Differential modulation of bombesin-stimulated phospholipase C beta and mitogen-activated protein kinase activity by [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P.
Mitogenic stimulation of Swiss 3T3 fibroblasts with bombesin results in receptor-mediated activation of a complex array of effectors, including phospholipase C beta and mitogen-activated protein (MAP) kinase. Incubation of Swiss 3T3 fibroblasts with the 11-amino acid [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P peptide inhibited bombesin-stimulated cell proliferation and phospholipase C beta activation even at high bombesin concentrations. The peptide did not inhibit the activation of phospholipase C beta by a GTPase-deficient form of the Gq-like protein, G16, indicating that the peptide does not inhibit phospholipase C beta and is acting at a point upstream of the activated form of the G protein alpha subunit. The peptide inhibited MAP kinase activation at low bombesin concentrations, but unlike phospholipase C beta, this inhibition could be overcome with 30 nM bombesin. In control Swiss 3T3 cells, bombesin did not measurably activate Ras or Raf-1 above basal levels. Following incubation of the cells with the [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P peptide, 50 nM bombesin activated Raf-1 4-6-fold over basal levels. Platelet-derived growth factor-stimulated activities of PLC, Ras, Raf-1, and MAP kinase were unaltered after incubation of Swiss 3T3 cells with the [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P peptide, as was platelet-derived growth factor-stimulated growth of the Swiss 3T3 cells. Thus, the peptide behaves as an antagonist that differentially inhibited phospholipase C beta and MAP kinase signal transduction pathways. The growth arrest observed with the peptide indicates that the bombesin-stimulated activation of MAP kinase is not sufficient to support mitogenesis in Swiss 3T3 cells.
Mitchell
F M
FM
Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206, USA.
Heasley
L E
LE
Qian
N X
NX
Zamarripa
J
J
Johnson
G L
GL
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001839:Bombesin / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D004789:Enzyme Activation; D051379:Mice; D008969:Molecular Sequence Data; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013373:Substance P / Q000031:analogs & derivatives* / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism*
D047428:Protein Kinase Inhibitors; D013373:Substance P; C045800:substance P, Phe(5)-Trp(7,9)-Leu(11)-; D011494:Protein Kinases; D010738:Type C Phospholipases; D001839:Bombesin