title: Regulation of C3a receptor signaling in human mast cells by G protein coupled receptor kinases.
abstract: BACKGROUND
  The complement component C3a activates human mast cells via its cell surface G protein coupled receptor (GPCR) C3aR. For most GPCRs, agonist-induced receptor phosphorylation leads to receptor desensitization, internalization as well as activation of downstream signaling pathways such as ERK1/2 phosphorylation. Previous studies in transfected COS cells overexpressing G protein coupled receptor kinases (GRKs) demonstrated that GRK2, GRK3, GRK5 and GRK6 participate in agonist-induced C3aR phosphorylation. However, the roles of these GRKs on the regulation of C3aR signaling and mediator release in human mast cells remain unknown.
  
  
  RESULTS
  We utilized lentivirus short hairpin (sh)RNA to stably knockdown the expression of GRK2, GRK3, GRK5 and GRK6 in human mast cell lines, HMC-1 and LAD2, that endogenously express C3aR. Silencing GRK2 or GRK3 expression caused a more sustained Ca(2+) mobilization, attenuated C3aR desensitization, and enhanced degranulation as well as ERK1/2 phosphorylation when compared to shRNA control cells. By contrast, GRK5 or GRK6 knockdown had no effect on C3aR desensitization, but caused a significant decrease in C3a-induced mast cell degranulation. Interestingly, GRK5 or GRK6 knockdown rendered mast cells more responsive to C3a for ERK1/2 phosphorylation.
  
  
  CONCLUSIONS
  This study demonstrates that GRK2 and GRK3 are involved in C3aR desensitization. Furthermore, it reveals the novel finding that GRK5 and GRK6 promote C3a-induced mast cell degranulation but inhibit ERK1/2 phosphorylation via C3aR desensitization-independent mechanisms. These findings thus reveal a new level of complexity for C3aR regulation by GRKs in human mast cells.
authors.affiliation: Department of Pathology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Guo
authors.forename: Hariharan
authors.initials: H
authors.lastname: Subramanian
authors.forename: Kshitij
authors.initials: K
authors.lastname: Gupta
authors.forename: Hydar
authors.initials: H
authors.lastname: Ali
mesh_terms: D015550:Cell Degranulation / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D015926:Complement C3a / Q000378:metabolism*; D054768:G-Protein-Coupled Receptor Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008407:Mast Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics; D011951:Receptors, Complement / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics
chemical_list: D034741:RNA, Small Interfering; D011951:Receptors, Complement; C085567:complement C3a receptor; D015926:Complement C3a; D054768:G-Protein-Coupled Receptor Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3