Sinnett-Smith J, Lehmann W, Rozengurt E
Imperial Cancer Research Fund, Lincoln's Inn Fields, London, U.K.
Biochem J. 1990 Jan 15;265(2):485-93. doi: 10.1042/bj2650485.
Bombesin-like neuropeptides, including mammalian gastrin-releasing peptide (GRP), are potent mitogens for Swiss 3T3 cells. In this study, we have characterized the bombesin receptor in membrane preparations from these cells. Addition of Mg2+ during cell homogenization was essential to preserve 125I-GRP binding activity in the resulting membrane preparation. The effect of Mg2+ was concentration dependent, with a maximum at 5 mM. Specific binding of 125I-GRP was saturable; Scatchard analysis indicated a single class of high-affinity sites of Kd = (2.1 +/- 0.3) x 10(-10) M at 15 degrees C and Kd = (1.9 +/- 0.4) x 10(-10) M at 37 degrees C, and a maximum binding capacity of 580 +/- 50 fmol/mg of protein (15 degrees C) or 604 +/- 40 fmol/mg of protein (37 degrees C). The kinetically derived dissociation constant was 1.5 x 10(-10) M. 125I-GRP binding was inhibited in a concentration-dependent manner by various peptides containing the highly conserved C-terminal heptapeptide of the bombesin family, including bombesin, GRP, neuromedin B and the 8-14 fragment of bombesin. In contrast, a variety of structurally unrelated mitogens and neuropeptides had no effect. The cross-linking agent ethyleneglycolbis(succinimidylsuccinate) covalently linked 125I-GRP to a single Mr 75 000-85 000 protein in membrane preparations of 3T3 cells. Affinity labelling of this molecule was specific and dependent on the presence of Mg2+ during membrane preparation. Finally, the non-hydrolysable GTP analogue guanosine-5'-[gamma-thio]triphosphate (GTP[S]) caused a concentration-dependent inhibition of 125I-GRP binding and cross-linking to 3T3 cell membranes [concentration giving half-maximal inhibition (IC50) approximately 0.2 microM]. The inhibitory effect was specific (GMP, ATP or ATP[S] had no effect at 10 microM) and was due to an increase in Kd from (1.7 +/- 0.2) x 10(-10) M to (4.3 +/- 0.6) x 10(-10) M in the presence of 10 microM-GTP[S]. This modulation of ligand affinity and cross-linking implies that the bombesin receptors that mediate mitogenesis in Swiss 3T3 cells are coupled to a guanine-nucleotide-binding-protein signal-transduction pathway.
蛙皮素样神经肽，包括哺乳动物促胃泌素释放肽（GRP），是瑞士3T3细胞的强效促有丝分裂原。在本研究中，我们对这些细胞的膜制剂中的蛙皮素受体进行了表征。细胞匀浆过程中添加Mg2+对于在所得膜制剂中保留125I-GRP结合活性至关重要。Mg2+的作用呈浓度依赖性，在5 mM时达到最大值。125I-GRP的特异性结合是可饱和的；Scatchard分析表明，在15℃时存在一类高亲和力位点，Kd = (2.1 +/- 0.3) x 10(-10) M，在37℃时Kd = (1.9 +/- 0.4) x 10(-10) M，最大结合容量为580 +/- 50 fmol/mg蛋白质（15℃）或604 +/- 40 fmol/mg蛋白质（37℃）。动力学推导的解离常数为1.5 x 10(-10) M。125I-GRP结合受到含有蛙皮素家族高度保守C末端七肽的各种肽的浓度依赖性抑制，包括蛙皮素、GRP、神经降压素B和蛙皮素的8-14片段。相比之下，多种结构不相关的促有丝分裂原和神经肽没有作用。交联剂乙二醇双（琥珀酰亚胺基琥珀酸酯）将125I-GRP共价连接到3T3细胞膜制剂中的一种单一的Mr 75 000-85 000蛋白质上。该分子的亲和标记是特异性的，并且取决于膜制备过程中Mg2+的存在。最后，不可水解的GTP类似物鸟苷-5'-[γ-硫代]三磷酸（GTP[S]）导致125I-GRP与3T3细胞膜的结合和交联受到浓度依赖性抑制[产生半数最大抑制（IC50）的浓度约为0.2 microM]。抑制作用是特异性的（10 microM时GMP、ATP或ATP[S]没有作用），并且是由于在10 microM-GTP[S]存在下Kd从(1.7 +/- 0.2) x 10(-10) M增加到(4.3 +/- 0.6) x 10(-10) M。配体亲和力和交联的这种调节意味着在瑞士3T3细胞中介导有丝分裂的蛙皮素受体与鸟嘌呤核苷酸结合蛋白信号转导途径偶联。
