Green S A, Spasoff A P, Coleman R A, Johnson M, Liggett S B
Department of Medicine (Pulmonary), University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA.
J Biol Chem. 1996 Sep 27;271(39):24029-35. doi: 10.1074/jbc.271.39.24029.
An inherent therapeutic limitation of many G protein-coupled receptor agonists is a short duration of action due to rapid dissociation from receptors. Salmeterol is a modified beta-adrenergic receptor (betaAR) agonist that has a long duration of action at the beta2AR (but not the beta1AR) both in vitro and in vivo and that is persistent despite extensive washout of the agonist. It has been proposed that salmeterol binds not only to the active site of the beta2AR (localized to receptor transmembrane spanning domains (TMDs) 3 and 5) but also to another site (termed the "exosite") that anchors it to the receptor and provides for repetitive active-site binding events. To identify the location of this exosite, we used site-directed mutagenesis to replace beta2AR amino acids 149-173 (within TMD4) with beta1AR sequence. The resulting constructs were then expressed in COS-7 cells for radioligand binding studies. Using this approach, when this domain was replaced with the analogous beta1AR sequence, the ability of salmeterol to persist at the receptor under washout conditions was reduced by 67%. The results from more selective mutants (S-(149-166), S-(164-173), and S-(149-158)) indicated that a limited 10-amino acid region (beta2AR residues 149-158), localized at the interface of the cytoplasm and the transmembrane domain, contains a critical determinant for exosite binding. Whereas CHW cells stably expressing wild-type beta2AR displayed persistent salmeterol-promoted cAMP accumulation despite agonist washout, substitution of beta2AR residues 149-158 with beta1AR sequence resulted in a 56% attenuation of salmeterol-promoted cAMP accumulation under identical washout conditions. A reverse chimera was also studied, which consisted of a substitution of beta2AR residues 152-156 into the beta1AR. This substitution was found to confer exosite binding to the beta1AR. None of these mutations decreased the affinity of salmeterol for the receptor at the active site as assessed in competition binding studies. Anchored binding to this motif thus represents a novel mechanism by which agonists like salmeterol can repetitively activate receptors. Conceivably, with other G protein-coupled receptors that have similar motifs, anchored ligands can be designed to provide for long durations of action by this mechanism.
许多G蛋白偶联受体激动剂存在一个固有的治疗局限性，即由于其与受体的快速解离而导致作用持续时间较短。沙美特罗是一种经过修饰的β-肾上腺素能受体（βAR）激动剂，在体外和体内对β2AR（而非β1AR）均具有长效作用，并且即便在大量冲洗激动剂后仍能持续存在。有人提出，沙美特罗不仅与β2AR的活性位点（定位于受体跨膜结构域（TMD）3和5）结合，还与另一个位点（称为“别构位点”）结合，该位点将其锚定在受体上，并支持重复性的活性位点结合事件。为了确定这个别构位点的位置，我们使用定点诱变技术，将β2AR的149 - 173位氨基酸（在TMD4内）替换为β1AR序列。然后将所得构建体在COS - 7细胞中表达，用于放射性配体结合研究。采用这种方法，当该结构域被类似的β1AR序列替换时，沙美特罗在冲洗条件下持续存在于受体上的能力降低了67%。来自更具选择性的突变体（S-(149 - 166)、S-(164 - 173)和S-(149 - 158)）的结果表明：位于细胞质与跨膜结构域界面的一个有限的10个氨基酸区域（β2AR的第(149 - 158)位残基）包含别构位点结合的关键决定因素。尽管冲洗激动剂，稳定表达野生型β2AR的CHW细胞仍显示出沙美特罗促进的cAMP持续积累，而在相同的冲洗条件下，将β2AR的(149 - 158)位残基替换为β1AR序列导致沙美特罗促进的cAMP积累减少了56%。还研究了一种反向嵌合体，它由将β2AR的(152 - 156)位残基替换到β1AR中组成。发现这种替换赋予了β1AR别构位点结合能力。在竞争结合研究中评估发现，这些突变均未降低沙美特罗对受体活性位点的亲和力。因此，与该基序的锚定结合代表了一种新机制，通过该机制，像沙美特罗这样的激动剂可以重复激活受体。可以想象，对于具有类似基序的其他G蛋白偶联受体，通过这种机制可以设计出具有长效作用的锚定配体。
