Crystal Structure of the Human Cannabinoid Receptor CB2

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Structural Features of β2 Adrenergic Receptor: Crystal Structures and Beyond

The beta2-adrenergic receptor (β2AR) family, which is the largest family of cell surface receptors in humans. Extra attention has been focused on the human GPCRs because they have been studied as important protein targets for pharmaceutical drug development. In fact, approximately 40% of marketed drugs directly work on GPCRs. GPCRs respond to various extracellular stimuli, such as sensory signals, neurotransmitters, chemokines, and hormones, to induce structural changes at the cytoplasmic surface, activating downstream signaling pathways, primarily through interactions with heterotrimeric G proteins or through G-protein independent pathways, such as arrestin. Most GPCRs, except for rhodhopsin, which contains covalently linked 11 cis-retinal, bind to diffusible ligands, having various conformational states between inactive and active structures. The first human GPCR structure was determined using an inverse agonist bound β2AR in 2007 and since then, more than 20 distinct GPCR structures have been solved. However, most GPCR structures were solved as inactive forms, and an agonist bound fully active structure is still hard to obtain. In a structural point of view, β2AR is relatively well studied since its fully active structure as a complex with G protein as well as several inactive structures are available. The structural comparison of inactive and active states gives an important clue in understanding the activation mechanism of β2AR. In this review, structural features of inactive and active states of β2AR, the interaction of β2AR with heterotrimeric G protein, and the comparison with β1AR will be discussed.     

Angiotensin Analogs with Divergent Bias Stabilize Distinct Receptor Conformations

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Conformational features of a synthetic model of the first extracellular loop of the angiotensin II AT1A receptor.

The angiotensin II AT1A receptor belongs to the G-protein coupled receptors (GPCRs). Like other membrane proteins, GPCRs are not easily amenable to direct structure determination by the currently available methods. The peptide encompassing the putative first extracellular loop of AT1A (residues Thr88-Leu100, el1) has been synthesized along with a cyclic model where the linear peptide has been covalently linked to a template designed to keep the distance between the peptide termini as expected in the receptor. The conformational features of the two molecules have been studied using circular dichroism and NMR techniques. The region W94PFG97 forms a type-II beta-turn and undergoes a Trp-Pro peptide bond cis-trans isomerization in both peptides confirming that these characteristics are intrinsic to el1. In addition, the presence of the spacer seems to modulate the flexibility of the peptide.     

High-Resolution Crystal Structure of Spectrin SH3 Domain Fused with a Proline-Rich Peptide

Abstract

A new chimeric protein, named WT-CIIA, was designed by connecting the proline-rich decapeptide PPPVPPYSAG to the C-terminus of the alpha-spectrin SH3 domain through a natural twelve-residue linker to obtain a single-chain model that would imitate intramolecular SH3-ligand interaction. The crystal structure of this fusion protein was determined at 1.7 Å resolution. The asymmetric unit of the crystal contained two SH3 globules contacting with one PPPVPPY fragment located between them. The domains are related by the twofold non-crystallographic axis and the ligand lies in two opposite orientations with respect to the conservative binding sites of SH3 domains.     

Photoaffinity scanning in the mapping of the peptide receptor interface of class II G protein-coupled receptors.

The family of G protein-coupled receptors constitutes about 50% of the therapeutic drug targets used in clinical medicine today, although the mechanisms of ligand binding, activation and signal transduction for G protein-coupled receptors are not yet well defined. This review discusses ongoing research using the photoaffinity scanning method to map the bimolecular interface between class II G protein-coupled receptors and their ligands. Furthermore the available computer model of class II peptide ligand docking into the receptor, based on the positional constraints imposed by the photoaffinity scanning analyses, will be discussed briefly. The ultimate goal of these efforts is to understand the molecular basis of receptor binding and therefore to generate a template for rational drug design.     

Structure of the Human Angiotensin II Type 1 (AT1) Receptor Bound to Angiotensin II from Multiple Chemoselective Photoprobe Contacts Reveals a Unique Peptide Binding Mode

Breakthroughs in G protein-coupled receptor structure determination based on crystallography have been mainly obtained from receptors occupied in their transmembrane domain core by low molecular weight ligands, and we have only recently begun to elucidate how the extracellular surface of G protein-coupled receptors (GPCRs) allows for the binding of larger peptide molecules. In the present study, we used a unique chemoselective photoaffinity labeling strategy, the methionine proximity assay, to directly identify at physiological conditions a total of 38 discrete ligand/receptor contact residues that form the extracellular peptide-binding site of an activated GPCR, the angiotensin II type 1 receptor. This experimental data set was used in homology modeling to guide the positioning of the angiotensin II (AngII) peptide within several GPCR crystal structure templates. We found that the CXC chemokine receptor type 4 accommodated the results better than the other templates evaluated; ligand/receptor contact residues were spatially grouped into defined interaction clusters with AngII. In the resulting receptor structure, a β-hairpin fold in extracellular loop 2 in conjunction with two extracellular disulfide bridges appeared to open and shape the entrance of the ligand-binding site. The bound AngII adopted a somewhat vertical binding mode, allowing concomitant contacts across the extracellular surface and deep within the transmembrane domain core of the receptor. We propose that such a dualistic nature of GPCR interaction could be well suited for diffusible linear peptide ligands and a common feature of other peptidergic class A GPCRs.     

Topological analysis of the complex formed between neurokinin A and the NK2 tachykinin receptor

Neurokinin A stimulates physiological responses in the peripheral and central nervous systems upon interacting primarily with the tachykinin NK2 receptor (NK2R). In this study, the structure of NKA bound to the NK2R is characterised by use of fluorescence resonance energy transfer. Four fluorescent NKA analogues with Texas red introduced at amino acid positions 1, 4, 7 and 10 were prepared. When bound to a NK2R carrying enhanced green fluorescent protein at the N-terminus, all peptides reduce green fluorescent protein fluorescence from 10% to 50% due to energy transfer. The derived donor-acceptor distances are 46, 55, 59 and 69 A for the fluorophore linked to positions 1-10, respectively. The monotonic increase in distance clearly indicates that the peptide adopts an extended structure when bound to its receptor. The present data are used, in combination with rhodopsin structure, fluorescence studies, photoaffinity labelling and site-directed mutagenesis data to design a computer model of the NKA-NK2R complex. We propose that the N-terminus of NKA is exposed and accessible to the extracellular medium. Subsequent amino acids of the NKA peptide become progressively more buried residues up to approximately one-third of the transmembrane-spanning domain.     

Peptide fragment of the m3 muscarinic acetylcholine receptor activates G(q) but not G(i2).

G(q), a heterotrimeric guanine nucleotide-binding protein, plays important roles such as the regulation of calcium mobilization and cell proliferation. This protein is considered as a promising drug target for the treatment of cardiac hypertrophy. Selective activation of G(q) would be quite useful for analyzing the role of G(q) in signaling pathways. We synthesized m3i3c-a peptide with 16 amino acid residues that corresponds to the junction between the C-terminus of the third intracellular loop and the sixth transmembrane helix (TM-VI) of human m3 muscarinic acetylcholine receptor, which couples to G(q) but not G(i2). At micromolar concentrations, this peptide was found to activate G(q) but not G(i2). This peptide is the first small compound that selectively activates G(q) but not G(i2). Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Regulation of angiotensinogen by angiotensin II in mouse primary astrocyte cultures

Astrocytes are the major source of angiotensinogen in the brain and play an important role in the brain renin-angiotensin system. Regulating brain angiotensinogen production alters blood pressure and fluid and electrolyte homeostasis. In turn, several physiological and pathological manipulations alter expression of angiotensinogen in brain. Surprisingly, little is known about the factors that regulate astrocytic expression of angiotensinogen. There is evidence that angiotensinogen production in both hepatocytes and cardiac myocytes can be positively regulated via the angiotensin type 1 receptor, but this effect has not yet been studied in astrocytes. Therefore, the aim of this project was to establish whether angiotensin II modulates angiotensinogen production in brain astrocytes. Primary astrocyte cultures, prepared from neonatal C57Bl6 mice, expressed angiotensinogen measured by immunocytochemistry and real-time PCR. Using a variety of approaches we were unable to identify angiotensin receptors on cultured astrocytes. Exposure of cultured astrocytes to angiotensin II also did not affect angiotensinogen expression. When astrocyte cultures were transduced with the angiotensin type 1A receptor, using adenoviral vectors, angiotensin II induced a robust down-regulation (91.4% ± 1.8%, p < 0.01, n = 4) of angiotensinogen gene expression. We conclude that receptors for angiotensin II are present in extremely low levels in astrocytes, and that this concurs with available data in vivo. The signaling pathways activated by the angiotensin type 1A receptor are negatively coupled to angiotensinogen expression and represent a powerful pathway for decreasing expression of this protein, potentially via signaling pathways coupled to Gα(q/11) .     

第一类鱼抗冻蛋白对冰晶生长界面层结构的影响

根据冰晶在水溶液中生长的基本热力学性质,应用多层界面模型,分别得到了冰晶在纯水及抗冻蛋白溶液中生长界面层的吉布斯自由能.由冰晶生长界面层的吉布斯自由能,分析了冰晶在三种不同第一类鱼抗冻蛋白分子溶液中,热平衡状态下生长界面层的微观平衡结构,发现冰晶在抗冻蛋白溶液中生长与其在纯水中生长相比,界面层结构有明显变化,结合抗冻蛋...     

Down-regulation of Cyclooxygenase-2 by the Carboxyl Tail of the Angiotensin II Type 1 Receptor

The enzyme cyclooxygenase-2 (COX-2) plays an important role in the kidney by up-regulating the production of the vasoconstrictor hormone angiotensin II (AngII), which in turn down-regulates COX-2 expression via activation of the angiotensin II type 1 receptor (AT₁) receptor. Chemical inhibition of the catalytic activity of COX-2 is a well-established strategy for treating inflammation but little is known of cellular mechanisms that dispose of the protein itself. Here we show that in addition to its indirect negative feedback on COX-2, AT₁ also down-regulates the expression of the COX-2 protein via a pathway that does not involve G-protein or β-arrestin-dependent signaling. Instead, AT₁ enhances the ubiquitination and subsequent degradation of the enzyme in the proteasome through elements in its cytosolic carboxyl tail (CT). We find that a mutant receptor that lacks the last 35 amino acids of its CT (Δ324) is devoid of its ability to reduce COX-2, and that expression of the CT sequence alone is sufficient to down-regulate COX-2. Collectively these results propose a new role for AT₁ in regulating COX-2 expression in a mechanism that deviates from its canonical signaling pathways. Down-regulation of COX-2 by a short peptide that originates from AT₁ may present as a basis for novel therapeutic means of eliminating excess COX-2 protein.     

人 Toll-like receptor 2 配基模拟肽的初步研究

TLR-2 (Toll-like receptor 2) 是介导天然免疫的重要模式识别分子,可参与识别多种病原体及其产物 . 为探索被 TLR-2 所识别配基的结构共性,以真核细胞表达的人 TLR-2 胞外段蛋白 (A26~T588) 为钓饵筛选噬菌体 12 肽库,获得一高度保守的阳性噬菌体克隆 P12-1 ,实验发现 P12-1 可与不同形式的 TLR-2 胞外段结合,并且可刺激细胞分泌 TNFα,提示 P12-1 可能模拟 TLR-2 配基的结构与生物学活性 .     

From G Protein-coupled Receptor Structure Resolution to Rational Drug Design

A number of recent technical solutions have led to significant advances in G protein-coupled receptor (GPCR) structural biology. Apart from a detailed mechanistic view of receptor activation, the new structures have revealed novel ligand binding sites. Together, these insights provide avenues for rational drug design to modulate the activities of these important drug targets. The application of structural data to GPCR drug discovery ushers in an exciting era with the potential to improve existing drugs and discover new ones. In this review, we focus on technical solutions that have accelerated GPCR crystallography as well as some of the salient findings from structures that are relevant to drug discovery. Finally, we outline some of the approaches used in GPCR structure based drug design.     

小鼠胚胎干细胞分化心肌细胞血管紧张素Ⅱ 1型和2型受体的表达特征

胚胎干细胞(ESC)具有无限增殖和分化为体内3个胚层来源的各种类型组织细胞的潜能,经过体外诱导能够分化为心肌细胞,亦称为胚胎干细胞分化心肌细胞(ESCM).本研究探讨了ESC诱导分化心肌细胞过程中血管紧张素Ⅱ受体(ATR)的亚型AT1R和AT2R的表达特征.10-4mol/L维生素C体外诱导小鼠R1胚胎干细胞分化为自发搏动的心肌细胞,用免疫荧光法检测分化后的细胞表达心肌细胞特异性标志物α辅肌动蛋白.小鼠胚胎干细胞在诱导分化为心肌细胞以后,逆转录聚合酶链反应(RT-PCR)和实时定量RT-PCR(Real-timeRT-PCR)方法检测到ESCM表达AT1R,并且呈时间依赖性逐渐增加的特点,在第14d达到高峰.Western印记法检测AT1R表达特征与RT-PCR结果相符.Western印记法的结果显示,血管紧张素Ⅱ(10-6mol/L)可作为AT1R激动剂激活AT1R,并使其下游的细胞外信号调节激酶(ERK1/2)磷酸化水平上调,预孵育AT1R抑制剂Losartan(10-6mol/L),此作用被抑制.RT-PCR方法显示,与新生小鼠心室肌细胞相比,ESCM的AT2R表达水平较低.     

Crystal structures and increased stabilization of the protein G variants with switched folding pathways NuG1 and NuG2

We recently described two protein G variants (NuG1 and NuG2) with redesigned first hairpins that were almost twice as stable, folded 100-fold faster, and had a switched folding mechanism relative to the wild-type protein. To test the structural accuracy of our design algorithm and to provide insights to the dramatic changes in the kinetics and thermodynamics of folding, we have now determined the crystal structures of NuG1 and NuG2 to 1.8 A and 1.85 A, respectively. We find that they adopt hairpin structures that are closer to the computational models than to wild-type protein G; the RMSD of the NuG1 hairpin to the design model and the wild-type structure are 1.7 A and 5.1 A, respectively. The crystallographic B factor in the redesigned first hairpin of NuG1 is systematically higher than the second hairpin, suggesting that the redesigned region is somewhat less rigid. A second round of structure-based design yielded new variants of NuG1 and NuG2, which are further stabilized by 0.5 kcal/mole and 0.9 kcal/mole.     

Structural insight into the role of the second intracellular loop of the bradykinin 2 receptor in signaling and internalization.

The second cytoplasmic loop (IC2) of the bradykinin B2 receptor plays a vital role in its dynamic life cycle including the activation, internalization, desensitization, and resensitization of this receptor. Here, we probe the structure and function of the IC2, with particular emphasis on threonine-137, which is crucial for signal transduction and internalization. Mutation of this threonine to proline (T137P) produces wild type (WT) signaling and complete inhibition of internalization. Incorporation of aspartate (T137D) leads to a marked reduction in receptor signaling but with WT receptor uptake. The T137D mutation coupled with serine to alanine substitution of S335 and S341 within the distal C-terminus recovers signaling, leading to an actually enhanced arachidonic acid release and phosphoinositide turnover compared to WT bradykinin B2 receptor (BKB2R). To provide a structural basis for the actions of this mutant, the conformational features of IC2 (both WT and mutant) were investigated by high-resolution NMR. The NMR analysis illustrated two prominent alpha-helices at the N- (L123-M138) and C-termini (A149-I156) of the IC2 receptor domain. Incorporating these structural characteristics into a model of BKB2R, we determined that the entire N-terminal helix of IC2 is incorporated as TM3, placing Y131 1.5 helical turns into TM3 and T137 at the membrane surface. The NMR data indicated no structural changes upon substitution of T137D. These results suggest that the altered signaling of the T137D mutant can be attributed to the introduction of a negative charge, indicating that phosphorylation of this residue takes place and participates in the life cycle of this receptor. Additionally, the return to WT signal capacity of the mutation T137D/S335A/S341A, to overcome the deleterious T137D substitution points to a functional interaction between the IC2 and the C-terminus.     

Crystal Structure of G Protein-coupled Receptor Kinase 5 in Complex with a Rationally Designed Inhibitor

G protein-coupled receptor kinases (GRKs) regulate cell signaling by initiating the desensitization of active G protein-coupled receptors. The two most widely expressed GRKs (GRK2 and GRK5) play a role in cardiovascular disease and thus represent important targets for the development of novel therapeutic drugs. In the course of a GRK2 structure-based drug design campaign, one inhibitor (CCG215022) exhibited nanomolar IC₅₀ values against both GRK2 and GRK5 and good selectivity against other closely related kinases such as GRK1 and PKA. Treatment of murine cardiomyocytes with CCG215022 resulted in significantly increased contractility at 20-fold lower concentrations than paroxetine, an inhibitor with more modest selectivity for GRK2. A 2.4 Å crystal structure of the GRK5·CCG215022 complex was determined and revealed that the inhibitor binds in the active site similarly to its parent compound GSK180736A. As designed, its 2-pyridylmethyl amide side chain occupies the hydrophobic subsite of the active site where it forms three additional hydrogen bonds, including one with the catalytic lysine. The overall conformation of the GRK5 kinase domain is similar to that of a previously determined structure of GRK6 in what is proposed to be its active state, but the C-terminal region of the enzyme adopts a distinct conformation. The kinetic properties of site-directed mutants in this region are consistent with the hypothesis that this novel C-terminal structure is representative of the membrane-bound conformation of the enzyme.     

Angiotensin II Type 1 Receptor mRNA Levels in the Brains of Normotensive and Spontaneously Hypertensive Rats

Abstract: The type 1 angiotensin II (All) receptor (AT₁-R) has been implicated in the physiological actions mediated by All in the brain. In view of the reported hyperactivity of the brain All system in the spontaneously hypertensive rat (SHR), we compared the expression of AT,-R mRNAs in the brains of normotensive [Wistar Kyoto (WKY)] and SHR animals. Northern blot analysis showed about three- and ∼20-fold increases in the levels of AT₁-R mRNAs from the hypothalamus and brainstem areas, respectively, of the SHR compared with the WKY rat brain. This was attributable to greater levels of both AT,_1A- and AT,_1B-R mRNA subtypes in these areas from the SHR. These observations suggest that increased All receptor levels in SHR brain may, in part, be a result of increased expression of the AT₁-R gene.