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.