Wu J M, Nitecki D E, Biancalana S, Feldman R I
Department of Protein Biochemistry and Biophysics, Berlex Biosciences, Richmond, California 94804-0099, USA.
Mol Pharmacol. 1996 Nov;50(5):1355-63.
Human bombesin receptor subtype 3 (BRS-3) was cloned based on its homology to the human gastrin-releasing peptide (GRP) receptor and neuromedin B (NMB) receptor. Some bombesin-like peptides were shown to activate BRS-3 expressed in Xenopus laevis oocytes, but only at relatively high concentrations, which suggests that BRS-3 is an orphan receptor. To study the pharmacology of BRS-3 in the context of a mammalian cell, we used BR2 cells, which are Balb/3T3 fibroblasts transfected with BRS-3 cDNA. A number of bombesin-like peptides found in mammals and amphibians stimulated calcium mobilization in BR2 cells but exhibited no effect on nontransfected parental Balb/3T3 cells. Of these peptides, NMB (EC50 approximately 1-10 microM) was the most active for stimulation of calcium mobilization. Testing of a series of NMB analogs truncated at the amino terminus and carboxyl terminus indicated that the minimal size of NMB required for retention of full activity was Ac-NMB(3-10). Systematically replacing each residue with alanine, or changing its chirality, demonstrated that the carboxyl-terminal residues His8, Phe9, and Met10 of NMB are important for optimal activity. We also tested whether a number of bombesin (BN) analogs that are potent pure or partial antagonists of the GRP receptor can activate BRS-3 in BR2 cells. One such analog, D-Phe6-BN(6-13) propyl amide, activated BRS-3-mediated calcium mobilization with an EC50 level of 84 nM. Through additional synthesis, we generated a significantly more potent analog, D-Phe6-Phe13-BN(6-13) propyl amide, which displayed an EC50 level of 5 nM for activation of BRS-3. Taken together, our data show that the core portions of bombesin-like peptides required for activation of BRS-3 are similar to those necessary for activation of the GRP and NMB receptors and thus provide pharmacological evidence that BRS-3 is in the BN receptor family. Furthermore, we have identified an agonist of BRS-3, namely D-Phe6-Phe13-BN(6-13) propyl amide, which is roughly 1000-fold more potent than BRS-3 agonists described previously.
人胃泌素释放肽受体亚型3（BRS - 3）是根据其与人胃泌素释放肽（GRP）受体和神经介素B（NMB）受体的同源性克隆得到的。一些胃泌素释放肽样肽已被证明可激活非洲爪蟾卵母细胞中表达的BRS - 3，但仅在相对较高的浓度下才能激活，这表明BRS - 3是一种孤儿受体。为了在哺乳动物细胞环境中研究BRS - 3的药理学特性，我们使用了BR2细胞，它是用BRS - 3 cDNA转染的Balb / 3T3成纤维细胞。在哺乳动物和两栖动物中发现的许多胃泌素释放肽样肽可刺激BR2细胞中的钙动员，但对未转染的亲代Balb / 3T3细胞没有影响。在这些肽中，NMB（半数有效浓度约为1 - 10 microM）对刺激钙动员最具活性。对一系列在氨基末端和羧基末端截短的NMB类似物进行测试表明，保留全部活性所需的NMB最小长度为Ac - NMB(3 - 10)。通过用丙氨酸系统地替换每个残基或改变其手性，证明NMB的羧基末端残基His8、Phe9和Met10对最佳活性很重要。我们还测试了一些作为GRP受体的强效纯拮抗剂或部分拮抗剂的胃泌素释放肽（BN）类似物是否能激活BR2细胞中的BRS - 3。其中一种类似物，D - Phe6 - BN(6 - 13)丙酰胺，以84 nM的半数有效浓度激活了BRS - 3介导的钙动员。通过进一步合成，我们得到了一种活性显著更高的类似物，D - Phe6 - Phe13 - BN(6 - 13)丙酰胺，其激活BRS - 3的半数有效浓度为5 nM。综上所述，我们的数据表明激活BRS - 3所需的胃泌素释放肽样肽的核心部分与激活GRP和NMB受体所需的部分相似，因此提供了药理学证据证明BRS - 3属于BN受体家族。此外，我们鉴定出了一种BRS - 3激动剂，即D - Phe6 - Phe13 - BN(6 - 13)丙酰胺，其效力比先前描述的BRS - 3激动剂高约1000倍。
