Importance of the 10-13 region of glucagon for its receptor interactions and activation of adenylate cyclase.
Importance of the 10-13 region of glucagon for its receptor interactions and activation of adenylate cyclase.
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胰高血糖素 10-13 区域对其受体相互作用和腺苷酸环化酶激活的重要性。
DOI：
10.1021/bi00361a014
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Hruby,VJ
中科院分区：
文献类型：
--
作者：
Krstenansky,JL;Trivedi,D;Hruby,VJ
亚利桑那大学化学系，图森，亚利桑那州85721摘要：通过合成胰高血糖素类似物[Phe13]胰高血糖素(2)、[Phe10]胰高血糖素(3)、[Phe10]胰高血糖素(4)、[Phe10, 13]胰高血糖素(5)、[Pro11]胰高血糖素(6)、[Pro11， Gly12]胰高血糖素(7)、[Alan]胰高血糖素(8)和[Oac11-13]胰高血糖素(9)，研究了胰高血糖素Tyr10-Sern-Lys12-Tyr13区在胰高血糖素受体结合相互作用和激活中的作用。这些类似物采用固相合成方法在对甲基苯并苯胺或具有保护的7V”-/eri-丁基羰基氨基酸的Merrifield树脂上合成。通过透析、阳离子交换色谱、凝胶过滤和制备反相高效液相色谱（HPLC）进行纯化，得到的产品经薄层色谱和高效液相色谱证明均质，并经氨基酸分析、测序和高效液相色谱分析证实均质。通过测量肝质膜腺苷酸环化酶的刺激和胰高血糖素从胰高血糖素受体的[125I]特异性位移来检测其生物活性。这些研究结果表明，虽然胰高血糖素的生物学“信息”区位于其他位置，但10-13区在胰高血糖素-胰高血糖素受体相互作用中具有多重作用：(1)该区域提供与受体直接结合相互作用的功能基团，(2)该区域与受体相互作用，使胰高血糖素的“转导信息”部分相互作用并激活受体。胰腺激素胰高血糖素的10-13区(1)（图1）通过半合成修饰胰高血糖素在胰高血糖素受体相互作用中的作用已被积极研究。这项工作最近得到了回顾（Hruby et al., 1985; Bromer, 1983）。对胰高血糖素的一种或两种酪氨酸残基进行硝化或碘化，可提高兔体内胰高血糖素释放葡萄糖的能力（Patterson & Bromer, 1973; Bromer et al., 1973）。然而，酪氨酸残基的胺化会导致效力的丧失。Lin等人（1976）提出，碘化增加了酪氨酸残基与受体的疏水相互作用，并证明酚基的电离会导致结合效力的降低。位置10的酪氨酸具有浓度依赖的pKa （Frank & Pekar, 1974）。在稀溶液中，pKa= 9.7，但在浓溶液中上升到10.1。Tyr13残基的pKa值与浓度无关，为10.4。这一发现推翻了Gratzer和heaven（1969）的早期建议，并导致Korn和Ottensmeyer（1983）提出Tyr10残基的pKa降低可能是由于其在分子内氢键中的相互作用。由于CD1研究表明，由于胰高血糖素分子的聚集，胰高血糖素的构象是浓度依赖的（Gratzer et al., 1967; Gratzer & heaven, 1979; sere & Brooks, 1969）， pKa对Tyr10残基浓度的依赖可能是由于分子内或分子间的相互作用。通过x射线分析，在碱性条件下结晶的胰高血糖素的晶体结构主要为a-螺旋三聚体结构（Sasaki et al., 1975）。该三聚体中的一个关联位点位于Trp25的侧链之间。本研究得到了美国公共卫生服务（AM 21085）、默克夏普公司（Merck Sharp & Dohme）和吉布森斯蒂芬斯神经制药公司的资助。
Department of Chemistry, University of Arizona, Tucson, Arizona 85721 Received November 7, 1985; Revised Manuscript Received February 13, 1986 abstract: The role of the Tyr10-Sern-Lys12-Tyr13 region of glucagon in the binding interaction and activation of the glucagon receptor was investigated by means of the synthetic glucagon analogues [Phe13] glucagonamide (2),[Phe10] glucagonamide (3),[Phe10] glucagon (4),[Phe10, 13] glucagon (5),[Pro11] glucagon (6),[Pro11, Gly12] glucagonamide (7),[Alan] glucagon (8), and [Oac11-13] glucagonamide (9). These analogues were synthesized by solid-phase peptide synthesis on p-methylbenzhydrylamine or Merrifield resins with protected 7V “-/eri-butyloxycarbonyl amino acids. Purification by dialysis, cation-exchange chromatography, gel filtration, and preparative reverse-phase high-performance liquid chromatography (HPLC) gave products that proved homogeneous by thin-layer chromatography and HPLC and on analysis by amino acid analysis, by sequencing, and by-chymotryptic peptide mappingwith HPLC. Biological activities were examined by measurement of the stimulation of liver plasma membrane adenylate cyclase and by specific displacement of [125I] glucagon from glucagon receptors. The results of these studies indicate that while the biological“message” region of glucagon is located elsewhere, the 10-13 region has multiple roles in the glucagon-glucagon receptor interaction:(1) this region provides functional groups for direct binding interaction with the receptor, and (2) this region interacts with the receptor in such a way as to allow the “transduction message” portion of glucagonto interact and activate the receptor. e 10-13 region of the pancreatic hormone glucagon (1)(Figure 1) hasbeen actively studied for its role in glucagon’s receptor interaction by means of semisynthetic modifications of glucagon. This work has recently been reviewed (Hruby et al., 1985; Bromer, 1983). Nitration or iodination of either or both of the tyrosine residues of glucagon increases the ability of the hormone to release glucose in rabbits (Patterson & Bromer, 1973; Bromer et al., 1973). However, amination of the tyrosine residues results in a loss of potency. Lin et al.(1976) have proposed that the iodination increases the hydrophobic interaction of the tyrosine residues with the receptor and have demonstrated that ionization of the phenolic groups results in a reduction in binding potency. The tyrosine at position 10 has a concentration-dependent pKa (Frank & Pekar, 1974). In dilute solution, the pKa= 9.7 but rises to a value of 10.1 in concentrated solutions. The pKa of the Tyr13 residue has a concentration-independent value of 10.4. This finding reversed earlier suggestions of Gratzer and Beaven (1969) and led Korn and Ottensmeyer (1983) topropose that the lowered pKa of the Tyr10 residue could be due to its interacting in an intramolecular hydrogen bond. Since CD1 studies show that the conformation of glucagon is concen-tration-dependent due to aggregation of the glucagon molecules (Gratzer et al., 1967; Gratzer & Beaven, 1979; Srere & Brooks, 1969), the pKa dependence on concentration of the Tyr10 residue could be due to either intra-or intermolecular interactions. The crystal structure of glucagon crystallized at alkaline pH determined by X-ray analysis is a largely a-helical trimeric structure (Sasaki et al., 1975). One of the sites of association in the trimer is between the side chains of Trp25, fThis research was supported by grants from the US Public Health Service (AM 21085), Merck Sharp & Dohme, and Gibson-Stephens Neuropharmaceuticals.
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