Direct evidence for two distinct G proteins coupling with thrombin receptors in human neuroblastoma SH-EP cells.
Thrombin receptor-G protein coupling was investigated in the human epithelial neuroblastoma cell line, SH-EP. In these cells, both alpha-thrombin and thrombin receptor peptides, SFLLRNP (one-letter amino-acid code), which are newly exposed following cleavage by alpha-thrombin, stimulated GTPase activity about 2-fold over basal activity. Pertussis toxin treatment only partially attenuated alpha-thrombin- and SFLLRNP-stimulated GTPase activity by 50%, whereas antibody raised against synthetic heptapeptide SFLLRNP blocked alpha-thrombin-stimulated phosphoinositide hydrolysis more than 80%. Immunoprecipitation studies using this antibody showed that both Gi2, a subtype of guanine nucleotide-binding regulatory proteins (G proteins) mediating inhibition of adenylyl cyclase, and Gq/G11, a G protein mediating stimulation of phospholipase C, were activated by alpha-thrombin. These data suggest that in these cells the thrombin receptor activates pertussis toxin-sensitive and pertussis toxin-insensitive G proteins simultaneously and directly couples to Gi2 and Gq/G11, which mediate different signaling pathways.
Ogino
Y
Y
Third Department of Internal Medicine, Teikyo University School of Medicine, Chiba, Japan.
Tanaka
K
K
Shimizu
N
N
D000906:Antibodies / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D009447:Neuroblastoma / Q000201:enzymology / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism; D011233:Precipitin Tests; D018179:Receptors, Thrombin / Q000378:metabolism*; D013268:Stimulation, Chemical; D013917:Thrombin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000906:Antibodies; D010446:Peptide Fragments; D010716:Phosphatidylinositols; D018179:Receptors, Thrombin; C086309:thrombin receptor peptide SFLLRNP; D013917:Thrombin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins