Regulation of G Protein-Coupled Receptor Kinase 2 in Brains of Opiate-Treated Rats and Human Opiate Addicts

Abstract: The effects of opiate drugs (heroin, morphine, and methadone) on the levels of G protein-coupled receptor kinase 2 (GRK2) were studied in rat and human brain frontal cortices. The density of brain GRK2 was measured by immunoblot assays in acute and chronic opiate-treated rats as well as in opiate-dependent rats after spontaneous or naloxone-precipitated withdrawal and in human opiate addicts who had died of an opiate overdose. In postmortem brains from human addicts, total GRK2 immunoreactivity was not changed significantly, but the level of the membrane-associated kinase was modestly but significantly increased (12%) compared with matched controls. In rats treated chronically with morphine or methadone modest increases of the enzyme levels (only significant after methadone) were observed. Acute treatments with morphine and methadone induced dose- and time-dependent increases (8–22%) in total GRK2 concentrations [higher increases were observed for the membrane-associated enzyme (46%)]. Spontaneous and naloxone-precipitated withdrawal after chronic morphine or methadone induced a marked up-regulation in the levels of total GRK2 in the rat frontal cortex (18–25%). These results suggest that GRK2 is involved in the short-term regulation of μ-opioid receptors in vivo and that the activity of this regulatory kinase in brain could have a relevant role in opiate tolerance, dependence, and withdrawal.     

Regulation of G Protein-Coupled Receptor Signaling by A-Kinase Anchoring Proteins

Specificity of transduction events is controlled at the molecular level by scaffold, anchoring, and adaptor proteins, which position signaling enzymes at proper subcellular localization. This allows their efficient catalytic activation and accurate substrate selection. A-kinase anchoring proteins (AKAPs) are group of functionally related proteins that compartmentalize the cAMP-dependent protein kinase (PKA) and other signaling enyzmes at precise subcellular sites in close proximity to their physiological substrate(s) and favor specific phosphorylation events. Recent evidence suggests that AKAP transduction complexes play a key role in regulating G protein-coupled receptor (GPCR) signaling. Regulation can occur at multiple levels because AKAPs have been shown both to directly modulate GPCR function and to act as downstream effectors of GPCR signaling. In this minireview, we focus on the molecular mechanisms through which AKAP-signaling complexes modulate GPCR transduction cascades.     

Structural Model of Ghrelin Bound to its G Protein-Coupled Receptor

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Reviews in Molecular Biology and Biotechnology: Transmembrane Signaling by G Protein-Coupled Receptors

As the most diverse type of cell surface receptor, the importance heptahelical G protein-coupled receptors (GPCRs) to clinical medicine cannot be overestimated. Visual, olfactory and gustatory sensation, intermediary metabolism, cell growth and differentiation are all influenced by GPCR signals. The basic receptor-G protein-effector mechanism of GPCR signaling is tuned by a complex interplay of positive and negative regulatory events that amplify the effect of a hormone binding the receptor or that dampen cellular responsiveness. The association of heptahelical receptors with a variety of intracellular partners other than G proteins has led to the discovery of potential mechanisms of GPCR signaling that extend beyond the classical paradigms. While the physiologic relevance of many of these novel mechanisms of GPCR signaling remains to be established, their existence suggests that the mechanisms of GPCR signaling are even more diverse than previously imagined.     

Desensitization of the δ-Opioid Receptor Correlates with Its Phosphorylation in SK-N-BE Cells: Involvement of a G Protein-Coupled Receptor Kinase

Abstract: Phosphorylation of G protein-coupled receptors is considered an important step during their desensitization. In SK-N-BE cells, recently presented as a pertinent model for the studies of the human δ-opioid receptor, pretreatment with the opioid agonist etorphine increased time-dependently the rate of phosphorylation of a 51-kDa membrane protein. Immunological characterization of this protein with an antibody, raised against the amino-terminal region of the cloned human δ-opioid receptor, revealed that it corresponded to the δ-opioid receptor. During prolonged treatment with etorphine, phosphorylation increased as early as 15 min to reach a maximum within 1 h. Phosphorylation and desensitization of adenylyl cyclase inhibition paralleled closely and okadaic acid inhibited the resensitization, a result strongly suggesting that phosphorylation of the δ-opioid receptor plays a prominent role in its rapid desensitization. The increase in phosphorylation of the δ-opioid receptor, as well as its desensitization, was not affected by H7, an inhibitor of protein kinase A and protein kinase C, but was drastically reduced by heparin or Zn²⁺, known to act as G protein-coupled receptor kinase (GRK) inhibitors. These results are the first to show, on endogenously expressed human δ-opioid receptor, that a close link exists between receptor phosphorylation and agonist-promoted desensitization and that desensitization involves a GRK.     

G蛋白偶联受体激酶的调控

G蛋白偶联受体激酶(G protein-coupled receptor kinase,GRK)特异地使活化的G蛋白偶联受体(G protein-coupled receptor,GPCR)发生磷酸化及脱敏化,从而终止后者介导的信号转导通路。研究表明,GRK的功能被高度调控,并具有下行调节GPCR的能力。调控GRK功能的机制包括两个层次：(1)多种途径调控激酶的亚细胞定位及活性,包括GPCR介导、G蛋白偶联、磷脂作用、Ca^2 结合蛋白调控、蛋白激酶C活化、MAPK反馈抑制、小窝蛋白抑制等;(2)调控GRK表达水平,主要体现在其与某些疾病的联系。     

Molecular Evolution of CXCR1, a G Protein-Coupled Receptor Involved in Signal Transduction of Neutrophils

Human neutrophils are a type of white blood cell, which forms an early line of defense against bacterial infections. Neutrophils are highly responsive to the chemokine, interleukin-8 (IL-8) due to the abundant distribution of CXCR1, one of the IL-8 receptors on the neutrophil cell surface. As a member of the GPCR family, CXCR1 plays a crucial role in the IL-8 signal transduction pathway in neutrophils. We sequenced the complete coding region of the CXCR1 gene in worldwide human populations and five representative nonhuman primate species. Our results indicate accelerated protein evolution in the human lineage, which was likely caused by Darwinian positive selection. The sliding window analysis and the codon-based neutrality test identified signatures of positive selection at the N-terminal ligand/receptor recognition domain of human CXCR1. [Reviewing Editor: Dr. Manyuan Long] The GenBank accession numbers of sequences reported herein are AY916760–AY916773.     

Atomic Structure of GRK5 Reveals Distinct Structural Features Novel for G Protein-coupled Receptor Kinases

G protein-coupled receptor kinases (GRKs) are members of the protein kinase A, G, and C families (AGC) and play a central role in mediating G protein-coupled receptor phosphorylation and desensitization. One member of the family, GRK5, has been implicated in several human pathologies, including heart failure, hypertension, cancer, diabetes, and Alzheimer disease. To gain mechanistic insight into GRK5 function, we determined a crystal structure of full-length human GRK5 at 1.8 Å resolution. GRK5 in complex with the ATP analog 5′-adenylyl β,γ-imidodiphosphate or the nucleoside sangivamycin crystallized as a monomer. The C-terminal tail (C-tail) of AGC kinase domains is a highly conserved feature that is divided into three segments as follows: the C-lobe tether, the active-site tether (AST), and the N-lobe tether (NLT). This domain is fully resolved in GRK5 and reveals novel interactions with the nucleotide and N-lobe. Similar to other AGC kinases, the GRK5 AST is an integral part of the nucleotide-binding pocket, a feature not observed in other GRKs. The AST also mediates contact between the kinase N- and C-lobes facilitating closure of the kinase domain. The GRK5 NLT is largely displaced from its previously observed position in other GRKs. Moreover, although the autophosphorylation sites in the NLT are >20 Å away from the catalytic cleft, they are capable of rapid cis-autophosphorylation suggesting high mobility of this region. In summary, we provide a snapshot of GRK5 in a partially closed state, where structural elements of the kinase domain C-tail are aligned to form novel interactions to the nucleotide and N-lobe not previously observed in other GRKs.     

The Amino Terminus with a Conserved Glutamic Acid of G Protein-Coupled Receptor Kinases Is Indispensable for Their Ability to Phosphorylate Photoactivated Rhodopsin

To investigate functions of the consensus amino terminus of G protein-coupled receptor kinases (GRKs), two amino terminus-truncated mutants (delta30 or delta15) and two single-amino-acid mutants of conserved acidic residues (D2A or E7A) of human GRK1 were constructed and expressed in human embryonic kidney 293 cells. It was shown that truncated mutations and one single-point mutation (E7A) greatly decreased GRK1's activity to phosphorylate photoactivated rhodopsin (Rho*), whereas the abilities of these mutants to phosphorylate a synthetic peptide substrate and to translocate from cytosol to rod outer segments on light activation were unaffected. Further experiments demonstrated that the same truncated mutations (delta30 or delta15) of GRK2, representative of another GRK subfamily, also abolished the kinase's activity toward Rho*. The similar single-point mutation (E5A) of GRK2 heavily impaired its phosphorylation of Rho* but did not alter its ability to phosphorylate the peptide, and the G329-rhodopsin-augmented peptide phosphorylation by GRK2 (E5A) remained unchanged. Our data, taken together, suggest that the amino terminus as well as a conserved glutamic acid in the region of GRKs appears essential for their ability to functionally interact with G protein-coupled receptors.     

Opiate Receptor: Multiple Effects of Metal Ions

Abstract: The opiate antagonist [³H]diprenorphine ([³H]dip), a universal ligand at the μ, δ, and k opiate receptor subtypes, was used to study the effects of Ca-II, Cu-II, Mg-II, Mn-II, and Na⁺ on the rat cerebral opiate receptor. Two categories of effects were observed: (a) those on the binding rate constants and (b) those on binding capacity. (a) Sodium ions increased on- and off-rates on [³H]dip with a rather small net change in receptor affinity. The effects of Na⁺ and the divalent ions Ca-II, Mg-II, and Mn-II were antagonistic to each other. Ca-II, Mg-II, and the more effective Mn-II decreased receptor association and dissociation rates, again with minimal changes in the overall binding affinity in washed membrane homogenates. Previous studies using equilibrium binding analysis alone failed to detect changes in [³H]dip binding kinetics caused by these metal ions. In untreated rat brain homogenates, however, Ca-II (and to a lesser extent Mg-II) decreased [³H]dip binding, an effect distinct from that on the binding rate constants in washed membrane homogenates. (b) In untreated, Tris-buffer homogenates not containing external metal ions, a gradual decline in [³H]dip binding was observed. Cu-II or an equivalent endogenous divalent metal ion was identified as a causative factor, and Mn-II partially reversed this effect. Moreover, the addition of Mn-II stabilized the [³H]dip binding sites at very low concentrations of the metal (nM to μM range) that did not change the binding rate constants and that were in the physiological range of Mn-II in rat brain. This unique effect of Mn-II may represent a physiological function in the regulation of the opiate receptor that is not shared by Mg-II and Ca-II. The opposite effects of Cu-II and Mn-II on the in vitro receptor stability may be related to their opposite pharmacological effect in vivo. Finally, multiple changes of the effects of the tested metal ions on [³H]dip binding were observed during in vitro membrane homogenate dilution, centrifugation, and washing. These changes indicate that the opiate receptor complex as it exists in vivo may lose some of its functions and control mechanisms in vitro.     

人源孤儿G蛋白偶联受体hGPCRc的分子克隆及其初步鉴定

孤儿G蛋白偶联受体 (orphanGprotein coupledreceptors ,oGPCRs)是最重要的潜在药物靶点 ,对于创新药物研究意义重大 .根据已有文献及相关基因数据库提供的信息 ,利用RT PCR从人结肠组织获得oGPCR某一成员的氨基酸编码序列 ,大小为 10 14bp ,而且与GenBank已登录序列(AB0 835 98)完全一致 ,称之为hGPCRc ;又用相同的引物以健康志愿者血液基因组DNA作为模板进行PCR扩增 ,亦得到同样大小的DNA序列 ,测序显示二者个别碱基不一致 ,但所对应氨基酸序列并无差异 .另外 ,RT PCR对人源部分组织及细胞系的检测结果显示 :hGPCRc在人脑组织表达最高 ,结肠次之 ,其它组织或细胞系如胃、血液、肝、肺、上皮未检测到该基因的表达 .利用相关软件对hGPCRc分别结果显示 :hGPCRc定位于人染色体 13q32 3,与小鼠、大鼠的对应物序列同源性高达85 % ,但与人源其他已知基因的同源性较低 ,对应的氨基酸序列组成了 7个跨膜区段的结构域 .因此 ,hGPCRc符合GPCR的结构特点 ,应为人类oGPCRs的新成员 .     

家鸡G蛋白偶联受体85基因的结构、序列与组织表达分析

G蛋白偶联受体(GPCR)超家族是细胞膜上广泛存在的一类受体,是细胞跨膜信号转导的一类重要受体分子,参与许多生理过程调节。它们中仍有很多至今尚未找到内源性配体,这类受体被称为孤儿型受体。G蛋白偶联受体85(GPR85)是GPCR超家族中孤儿型受体的一员。目前,在非哺乳类脊椎动物中,针对GPR85的研究极少。本研究以家鸡Gallus gallus domesticus为模型,通过反转录PCR和RACE-PCR等方法从脑中克隆到GPR85基因的cDNA全长序列,揭示其基因结构,并用实时荧光定量PCR(qPCR)方法探究了该基因在家鸡各组织中的表达情况。结果显示:家鸡GPR85基因位于1号染色体上,由2个外显子组成,其编码区位于第2个外显子上,长为1 113 bp,可编码1个370个氨基酸的7次跨膜受体蛋白。家鸡GPR85与其他脊椎动物(人Homo sapiens、小鼠Mus musculus、大鼠Rattus norvegicus、热带爪蟾Xenopus tropicalis和斑马鱼Danio rerio)的GPR85具有高度的氨基酸序列一致性(>93%)。qPCR分析发现,GPR85基因mRNA在家鸡全脑、垂体、肾上腺、精巢中有较高表达,而在所检测的其他外周组织中表达极低。本研究首次揭示了家鸡GPR85基因的结构与表达特征,为后续探究GPR85基因在家鸡等非哺乳类中的生理功能奠定基础。     

G protein-coupled receptor dimerisation: Molecular basis and relevance to function

The belief that G protein-coupled receptors exist and function as monomeric, non-interacting species has been largely supplanted in recent years by evidence, derived from a range of approaches, that indicate they can form dimers and/or higher-order oligomeric complexes. Key roles for receptor homo-dimerisation include effective quality control of protein folding prior to plasma membrane delivery and interactions with hetero-trimeric G proteins. Growing evidence has also indicated the potential for many co-expressed G protein-coupled receptors to form hetero-dimers/oligomers. The relevance of this to physiology and function is only beginning to be unravelled but may offer great potential for more selective therapeutic intervention.     

人孤儿受体GPR81分子克隆、组织分布及表达工程细胞株的建立

采用PCR技术分别从人全血基因组DNA及引产胚胎肾组织cDNA中扩增得到gpr81的全长cDNA序列(1041bp),运用生物信息学手段绘制该基因的分子进化树,显示该基因的氨基酸序列与烟酸受体同源性最高;然后,采用RT-PCR法分析该基因表达的组织分布,组织表达谱显示该基因在多种组织均有表达,以心脏及肝脏组织为最高;利用分子克隆手段构建含6×组氨酸(His)标签蛋白的真核表达载体pcDNA3·1(-)/his-myc-A-gpr81,通过脂质体介导,将该重组质粒转染CHO-K1细胞,RT-PCR证实该基因已整合入CHO-K1细胞的基因组中,Western-blot表明GPR81在CHO-GPR81工程细胞株中有表达。组织表达谱的检测和工程细胞株的建立为进一步研究该受体的生物学功能奠定了基础。     

孤儿G蛋白偶联受体hGPCRc的亚细胞定位及组织分布

利用相关生物信息学软件,对从人结肠组织克隆所得某一孤儿G蛋白偶联受体(orphanGprotein_coupledreceptors ,oGPCRs)成员hGPCRc的氨基酸序列进行分析显示,hGPCRc对应的氨基酸序列组成了七个跨膜区段的结构域,具备GPCR的结构特征;然后,将hGPCRc之cDNA与绿色荧光载体pEGFP-N₁ 构建GFP_hGPCRc表达载体,以空白质粒pEGFP-N₁ 作对照,转染CHO-K₁ 细胞,在激光扫描共聚焦显微镜下观察到空白质粒pEGFP-N₁ 转染的细胞表达了GFP并均匀分布于整个细胞,而GFP_hGPCRc转染的细胞观察到荧光清晰聚集于细胞膜和各细胞器质膜上,因而hGPCRc蛋白定位于膜上并稳定表达,与软件分析结果相一致;最后,以RT_PCR检测hGPCRc在2 0周龄胎儿重要组织器官及部分成人组织中的表达情况,结果显示hGPCRc在人心、肾、小脑及结肠等组织均有表达,但在肝、大脑、小肠及肌肉等组织里未检测到表达。该表达谱对于进一步认识hGPCRc在胚胎发育中的作用及生理功能提供了线索。     

Protease Signaling to G Protein-Coupled Receptors: Implications for Inflammation and Pain

Proteases, like thrombin, trypsin, cathepsins, or tryptase, can signal to cells by cleaving in a specific manner, a family of G protein-coupled receptors, the protease-activated receptors (PARs). Proteases cleave the extracellular N-terminal domain of PARs to reveal tethered ligand domains that bind to and activate the receptors. Recent evidence has supported the involvement of PARs in inflammation and pain. Activation of PAR₁, PAR₂, and PAR₄ either by proteinases or by selective agonists causes inflammation inducing most of the cardinal signs of inflammation: swelling, redness, and pain. Recent studies suggest a crucial role for the different PARs in innate immune response. The role of PARs in the activation of pain pathways appears to be dual. Subinflammatory doses of PAR₂ agonists induce hyperalgesia and allodynia, and PAR₂ activation has been implicated in the generation of inflammatory hyperalgesia. In contrast, subinflammatory doses of PAR₁ or PAR₄ increase nociceptive threshold, inhibiting inflammatory hyperalgesia, thereby acting as analgesic mediators. PARs have to be considered as an additional subclass of G protein-coupled receptors that are active participants to inflammation and pain responses and that could constitute potential novel therapeutic targets.     

Internalization of G Protein-Coupled Receptors in Single Olfactory Receptor Neurons

Abstract : Desensitization of many G protein-coupled receptors after ligand binding generally involves phosphorylation of the receptors and internalization of the ligandbound, phosphorylated receptors by a clathrin-mediated endocytic pathway. Olfactory receptor neurons from the channel catfish ( Ictalurus punctatus ) express the G protein-coupled odorant receptors and metabotropic glutamate receptors. To determine whether a clathrin-dependent receptor internalization pathway exists in olfactory receptor neurons, western blotting and immunocytochemistry were used to identify and localize clathrin and dynamin in isolated olfactory neurons. Clathrin and dynamin immunoreactivity was found in the cell bodies, dendrites, and dendritic knobs of the neurons. Using the activity-dependent fluorescent dye FM1-43 to monitor receptor internalization, we show that single olfactory neurons stimulated with the odorant amino acid l -glumate internalized the dye. Odorant-stimulated neurons showed a consistent pattern of internalized FM1-43 fluorescence localized in the cell bodies and dendritic knobs. Odorant-stimulated internalization was unaffected by the caveolae activator okadaic acid and was significantly decreased by a metabotropic glutamate receptor antagonist, suggesting that a functional, clathrindependent, receptor-mediated internalization pathway exists in olfactory receptor neurons.     

Key Residues Defining the μ-Opioid Receptor Binding Pocket: A Site-Directed Mutagenesis Study

Abstract: Structural elements of the rat μ-opioid receptor important in ligand receptor binding and selectivity were examined using a site-directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the μ, δ, and κ receptors (Asn¹⁵⁰ to Ala, His²⁹⁷ to Ala, and Tyr³²⁶ to Phe) and two designed to test for μ/δ selectivity (Ile¹⁹⁸ to Val and Val²⁰² to Ile). Mutation of His²⁹⁷ in transmembrane domain 6 (TM6) resulted in no detectable binding with [³H]DAMGO (³H-labeled d -Ala², N -Me-Phe⁴,Gly-ol⁵-enkephalin), [³H]bremazocine, or [³H]ethylketocyclazocine. Mutation of Asn¹⁵⁰ in TM3 produces a three- to 20-fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, β-endorphin_1–31, JOM-13, deltorphin II, dynorphin_1–13, and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor-binaltorphamine. In contrast, the Tyr³²⁶ mutation in TM7 resulted in a decreased affinity for a wide spectrum of μ, δ, and κ agonists and antagonists. Altering Val²⁰² to Ile in TM4 produced no change on ligand affinity, but Ile¹⁹⁸ to Val resulted in a four- to fivefold decreased affinity for the μ agonists morphine and DAMGO, with no change in the binding affinities of κ and δ ligands.     

Effects of 5,5''-Dithiobis-(2-Nitrobenzoic Acid) on Opiate Binding to Both the Membrane-Bound Receptor and the Partially Purified Opiate Receptor

Pretreatment of partially purified opiate receptor from rat brains with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) decreased opiate agonist binding more effectively than that of antagonist. This agent, at a concentration that inhibits only 3H-agonist binding, increases the IC50 values of agonists but not those of antagonists. We also observed similar effects of DTNB on opiate binding to the membrane-bound receptor that are in good agreement with the published data. Moreover, there was an excellent correlation between the IC50 values of the two different preparations. However, opiate binding to the partially purified receptor was about a thousandfold more sensitive to DTNB than binding to this membrane-bound receptor. Dithiothreitol, a sulfide bond reducing agent, reversed the effects of DTNB on the opiate binding.     

Heterotrimeric G Proteins and the Single-Transmembrane Domain IGF-II/M6P Receptor: Functional Interaction and Relevance to Cell Signaling

The G protein-coupled receptor (GPCR) family represents the largest and most versatile group of cell surface receptors. Classical GPCR signaling constitutes ligand binding to a seven-transmembrane domain receptor, receptor interaction with a heterotrimeric G protein, and the subsequent activation or inhibition of downstream intracellular effectors to mediate a cellular response. However, recent reports on direct, receptor-independent G protein activation, G protein-independent signaling by GPCRs, and signaling of nonheptahelical receptors via trimeric G proteins have highlighted the intrinsic complexities of G protein signaling mechanisms. The insulin-like growth factor-II/mannose-6 phosphate (IGF-II/M6P) receptor is a single-transmembrane glycoprotein whose principal function is the intracellular transport of lysosomal enzymes. In addition, the receptor also mediates some biological effects in response to IGF-II binding in both neuronal and nonneuronal systems. Multidisciplinary efforts to elucidate the intracellular signaling pathways that underlie these effects have generated data to suggest that the IGF-II/M6P receptor might mediate transmembrane signaling via a G protein-coupled mechanism. The purpose of this review is to outline the characteristics of traditional and nontraditional GPCRs, to relate the IGF-II/M6P receptor’s structure with its role in G protein-coupled signaling and to summarize evidence gathered over the years regarding the putative signaling of the IGF-II/M6P receptor mediated by a G protein.