Befort K, Tabbara L, Bausch S, Chavkin C, Evans C, Kieffer B
Ecole Supérieure de Biotechnologie, Parc d'Innovation, Illkirch-Graffenstaden, France.
Mol Pharmacol. 1996 Feb;49(2):216-23.
Opioids are cationic compounds that mediate their biological action through three highly homologous receptors (mu, delta, and kappa) known to belong to the G protein-coupled receptor (GPR) family. The third putative transmembrane domain of opioid receptors contains a conserved aspartate residue that is typically found in biogenic amine binding GPRs and is generally believed to form an ion pair with the cationic neurotransmitters. Using site-directed mutagenesis, we investigated the possibility of an identical role for this residue (Asp128) in the mouse delta-opioid receptor. Removal of the carboxylate group via an aspartate-to-alanine mutation did not modify binding affinity of a representative set of opioid compounds, including bremazocine, diprenorphine, naloxone, Tyr-D-Thr-Gly-Phe-Leu-Thr, [D-Ala2,D-Leu5]enkephalin, cyclic[D-penicillamine2,D-penicillamine5]enkephalin, deltorphin II, (+/-)-4-[(a-R*)-a-[(2S*,5R*)-4-allyl-2,5-di-methyl-1- piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide, and naltrindole. It nevertheless decreased receptor expression level and affected the binding of three agonists ([D-Ala2,D-Leu5]enkephalin, Tyr-D-Thr-Gly-Phe-Leu-Thr, and (+/-)-4-[(a-R*)-a-[(2S*,5R*)-4-allyl-2,5-di- methyl-1-piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide) when the receptor was under Na(+)-induced low affinity state. On the other hand, the aspartate-to-asparagine mutation strongly impaired the binding of all of the above ligands and highlighted differential modes of interaction for alkaloids and peptides. Finally, removal of the homologous carboxylate group in the mouse mu receptor had distinct effects because it dramatically reduced the binding potency of some, but not all, tested ligands. Taken together, these results demonstrate that (i) the direct ligand/receptor interaction previously demonstrated for the beta-adrenergic receptor does not take place in the delta receptor, (ii) Asp128 nevertheless contributes to stabilization of the spatial conformation of the binding pocket, and (iii) these conclusions cannot be extended to the closely related mu receptor.
阿片类药物是阳离子化合物，它们通过三种高度同源的受体（μ、δ和κ）介导其生物学作用，这些受体属于G蛋白偶联受体（GPR）家族。阿片受体的第三个假定跨膜结构域包含一个保守的天冬氨酸残基，该残基通常存在于生物胺结合GPR中，一般认为它与阳离子神经递质形成离子对。我们使用定点诱变技术研究了小鼠δ阿片受体中这个残基（Asp128）是否具有相同的作用。通过将天冬氨酸突变为丙氨酸去除羧基，并未改变包括布马佐辛、二丙诺啡、纳洛酮、酪氨酰-D-苏氨酰-甘氨酰-苯丙氨酰-亮氨酰-苏氨酸、[D-丙氨酸2，D-亮氨酸5]脑啡肽、环[D-青霉胺2，D-青霉胺5]脑啡肽、强啡肽II、（±）-4-[（α-R*）-α-[(2S*，5R*)-4-烯丙基-2，5-二甲基-1-哌嗪基]-3-羟基苄基]-N，N-二乙苯甲酰胺和纳曲吲哚在内的一组代表性阿片类化合物的结合亲和力。然而，当受体处于Na(+)诱导的低亲和力状态时，它降低了受体表达水平并影响了三种激动剂（[D-丙氨酸2，D-亮氨酸5]脑啡肽、酪氨酰-D-苏氨酰-甘氨酰-苯丙氨酰-亮氨酰-苏氨酸和（±）-4-[（α-R*）-α-[(2S*，5R*)-4-烯丙基-2，5-二甲基-1-哌嗪基]-3-羟基苄基]-N，N-二乙苯甲酰胺）的结合。另一方面，天冬氨酸突变为天冬酰胺的突变强烈损害了上述所有配体的结合，并突出了生物碱和肽的不同相互作用模式。最后，去除小鼠μ受体中的同源羧基有不同的影响，因为它显著降低了一些（但不是所有）测试配体的结合效力。综上所述，这些结果表明：（i）先前在β肾上腺素能受体中证明的直接配体/受体相互作用在δ受体中不发生；（ii）Asp128仍然有助于结合口袋空间构象的稳定；（iii）这些结论不能扩展到密切相关的μ受体。
