Galpha(q) and Gbetagamma regulate PAR-1 signaling of thrombin-induced NF-kappaB activation and ICAM-1 transcription in endothelial cells.
As thrombin binding to the G protein-coupled proteinase activated receptor-1 (PAR-1) induces endothelial adhesivity to leukocytes through NF-kappaB activation and intercellular adhesion molecule-1 (ICAM-1) expression, we determined the signaling pathways mediating the response. Studies showed that the heterotrimeric G proteins, Galpha(q), and the Gbetagamma dimer were key determinants of the PAR-1 agonist peptide (TFLLRNPNDK)-induced NF-kappaB activation and ICAM-1 expression in endothelial cells. Cotransfection of RGS3T, a regulator of G-protein signaling that inhibits Galpha(q), or alpha-transducin (Galpha(t)), a scavenger of the Gbetagamma, markedly decreased NF-kappaB activity induced by PAR-1 activation. We determined the downstream signaling targets activated by Galpha(q) and Gbetagamma that mediate NF-kappaB activation. Expression of the kinase-defective protein kinase C (PKC)-delta mutant inhibited NF-kappaB activation induced by the constitutively active Galpha(q) mutant, but had no effect on NF-kappaB activity induced by Gbeta(1)gamma(2). In related experiments, NF-kappaB as well as ICAM-1 promoter activation induced by Gbeta(1)gamma(2) were inhibited by the expression of the dominant-negative mutant of 85-kDa regulatory subunit of PI 3-kinase; however, the expression of this mutant had no effect on the response induced by activated Galpha(q). Cotransfection of the catalytically inactive Akt mutant inhibited the NF-kappaB activation induced by the constitutively active PI 3-kinase mutant as well as that by the activated forms of Galpha(q) and PKC-delta. These results support a model in which ligation of PAR-1 induces NF-kappaB activation and ICAM-1 transcription by the engagement of parallel Galphaq/PKC-delta and Gbetagamma/PI3-kinase pathways that converge at Akt.
Rahman
Arshad
A
Department of Pharmacology, College of Medicine, The University of Illinois, Chicago, Ill 60612-7343, USA. ARahman@uic.edu
True
Andrea L
AL
Anwar
Khandaker N
KN
Ye
Richard D
RD
Voyno-Yasenetskaya
Tatyana A
TA
Malik
Asrar B
AB
D000595:Amino Acid Sequence; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D006801:Humans; D051550:I-kappa B Kinase; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics*; D007527:Isoenzymes / Q000378:metabolism; D009154:Mutation; D016328:NF-kappa B / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism; D051745:Protein Kinase C-delta; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects
D020962:Heterotrimeric GTP-Binding Proteins; D051550:I-kappa B Kinase; D018799:Intercellular Adhesion Molecule-1; D007527:Isoenzymes; D016328:NF-kappa B; D009842:Oligopeptides; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D013917:Thrombin; C494918:AKT1 protein, human; C496559:CHUK protein, human; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C497258:PRKCD protein, human