MOLECULAR CLONING OF THE BOMBESIN RECEPTOR

哺乳动物蛙皮素肽胃泌素释放肽（GRP）和 神经介肽B（NMB）介导一系列正常人的生物学反应， 细胞，包括促进一些人的旁分泌或自分泌生长 小细胞肺癌（SCLC）细胞。最近我们报道了克隆 三种不同的啮齿动物和人类 铃蟾肽受体（胃泌素释放肽受体（GRP-R）， 神经调节素-B受体（NMB-R）和铃蟾肽受体亚型3（BRS-3））， 在正常和恶性细胞中具有不同的表达模式。这些 受体由七个假定的跨膜结构域组成， 短的细胞内和细胞外环，其与 磷脂酶C活化、磷酸肌醇代谢和钙 通过百日咳毒素不敏感的G蛋白进行动员。GRP-R结合 GRP以高亲和力结合，NMB-R以高亲和力结合NMB，BRS-3以高亲和力结合 两种肽的亲和力都相对较低。建设和 GRP-R/NMB-R嵌合受体的表征表明， 跨膜结构域5对于NMB的高亲和力结合是关键的， NMB-R，特别是Ile 216。定点突变研究强调 丝氨酸和苏氨酸在C-末端尾部结构域的重要性， GRP-R用于快速和有效的受体内化。类似 受体突变分析表明，15个氨基酸直接C- 跨膜结构域7末端形成了 效应子偶联，与第二个中的特定氨基酸结合， 和第三细胞内环。

The mammalian bombesin peptides gastrin releasing peptide (GRP) and neuromedin B (NMB) mediate a range of biological responses in normal cells, including promotion of paracrine or autocrine growth in some human small cell lung carcinoma (SCLC) cells. Recently we reported the cloning and characterization of three pharmacologically distinct rodent and human bombesin peptide receptors (gastrin-releasing peptide receptor (GRP-R), neuromedin-B receptor (NMB-R), and bombesin receptor subtype 3 (BRS-3)), with distinct patterns of expression in normal and malignant cells. These receptors consist of seven putative membrane-spanning domains separated by short intracellular and extracellular loops, which are coupled to phospholipase C activation, inositol phosphate metabolism, and calcium mobilization through pertussis toxin insensitive G-proteins. GRP-R binds GRP at high affinity, NMB-R binds NMB at high affinity, and BRS-3 binds both peptides at relatively low affinity. Construction and characterization of GRP-R/NMB-R chimeric receptors indicates that transmembrane domain 5 is critical for high affinity binding of NMB by NMB-R, in particular Ile216. Site directed mutagenesis studies highlight the importance of serines and threonines in the C-terminal tail domain of the GRP-R for rapid and efficient receptor internalization. Similar receptor mutagenesis analysis show that 15 amino acids immediately C- terminal to transmembrane domain 7 form a structural motif essential for effector coupling, in conjunction with specific amino acids in the second and third intracellular loops.