G蛋白偶联受体固有活性研究进展与新药开发

G蛋白偶联受体(G-prote in-coup led receptor,GPCR)是与G蛋白有信号连接的一大类受体家族,是人体内最大的膜受体蛋白家族,是一类具有7个跨膜螺旋的跨膜蛋白受体。GPCR的结构特征和在信号传导中的重要作用决定了其可以作为很好的药物靶标。目前世界药物市场上有三分之一的小分子药物是GPCR的激活剂(agon ist)或拮抗剂(antagon ist)。以其为靶点的药物在医药产业中占据显著地位。在当今前50种最畅销的上市药物中,20%属于G蛋白受体相关药物。近来的研究发现,大多数G蛋白偶联受体具有一个很重要的特性,就是具有固有活性(Constitutive ac-tivity),即无激动剂条件受体自发的维持激活并维持下游信号传导通路的活性。固有活性涉及受体、G蛋白及下游信号通路之间的关系。该文就G蛋白偶联受体固有活性概念、研究进展、反相激动剂与固有活性研究、固有活性与新药开发4个方面,进行以下论述。     

G蛋白偶联受体：激活B类GPCR的新开关

胰高血糖素样肽1（GLP1）受体属于难以被小分子药物靶向作用的G蛋白偶联受体（GPCR）。Bjerre-Knudsen等发现了一个能刺激胰高血糖素依赖性胰岛素释放的小分子GLP1受体激动剂，这可能对研究治疗糖尿病的口服GLP1激动剂有帮助。     

G蛋白信号蛋白的调节剂：新的多功能药物靶点

G蛋白信号调节（RGS）是蛋白新近发现的能调节G蛋白功能的蛋白家族。RGS能通过加速鸟苷三磷酸（GTP）水解抑制G蛋白信号，即其GTP酶活化蛋白（GAP）功能。此外，RGS还能通过其RGS区域和非RGS区域产生非GAP功能。此类蛋白的组织分布特异，受到信号传递物质的调节并能在活细胞产生特定功能，也因而 而成为新的药理和治疗靶点。以RGS蛋白为靶点的药物分为5类：增强内源性激动剂功能的药物；阻断外源性G蛋白偶联受体（GPCR）激动剂脱敏的药物；增强外源性激动剂功能的药物，RGS蛋白效应器信号抑制剂；RGS激动剂。     

组成型激活突变体在G蛋白偶联受体研究中的应用

G蛋白偶联受体(GPCR)组成型活性概念的提出，使组成型激活突变体(CAM)成为研究GPCR的新方法。CAM促进了对GPCR的结构、激活机制、活性调节及病理生理的研究。通过对CAM的研究发现了GPCR激活的关键结构区域，提出配体受体相互作用的”扩充的三元复合物模型”和GPCR激活过程中存在多个中间激活态的观点。CAM在一定程度上模拟了野生型受体激活的失敏过程，并为疾病的研究提供新的思路。     

针对G蛋白偶联受体的药物筛选新方法

G蛋白偶联受体（GPCR）为具有7个跨膜螺旋的蛋白质受体，是人体内最大的蛋白质家族，其为极重要的药物靶点。本文针对GPCR的固有激活和变构效应的药物筛选模型开发新进展和高内涵药物筛选新技术进行综述。     

肾脏中G蛋白偶联受体信号转导的多样性

G蛋白偶联受体(GPCR)是最重要的药物靶点之一;临床有超过30%处方药是直接作用在GPCR上的。在肾脏中,升压素受体、血管紧张素受体、内皮素受体、前列腺素受体和嘌呤受体等都对肾脏的多种功能有重要的调控作用,也是重要的治疗肾病的药物靶点。多种靶向这些肾脏GPCR的激动剂或者拮抗剂已经进入临床应用或者临床测试阶段。然而,这些GPCR药物的设计主要以激动剂和拮抗剂进行区分,与GPCR的功能多样性存在着一定的鸿沟。我们最近在研究靶向血管紧张素受体(AT1R)的药理学研究过程中,不仅发现了高同型半胱氨酸是血管紧张素受体的内源性配体,还发现Arrestin偏向性信号途径不仅可以介导传统的第二波信号途径,还可以在时序上进行第一波信号转导,通过激活TRPC3来促进肾上腺素的释放,从而产生在治疗心血管疾病时的有害作用。我们据此提出了更合理的靶向AT1R开发药物的方法。不仅如此,我们还针对升压素受体的磷酸化编码,阐明了Arrestin对GPCR的磷酸化编码的识别机制,Arrestin的多聚脯氨酸码头的分选机制,以及配体通过操控受体7此跨膜核心与Arrestin的相互作用来指导Arrestin功能的机制。这些研究工作为以后特异性的靶向GPCR的Arrestin信号通路开发药物奠定了基础。     

G蛋白偶联受体的结合在镇痛中的作用[英]

体内组织特异性G蛋白偶联受体（GPCR）对于镇痛药物的发展非常重要，μ-和δ-阿片受体就属于此类。Daniels等的研究表明，同时作用于这两种受体的二价配体有与吗啡相似的镇痛效果，且无副作用。δ-阿片受体与μ-阿片受体激动剂耐受性和依赖性的形成有关，因而研究者提出同时调节两种受体的二价配体可能改善止痛药物的副作用，同时也可为研究受体的协同作用提供有力的工具。     

G蛋白信号调节蛋白作为中枢神经系统药物新靶点研究进展

G蛋白偶联受体(G-prote in-coup led receptors,GPCR)是许多治疗药物的作用靶点。G蛋白信号调节蛋白(regu latorof G-prote in signaling,RGS)属一类新发现的蛋白家族,它们在GPCR信号传导中起重要作用。一般来说RGS可加速G蛋白失活进而终止GPCR信号传导,但也有些RGS同时具有效应分子和信号传递功能。兼具GPCR激动和RGS抑制功能的药物将大大增强信号传导,同时还能增加激动剂的区域特异性。由于RGS的多样性,组织分布特异性以及较强的调节活性,RGS很可能成为寻找新型中枢神经系统疾病治疗药物的新靶点。     

5-HT2C受体去磷酸化：治疗药物成瘾的新靶标

5-羟色胺（5-HT）是中枢神经系统中调节情绪的神经递质，通过对中脑-边缘多巴胺系统的调节和与其受体相互作用影响大脑功能。研究表明，5-HT2C与药物成瘾性密切相关，其受体激动剂可以阻断药物成瘾时的强制性行为，但具有焦虑、厌食、情绪低落，运动功能紊乱，阴茎勃起等副作用，但最新研究数据表明，5-HT2C受体激动剂可以在细胞内被诱导，从而减轻其副作用。     

何谓药物的内在活性?它与药物效应的关系如何?

<正> 通过受体发挥作用的药物,据其效应,可分为激动剂、部分激动剂和拮抗剂。其中,激动药能激活受体,产生生物效应;拮抗剂不能引起效应,却可阻滞激动剂(递质或药物)的作用。那么,决定药物归属的因素是什么?是药物本身的固有特性,即内在活性,或叫药物与受体结合后激动受体的能力。 按照Ariens修改的受体占领学说,在理论上,完全激动剂具有强的内在活性,以1表示;部分激动剂在0～1之间;拮抗剂内在活性为0。与完全激动剂不同,部分激动剂具有激动剂和拮抗剂两种特性。部分激动剂与完全激动剂相互作     

Constitutive activity of G-protein coupled receptors: emphasis on serotonin receptors

Several lines of evidence indicate that G-protein coupled receptors (GPCR) may exist in a state that allows a tonic level of stimulation in vivo (constitutive activity). Several native forms of GPCR, when expressed in recombinant cell lines, display significant signal transduction stimulation in the absence of activating ligand. Many GPCR, including three serotonin receptors, display robust constitutive activation upon the mutation of a single amino acid, indicating mutations producing inappropriate constitutive activation may be etiological factors in diseases. If constitutive activity of GPCR is as common a phenomenon as some researchers suspect, this would suggest significant alterations in the classical model of ligand-receptor interactions. One of the most significant implications of constitutive activity for pharmacologists and medicinal chemists, is the possibility of developing drugs that lower the level of constitutive activity. Such compounds have been termed inverse agonists . These drugs, in theory, would have different physiological effects, and therefore possibly different therapeutic potential, than classical competitive receptor antagonists ( neutral antagonists ). Theoretical issues concerning constitutive activity in the GPCR family and some of the evidence supporting the existence of constitutive activity in the GPCR family is reviewed. Studies are presented demonstrating the procedures for producing and characterizing constitutive activated forms of serotonin receptors, including the demonstration of inverse agonist activity of drugs on these receptors.     

Constitutive activity of G-protein-coupled receptors: cause of disease and common property of wild-type receptors

The aim of this review is to provide a systematic overview on constitutively active G-protein-coupled receptors (GPCRs), a rapidly evolving area in signal transduction research. We will discuss mechanisms, pharmacological tools and methodological approaches to analyze constitutive activity. The two-state model defines constitutive activity as the ability of a GPCR to undergo agonist-independent isomerization from an inactive (R) state to an active (R*) state. While the two-state model explains basic concepts of constitutive GPCR activity and inverse agonism, there is increasing evidence for multiple active GPCR conformations with distinct biological activities. As a result of constitutive GPCR activity, basal G-protein activity increases. Until now, constitutive activity has been observed for more than 60 wild-type GPCRs from the families 1-3 and from different species including humans and commonly used laboratory animal species. Additionally, several naturally occurring and disease-causing GPCR mutants with increased constitutive activity relative to wild-type GPCRs have been identified. Alternative splicing, RNA editing, polymorphisms within a given species, species variants and coupling to specific G-proteins all modulate the constitutive activity of GPCRs, providing multiple regulatory switches to fine-tune basal cellular activities. The most important pharmacological tools to analyze constitutive activity are inverse agonists and Na(+) that stabilize the R state, and pertussis toxin that uncouples GPCRs from G(i)/G(o)-proteins. Constitutive activity is observed at low and high GPCR expression levels, in native systems and in recombinant systems, and has been reported for GPCRs coupled to G(s)-, G(i)- and G(q)-proteins. Constitutive activity of neurotransmitter GPCRs may provide a tonic support for basal neuronal activity. For the majority of GPCRs known to be constitutively active, inverse agonists have already been identified. Inverse agonists may be useful in the treatment of neuropsychiatric and cardiovascular diseases and of diseases caused by constitutively active GPCR mutants.     

Physiological relevance of constitutive activity of 5-HT2A and 5-HT2C receptors

It is generally accepted that seven-transmembrane receptors have the capacity to regulate cellular signaling systems in the absence of occupancy by a ligand (i.e. the receptors display constitutive activity). Drugs can increase (agonists), decrease (inverse agonists) or not change (antagonists) receptor activity towards a cellular effector. Moreover, some drugs (protean ligands) have multiple pharmacological properties (e.g. agonism towards one response and inverse agonism towards another response coupled to the same receptor and measured from the same cells, simultaneously). In this article, we describe response-dependent constitutive activity and ligand pharmacology for 5-HT2A and 5-HT2C receptors in vitro. Moreover, we provide evidence that 5-HT2A and 5-HT2C receptor constitutive activity is physiologically relevant in vivo and suggest that strong consideration should be given to the impact of constitutive receptor activity on disease and the therapeutic potential of inverse agonism.     

Constitutive activity and inverse agonism at the alpha1adrenoceptors

Mutations of G protein-coupled receptors (GPCR) can increase their constitutive (agonist-independent) activity. Some of these mutations have been artificially introduced by site-directed mutagenesis, others occur spontaneously in human diseases. The alpha(1B)adrenoceptor was the first GPCR in which point mutations were shown to trigger receptor activation. This article briefly summarizes some of the findings reported in the last several years on constitutive activity of the alpha(1)adrenoceptor subtypes, the location where mutations have been found in the receptors, the spontaneous activity of native receptors in recombinant as well as physiological systems. In addition, it will highlight how the analysis of the pharmacological and molecular properties of the constitutively active adrenoceptor mutants provided an important contribution to our understanding of the molecular mechanisms underlying the mechanism of receptor activation and inverse agonism.     

Constitutive receptor activity series: endogenous inverse agonists and constitutive receptor activity in the melanocortin system

The recent discovery of melanocortin receptor mutations that selectively decrease constitutive receptor activity in obese individuals supports the physiological relevance of constitutive melanocortin receptor activity and its control by an endogenous inverse agonist. Furthermore, studies using mice that lack endogenous melanocortin receptor agonists show that differences in coat color are caused by different degrees of constitutive melanocortin receptor signaling regulated by an endogenous inverse agonist. Thus, the regulation of constitutive activity of melanocortin receptors is important for the normal control of pigmentation and body-weight homeostasis.     

Inverse agonist activity at the alpha(2A)-adrenergic receptor

Constitutive activation of G protein-coupled receptors (GPCRs) is now well recognized and many classical GPCR antagonists have been found to be inverse agonists. For the alpha(2A)-adrenergic receptor (AR) we determine the relative inverse efficacies of a series of antagonists and utilize the extended ternary complex model to estimate the fraction of constitutively active mutant (CAM) receptors in the active state. Stable Chinese hamster ovary cell lines expressing the porcine alpha(2A)-AR in its wild-type (WT) and constitutively activated (CAM-T373K) form were isolated. Activation of both G(i) and G(s) was enhanced for CAM receptors. cAMP production was suppressed in cells with the CAM alpha(2A)-AR and this suppression was reversed by alpha(2)-adrenergic antagonists with an order of inverse efficacy of rauwolscine > yohimbine > RX821002 > MK912, whereas phentolamine and idazoxan were essentially neutral antagonists. This striking difference in inverse efficacy between idazoxan and RX821002 may account for in vivo pharmacological differences between these two alpha(2)-adrenergic antagonists. Agonist binding affinity to the non-G protein-coupled CAM receptor was 3- to 9-fold higher than to WT, whereas binding of the most efficacious inverse agonists, yohimbine and rauwolscine, was 1.7- and 2.1-fold weaker. Analysis of this difference by the extended ternary complex model indicates that approximately 50% of the CAM alpha(2A)-AR is in the active (R*) state although there is no detectable constitutive activity of the WT receptor in the absence of agonist.     

G-protein-coupled receptor pharmacology: examining the edges between theory and proof

Over the past year, several salubrious concepts in the field of G-protein-coupled receptors (GPCRs) have been brought to the forefront of GPCR research, and theoretical models that describe their activation continue to be amended to accommodate new experimental data. Pharmacologists have traditionally divided ligands into agonists, which stimulate receptors, and antagonists, which block receptor activation. More recently, however, scientists have come to realize that GPCRs can also exhibit basal activity, and with this knowledge they have come to the understanding that many drugs previously classified as neutral antagonists, actually demonstrate 'negative efficacies'. Given the breadth of the subject matter, and the large number of publications on GPCRs produced each year, this review will highlight only the following topics: (i) allosteric modulators of GPCRs; (ii) functional selectivity and its relationship to drug efficacy; (iii) the prominence of inverse agonists as therapeutics; and (iv) new technologies that are being introduced that may allow us to better understand the 'when and how' of GPCR signaling.     

G protein-coupled receptors and their signaling pathways: classical therapeutical targets susceptible to novel therapeutic concepts

In recent years, new strategies in cancer therapy have been developed targeting key signaling molecules in the receptor tyrosine kinase signal transduction pathway. In contrast, most therapeutical concepts to manipulate G protein-coupled receptors (GPCR)-mediated disorders are still limited to the use of receptor-specific agonists or antagonists. Visible progress in the understanding of GPCR signaling complexity, especially the detection of several families of highly target- and cell-specific regulator proteins of GPCRs, G proteins, and effector components may open new horizons to develop novel therapeutical concepts targeting GPCR signaling elements. Thus, this review will focus on different molecular levels that may be of particular interest in terms of new drug development such as: (i) GPCR subtypes, allosteric binding sites, dimerization and constitutive activity, the use of RAMPs (receptor-activity-modifying proteins) and RASSLs (receptor activated solely by synthetic ligands); (ii) AGS (activators of G protein signaling) and RGS (regulators of G protein signaling) proteins which modify G protein activity; (iii) the high diversity of isozymes involved in the generation, signal transmission, and degradation of second messenger molecules.     

The effect of mutations in the DRY motif on the constitutive activity and structural instability of the histamine H(2) receptor

In previous studies we showed that the wild-type histamine H(2) receptor stably expressed in Chinese hamster ovary cells is constitutively active. Because constitutive activity of the H(2) receptor is already found at low expression levels (300 fmol/mg protein) this receptor is a relatively unique member of the G-protein-coupled receptor (GPCR) family and a useful tool for studying GPCR activation. In this study the role of the highly conserved DRY motif in activation of the H(2) receptor was investigated. Mutation of the aspartate 115 residue in this motif resulted in H(2) receptors with high constitutive activity, increased agonist affinity, and increased signaling properties. In addition, the mutant receptors were shown to be highly structurally instable. Mutation of the arginine 116 residue in the DRY motif resulted also in a highly structurally instable receptor; expression of the receptor could only be detected after stabilization with either an agonist or inverse agonist. Moreover, the agonist affinity at the Arg-116 mutant receptors was increased, whereas the signal transduction properties of these receptors were decreased. We conclude that the Arg-116 mutant receptors can adopt an active conformation but have a decreased ability to couple to or activate the G(s)-protein. This study examines the pivotal role of the aspartate and arginine residues of the DRY motif in GPCR function. Disruption of receptor stabilizing constraints by mutation in the DRY motif leads to the formation of active GPCR conformations, but concomitantly to GPCR instability.