Chen L, Hoeger C, Rivier J, Fitzpatrick V D, Vandlen R L, Tashjian A H
Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts, 02115, USA.
Biochem Biophys Res Commun. 1999 May 19;258(3):689-94. doi: 10.1006/bbrc.1999.0699.
The availability of subtype-specific agonists and antagonists for somatostatin (SS) receptors (SSTRs) will be important for elucidation of the function of each receptor isoform in vivo. A SS analog, des-AA1,2,5-[D-Trp8, IAmp9]SS (CH275), has been shown previously to bind preferentially to SSTR1. In this report, we identify structural determinants in the ligand and receptor responsible for the selective binding of CH275 to SSTR1 by modifying both the ligand and the receptor. We propose that IAmp9 in CH275, like Lys9 in SS, interacts with Asp137 in the middle of the third transmembrane domain of SSTR1 to form an ion pair, while other residues unique to SSTR1 conbribute to binding selectivity of CH275 for SSTR1. Replacement of Asp137 with Asn resulted in loss of binding of radiolabeled SS and decreased potencies of both SS and CH275 to induce a change in the extracellular acidification rate measured by microphysiometry. The structural determinants for specific binding to SSTR1 were mapped in chimeric SSTR1/SSTR2 receptors. One chimera, 2beta, with the N-terminus to second transmembrane domain (TM2) from SSTR2 and the remainder of the receptor from SSTR1, had low affinity for CH275. Furthermore, when a single residue, Leu107, in TM2 of SSTR1 was replaced with Phe, the corresponding residue in SSTR2, a 20-fold decrease in affinity for CH275 with no significant change in affinity for SS was observed. A reciprocal change from Phe to Leu in the chimeric receptor 2beta resulted in a 10-fold increase in affinity for CH275. Thus, Leu107 is an important determinant for CH275 binding to SSTR1. To identify the moiety in CH275 which could interact with Leu107, a new analog des-AA1,2,5-[D-Trp8, Amp9]SS was prepared. This analog bound to both SSTR1 and SSTR2 with similar affinities; thus, subtype selectivity was lost. Collectively, these data support a binding model for CH275 in which the positively charged IAmp interacts with the negatively charged Asp137 in TM3 of SSTR1 and the isopropyl group of IAmp forms a hydrophobic interaction with Leu107 in TM2.
生长抑素（SS）受体（SSTRs）亚型特异性激动剂和拮抗剂的可得性对于阐明每种受体亚型在体内的功能至关重要。一种SS类似物，去AA1,2,5-[D-色氨酸8，异亮氨酰胺9]SS（CH275），先前已被证明优先与SSTR1结合。在本报告中，我们通过修饰配体和受体来确定配体和受体中负责CH275与SSTR1选择性结合的结构决定因素。我们提出，CH275中的异亮氨酰胺9，就像SS中的赖氨酸9一样，与SSTR1第三个跨膜结构域中间的天冬氨酸137相互作用形成离子对，而SSTR1特有的其他残基有助于CH275对SSTR1的结合选择性。将天冬氨酸137替换为天冬酰胺导致放射性标记SS的结合丧失，并且SS和CH275诱导通过微生理测定法测量的细胞外酸化率变化的效力降低。在嵌合SSTR1/SSTR2受体中绘制了与SSTR1特异性结合的结构决定因素。一种嵌合体2β，其N端至第二个跨膜结构域（TM2）来自SSTR2，受体的其余部分来自SSTR1，对CH275具有低亲和力。此外，当SSTR1的TM2中的单个残基亮氨酸107被SSTR2中的相应残基苯丙氨酸取代时，观察到对CH275的亲和力降低20倍，而对SS的亲和力没有显著变化。在嵌合受体2β中从苯丙氨酸到亮氨酸的反向变化导致对CH275的亲和力增加10倍。因此，亮氨酸107是CH275与SSTR1结合的重要决定因素。为了确定CH275中可与亮氨酸107相互作用的部分，制备了一种新的类似物去AA1,2,5-[D-色氨酸8，氨酰胺9]SS。该类似物以相似的亲和力与SSTR1和SSTR2结合；因此，亚型选择性丧失。总体而言，这些数据支持CH275的一种结合模型，其中带正电荷的异亮氨酰胺与SSTRl的TM3中带负电荷的天冬氨酸137相互作用，并且异亮氨酰胺的异丙基与TM2中的亮氨酸107形成疏水相互作用。
