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

• Published in: European journal of immunology Vol. 33; no. 4; pp. 920 - 927
• Main Authors: Settmacher, Britta, Rheinheimer, Claudia, Hamacher, Henning, Ames, Robert S., Wise, Alan, Jenkinson, Lesley, Bock, Daniel, Schaefer, Myriam, Köhl, Jörg, Klos, Andreas
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY‐VCH Verlag 01.04.2003
• Subjects: Animals; Calcium - metabolism; Cell Line; Complement; Endocytosis; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gq-G11; Heterotrimeric GTP-Binding Proteins - metabolism; Humans; Inflammation; Internalization; Kinetics; Membrane Proteins; Mutation; Rats; Receptor; Receptors, Complement - chemistry; Receptors, Complement - genetics; Receptors, Complement - physiology; Regulation; Serine - genetics; Serine - physiology; Signal Transduction; Structure-Activity Relationship; Threonine - genetics; Threonine - physiology
• ISSN: 0014-2980; 1521-4141
• DOI: 10.1002/eji.200323293

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 Ca2+ assay and [35S]GTPγS‐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.