Swaminath Gayathri, Lee Tae Weon, Kobilka Brian
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford Medical Center, Stanford, Palo Alto, California 94305, USA.
J Biol Chem. 2003 Jan 3;278(1):352-6. doi: 10.1074/jbc.M206424200. Epub 2002 Oct 29.
The activity of G protein-coupled receptors (GPCRs) can be modulated by a diverse spectrum of drugs ranging from full agonists to partial agonists, antagonists, and inverse agonists. The vast majority of these ligands compete with native ligands for binding to orthosteric binding sites. Allosteric ligands have also been described for a number of GPCRs. However, little is known about the mechanism by which these ligands modulate the affinity of receptors for orthosteric ligands. We have previously reported that Zn(II) acts as a positive allosteric modulator of the beta(2)-adrenergic receptor (beta(2)AR). To identify the Zn(2+) binding site responsible for the enhancement of agonist affinity in the beta(2)AR, we mutated histidines located in hydrophilic sequences bridging the seven transmembrane domains. Mutation of His-269 abolished the effect of Zn(2+) on agonist affinity. Mutations of other histidines had no effect on agonist affinity. Further mutagenesis of residues adjacent to His-269 demonstrated that Cys-265 and Glu-225 are also required to achieve the full allosteric effect of Zn(2+) on agonist binding. Our results suggest that bridging of the cytoplasmic extensions of TM5 and TM6 by Zn(2+) facilitates agonist binding. These results are in agreement with recent biophysical studies demonstrating that agonist binding leads to movement of TM6 relative to TM5.
G蛋白偶联受体（GPCRs）的活性可被多种药物调节，这些药物范围从完全激动剂到部分激动剂、拮抗剂和反向激动剂。这些配体中的绝大多数与天然配体竞争结合正构结合位点。也已针对多种GPCRs描述了变构配体。然而，对于这些配体调节受体对正构配体亲和力的机制知之甚少。我们先前曾报道，锌离子（Zn(II)）作为β2 -肾上腺素能受体（β2AR）的正变构调节剂。为了确定负责增强β2AR中激动剂亲和力的锌离子（Zn(2+)）结合位点，我们对位于连接七个跨膜结构域的亲水性序列中的组氨酸进行了突变。组氨酸269（His-269）的突变消除了锌离子（Zn(2+)）对激动剂亲和力的影响。其他组氨酸的突变对激动剂亲和力没有影响。对与组氨酸269相邻残基的进一步诱变表明，半胱氨酸265（Cys-265）和谷氨酸225（Glu-225）对于实现锌离子（Zn(2+)）对激动剂结合的完全变构效应也是必需的。我们的结果表明，锌离子（Zn(2+)）桥接跨膜结构域5（TM5）和跨膜结构域6（TM6）的胞质延伸促进了激动剂结合。这些结果与最近的生物物理研究一致，该研究表明激动剂结合导致跨膜结构域6（TM6）相对于跨膜结构域5（TM5）的移动。
