:embryology; D015471:Leukemia, Basophilic, Acute / Q000378:metabolism; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000494:pharmacology; D037342:Pertussis Toxin; D011485:Protein Binding; D047428:Protein Kinase Inhibitors; D051381:Rats; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000819:agonists
D015703:Antigens, CD; D018076:DNA, Complementary; D007457:Iodine Radioisotopes; D008024:Ligands; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D010566:Virulence Factors, Bordetella; D015936:Complement C5a; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate