Mitchell F M, Heasley L E, Qian N X, Zamarripa J, Johnson G L
Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206, USA.
J Biol Chem. 1995 Apr 14;270(15):8623-8. doi: 10.1074/jbc.270.15.8623.
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.
用铃蟾肽对瑞士3T3成纤维细胞进行促有丝分裂刺激会导致受体介导的一系列效应器激活，包括磷脂酶Cβ和丝裂原活化蛋白（MAP）激酶。用11个氨基酸的[D-精氨酸1，D-苯丙氨酸5，D-色氨酸7,9，亮氨酸11]P物质肽孵育瑞士3T3成纤维细胞，即使在高铃蟾肽浓度下，也能抑制铃蟾肽刺激的细胞增殖和磷脂酶Cβ激活。该肽不抑制Gq样蛋白G16的GTP酶缺陷形式对磷脂酶Cβ的激活，这表明该肽不抑制磷脂酶Cβ，而是作用于G蛋白α亚基活化形式的上游位点。在低铃蟾肽浓度下，该肽抑制MAP激酶激活，但与磷脂酶Cβ不同的是，30 nM铃蟾肽可克服这种抑制作用。在对照瑞士3T3细胞中，铃蟾肽在基础水平之上未显著激活Ras或Raf-1。在用[D-精氨酸1，D-苯丙氨酸5，D-色氨酸7,9，亮氨酸11]P物质肽孵育细胞后，50 nM铃蟾肽使Raf-1的激活水平比基础水平高4至6倍。用[D-精氨酸1，D-苯丙氨酸5，D-色氨酸7,9，亮氨酸11]P物质肽孵育瑞士3T3细胞后，血小板衍生生长因子刺激的PLC、Ras、Raf-1和MAP激酶活性未改变，瑞士3T3细胞的血小板衍生生长因子刺激的生长也未改变。因此，该肽表现为一种拮抗剂，可差异性抑制磷脂酶Cβ和MAP激酶信号转导途径。用该肽观察到的生长停滞表明，铃蟾肽刺激的MAP激酶激活不足以支持瑞士3T3细胞的有丝分裂。
