Functional Coupling of G Proteins to Endothelin Receptors Is Ligand and Receptor Subtype Specific

1. The aims of the present study were (a) to determine the identity of the G proteins with which the endothelin receptor interacts and whether this interaction is subtype specific and (b) to determine whether agonist exposure can result in specific coupling between the endothelin receptor and G proteins.2. Coupling between endothelin A (ET_A) or endothelin B (ET_B) receptors and G proteins was assessed in two fibroblast cell lines, each expressing one receptor subtype. Four ligands, ET-1, ET-3, SRTXb, and SRTXc, were used for receptor stimulation. The G protein -subunit coupled to the receptor was identified by immunoprecipitation with an antibody against the endothelin receptor and immunoblotting with specific antibodies against different G protein -subunits.3. Unstimulated ET_A and ET_B receptors (ET_AR and ET_BR, respectively) were barely coupled to G_o. The unstimulated ET_AR coimmunoprecipitated with G_i3, whereas the unstimulated ET_BR was much less strongly coupled to G_i3. The coupling of ET_BR to G_i1G_i2 -subunits was much stronger than the coupling of ET_AR to these -subunits. Stimulation with the different ET agonists also resulted in differential coupling of G proteins to the receptor subtypes. All four ligands caused a strong increase in coupling of the ET_BR to G_i3, whereas coupling of the ET_AR to this subunit was not affected by ET-1 and was even decreased by SRTXc. On the other hand, all four ligands caused a much greater increase in the coupling of ET_AR to G_q/G₁₁ than in the coupling of ET_BR to these -subunits. Ligand-induced coupling between the receptors and the G_i1 and G_i2 -subunits is similar for the two receptor subtypes. The same was true for ligand-induced coupling of the receptors to G_o, except that ET-3 increased the coupling of this -subunit to ET_BR and decreased the coupling to ET_AR. Taken together, the results of this study show that coupling between ET receptors and G proteins is ligand and receptor subtype specific.4. It remains to be established whether this diversity of receptor–G protein coupling is of relevance for the various endothelin signaling pathways and/or pathological states.     

Retrovirus-Mediated Expression of the Galr1 Galanin Receptor: Implication for Efficient Stable Expression of Functional G Protein-Coupled Receptors

Abstract

The rat GalR1 galanin receptor was used as a prototypic G protein-coupled receptor to test the feasibility of heterologous expression in a retrovirus-based system. The system utilizes an independent retroviral vector pMX, a virus-packaging cell line BOSC23 and a pre-B cell line BA/F3 as the host for expression. A polyclonal cell population that expresses high ligand affinity (K_D = 0.18 nM) and high level (7 pmol/mg) of GalR1 was generated within days with no drug sensitivity-based selection. The expression represented a 20-fold increase over the expression level of GalR1 achieved in CHO cells. The affinity of galanin for the expressed receptor was decreased by 19-fold in the presence of GTP-γ-S, suggesting that the expression system can produce active galanin receptor functionally coupled to G proteins. The fast and efficient method to generate stable cell lines and to prepared large quantities of receptors may provide a general application for expression of other G protein-coupled receptors.     

G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

We have recently reported that cannabinoid agonists can up-regulate and enhance the activity of serotonin 2A (5-HT_2A) receptors in the prefrontal cortex (PFCx). Increased expression and activity of cortical 5-HT_2A receptors has been associated with neuropsychiatric disorders, such as anxiety and schizophrenia. Here we report that repeated CP55940 exposure selectively up-regulates GRK5 proteins in rat PFCx and in a neuronal cell culture model. We sought to examine the mechanism underlying the regulation of GRK5 and to identify the role of GRK5 in the cannabinoid agonist-induced up-regulation and enhanced activity of 5-HT_2A receptors. Interestingly, we found that cannabinoid agonist-induced up-regulation of GRK5 involves CB₂ receptors, β-arrestin 2, and ERK1/2 signaling because treatment with CB₂ shRNA lentiviral particles, β-arrestin 2 shRNA lentiviral particles, or ERK1/2 inhibitor prevented the cannabinoid agonist-induced up-regulation of GRK5. Most importantly, we found that GRK5 shRNA lentiviral particle treatment prevented the cannabinoid agonist-induced up-regulation and enhanced 5-HT_2A receptor-mediated calcium release. Repeated cannabinoid exposure was also associated with enhanced phosphorylation of CB₂ receptors and increased interaction between β-arrestin 2 and ERK1/2. These latter phenomena were also significantly inhibited by GRK5 shRNA lentiviral treatment. Our results suggest that sustained activation of CB₂ receptors, which up-regulates 5-HT_2A receptor signaling, enhances GRK5 expression; the phosphorylation of CB₂ receptors; and the β-arrestin 2/ERK interactions. These data could provide a rationale for some of the adverse effects associated with repeated cannabinoid agonist exposure.     

Functional Properties of the Nicotinic and Glutamatergic Receptors

Abstract

Several important physiological processes such as plasticity, memory, cell death, and rhythmic firing involve the N-methyl-D-aspartate (NMDA)-type of glutamatergic receptor. Nicotinic acetylcholine receptors (AChR), recently demonstraied in the central nervous system (CNS), are also of great interest. We have used several ligands to study the physiology and pharmacology of the agonist recognition sites of these receptors and kinetic properties of associated ion channels using whole-cell, cell-attached or outside-out variants of the patchclamp technique. Enzymatically dissociated frog interosseal muscles were used to study peripheral AChRs, and tissue cultured or acutely dissociated hippocampal neurons and retinal ganglion cells (RGCs) for CNS receptors. For reproducible and fast solution changes when recording in the whole-cell configuration, we modified the “U”-shaped tube system to obtain different outputs from the same outflow port. We used fluorescent rhodamine-labeled latex microspheres to identify RGCs. Our studies provide important information regarding the molecular mechanisms of several clinically used agents. Additionally, similar actions of noncompetitive agents on the ion channels of the nicotinic ACh and NMDA receptors support the concept of a receptor ion channel superfamily.     

Characterization of CB1 Receptors on Rat Neuronal Cell Cultures: Binding and Functional Studies Using the Selective Receptor Antagonist SR 141716A

Abstract: This study was undertaken to characterize further the central cannabinoid receptors in rat primary neuronal cell cultures from selected brain structures. By using [³H]SR 141716A, the specific CB1 receptor antagonist, we demonstrate in cortical neurons the presence of a high density of specific binding sites ( B _max = 139 ± 9 fmol/mg of protein) displaying a high affinity ( K _D = 0.76 ± 0.09 n M ). The two cannabinoid receptor agonists, CP 55940 and WIN 55212-2, inhibited in a concentration-dependent manner cyclic AMP production induced by either 1 µ M forskolin or isoproterenol with EC₅₀ values in the nanomolar range (4.6 and 65 n M with forskolin and 1.0 and 5.1 n M with isoproterenol for CP 55940 and WIN 55212-2, respectively). Moreover, in striatal neurons and cerebellar granule cells, CP 55940 was also able to reduce the cyclic AMP accumulation induced by 1 µ M forskolin with a potency similar to that observed in cortical neurons (EC₅₀ values of 3.5 and 1.9 n M in striatum and cerebellum, respectively). SR 141716A antagonized the CP 55940- and WIN 55212-2-induced inhibition of cyclic AMP accumulation, suggesting CB1 receptor-specific mediation of these effects on all primary cultures tested. Furthermore, CP 55940 was unable to induce mitogen-activated protein kinase activation in either cortical or striatal neurons. In conclusion, our results show nanomolar efficiencies for CP 55940 and WIN 55212-2 on adenylyl cyclase activity and no effect on any other signal transduction pathway investigated in primary neuronal cultures.     

降钙素基因相关肽家族受体及其受体活性修饰蛋白

降钙素基因相关肽家族是一类多功能的激素家族 ,参与人体的多种生物学功能 ,与多种疾病有关。降钙素基因相关肽受体包括降钙素受体 (CTR)和降钙素受体样受体 (CRLR) ,CTR可以独自与降钙素结合 ,而CRLR必须与一组称作受体活性修饰蛋白 (RAMPs)的蛋白质共同作用才能发挥生物学功能。综述CTR的研究概况及CRLR与RAMPs相互作用的机制和表达调控 ,以期为人们设计新型药物提供参考。     

Functional Interaction of the tonA/tonB Receptor System in Escherichia coli

Host range mutants of phage T1 (T1h), which productively infected tonB mutants of Escherichia coli, were isolated. The phage mutants were inactivated by isolated outer membranes of E. coli in contrast to the wild-type phage, which only adsorbed reversibly. For the infection process, the tonB function is apparently only required for the irreversible adsorption of the phage T1, but not for the transfer of the phage DNA through the outer membrane and the cytoplasmic membrane of the cell. Mutants of the tonA gene expressing normal amounts of outer membrane receptor proteins were isolated and found to be partially sensitive to phage T5 and resistant to the phages T1 and T1h, colicin M, and albomycin and unable to take up iron as a ferrichrome complex. One tonA mutant remained partially sensitive to T5, colicin M, and albomycin and supported growth of T1h (not of T1) with the same plating efficiency as the parent strain. Only a small region of the tonA receptor protein seems to function for all the very different substrates. A newly isolated host range mutant of T5 (T5h) adsorbed faster to tonA(+) cells than did wild-type T5 and infected tonA missense mutants resistant to wild-type T5. The interplay of the tonA with the tonB function was observed with phage T5 infection, although T5 required only the tonA receptor. Ferrichrome inhibited plaque formation of T5 only when plated on tonB mutants. Adsorption of T5 to cells in liquid medium was influenced by ferrichrome as follows: complete inhibition by 0.1 muM ferrichrome with tonB mutants, not more than 35% inhibition by 1 to 100 muM ferrichrome with the tonB(+) parent strain in the presence of glucose as energy source, and 90% inhibition by 1 muM ferrichrome with partially starved parent cells. We conclude that there exist different functional states of the receptor protein that depend on the energy state of the cell and the tonB function. The latter seems to be required only for translocation processes with outer membrane proteins involved.     

A Silver Impregnation Technique for the Study of Steroid Receptors in Central Nervous System Tissue Prepared for Autoradiography

The commonly used silver stains were found to be unsatisfactory for nervous tissue processed for autoradiography. A silver impregnation procedure for central nervous system tissues prepared for the autoradiographic study of steroid receptors is described. The procedure is a combination of several silver and reticular stains made up in solutions containing dimethylsulfoxide. The technique clearly distinguishes perikarya of neurons, brain nuclei and fiber tracts without substantial loss of silver grains, and thus greatly facilitates the identification of steroid receptor nuclei at all levels of the central nervous system.     

Evolutionary Analysis for Functional Divergence of the Toll-Like Receptor Gene Family and Altered Functional Constraints

The Toll-like receptor (TLR) gene family consists of type 1 transmembrane receptors, which play essential roles in both innate immunity and adaptive immune response by ligand recognition and signal transduction. Using all available vertebrate TLR protein sequences, we inferred the phylogenetic tree and then characterized critical amino acid residues for functional divergence by detecting altered functional constraints after gene duplications. We found that the extracellular domain of TLR genes showed higher functional divergence than that of the cytoplasmic domain, particularly in the region between leucine-rich repeat (LRR) 10 and LRR 15 of TLR 4. Our finding supports the concept that sequence evolution in the extracellular domain may be responsible for the broad diversity of TLR ligand-binding affinity, providing a testable hypothesis for potential targets that could be verified by further experimentation.     

Hsp90伴侣复合体装配及对类固醇受体调节

真核细胞中近100种蛋白质都受Hsp90的调节。这些蛋白质多与信号转导作用有关,它们与Hsp90一起进入一个以Hsp90/Hsp70为主的伴侣复合体,在复合体内完成信号转导作用。Hsp90除了和蛋白质的伴侣位点结合以外,还在其他位点与辅助因子连接,这是Hsp90能与蛋白质及辅助因子组装成复合体,并进而调节其信号作用的结构基础。类固醇受体等蛋白质的信号转导作用是在Hsp70、Hsp90为基础的5种蛋白质(Hsp90,Hsp70,Hop,Hsp40和p23)组成的复合体中进行的。这个系统可以帮助理解在真核细胞中,Hsp70和Hsp90怎样联合作用,改变底物蛋白构象,以及怎样应答信号作用。     

重组人白介素6受体功能区片段的功能鉴定

用生物素标记重组人白介素6受体功能区片段rIL6R-28及其二联体蛋白rIL6R-53．竞争ELISA表明重组蛋白可以与配基IL-6特异结合．流式细胞术检测结果表明IL-6与生物素标记的重组蛋白所形成的复合物能够与7TD1细胞表面的gp130结合．而7TD1细胞生长分析则表明,重组蛋白可以增强IL-6对7TD1．细胞的生长刺激作用．     

Macrophage Receptors for Influenza A Virus: Role of the Macrophage Galactose-Type Lectin and Mannose Receptor in Viral Entry

Although sialic acid has long been recognized as the primary receptor determinant for attachment of influenza virus to host cells, the specific receptor molecules that mediate viral entry are not known for any cell type. For the infection of murine macrophages by influenza virus, our earlier study indicated involvement of a C-type lectin, the macrophage mannose receptor (MMR), in this process. Here, we have used direct binding techniques to confirm and characterize the interaction of influenza virus with the MMR and to seek additional macrophage surface molecules that may have potential as receptors for viral entry. We identified the macrophage galactose-type lectin (MGL) as a second macrophage membrane C-type lectin that binds influenza virus and is known to be endocytic. Binding of influenza virus to MMR and MGL occurred independently of sialic acid through Ca²⁺-dependent recognition of viral glycans by the carbohydrate recognition domains of the two lectins; influenza virus also bound to the sialic acid on the MMR. Multivalent ligands of the MMR and MGL inhibited influenza virus infection of macrophages in a manner that correlated with expression of these receptors on different macrophage populations. Influenza virus strain A/PR/8/34, which is poorly glycosylated and infects macrophages poorly, was not recognized by the C-type lectin activity of either the MMR or the MGL. We conclude that lectin-mediated interactions of influenza virus with the MMR or the MGL are required for the endocytic uptake of the virus into macrophages, and these lectins can thus be considered secondary or coreceptors with sialic acid for infection of this cell type.Infection of host cells by influenza virus is initiated by attachment of virus to sialic acid residues on the host cell surface through the receptor-binding site at the distal tip of the viral hemagglutinin (HA) (43). After attachment, the virus is internalized by endocytosis, and acidification of the endosome triggers a conformational change in viral HA that results in fusion of the viral envelope and host cell membrane (34). At the cell surface, sialic acid residues are commonly found at the termini of oligosaccharide chains that are attached in O or N linkage to cell surface proteins; they are also an essential component of acidic glycosphingolipids (gangliosides) that are present in all mammalian cell membranes. Although the abundance of sialic acid on mammalian cells provides influenza virus with multiple potential receptors, virus attachment does not always lead to virus entry (5, 8, 46). Furthermore, sialic acid-independent infection of Madin-Darby canine kidney (MDCK) cells by influenza virus has been reported (35). The specific host cell molecules that serve as functional receptors (or coreceptors) for the infectious entry of influenza virus have yet to be defined.We have studied the infectious entry of influenza virus into macrophages (Mφ), which represents an early event in recognition of the virus by the innate immune system (23, 44). After intranasal infection of mice, influenza virus replicates productively in cells of the respiratory epithelium. Mφ are also infected and viral proteins are produced, but replication is abortive and no live progeny are released (32); infection of Mφ is thus a dead-end for the virus leading to a reduction in viral load. In addition, influenza virus infection of Mφ stimulates production and release of proinflammatory cytokines and alpha/beta interferon (28), which may assist in further limiting viral replication and spread within the respiratory tract. Depletion of airway Mφ from mice prior to intranasal influenza virus infection leads to increased virus titers in the lung, attesting to the important role of Mφ in early host defense against the virus (38, 44).We observed in a previous study (30) that influenza A virus strains differed in their ability to infect murine Mφ, strains carrying a more highly glycosylated hemagglutinin (HA) molecule being more efficient at infecting Mφ than less glycosylated strains, although binding of viruses to the Mφ cell surface was equivalent. Our investigation of this phenomenon indicated involvement of the Mφ mannose receptor MMR (CD206), a C-type lectin, in infectious viral entry (29, 30). The involvement of other receptors was not excluded, and our subsequent observation that influenza virus can infect the RAW 264.7 Mφ cell line, which does not express the MMR, indeed points to the existence of other routes of infectious entry of the virus into Mφ.In the present study we used direct binding methods to confirm and characterize the interaction of influenza virus with the MMR and to seek additional Mφ surface molecules that may have potential as receptors for viral entry. We identify the Mφ galactose-type lectin (MGL) as a second Mφ membrane C-type lectin that binds influenza virus and investigate its involvement in the infectious process.     

A Computer Generated Model of Adenosine Receptors Rationalising Binding and Selectivity of Receptor Ligands

Abstract

Computer graphic analyses on a broad spectrum of adenosine receptor ligands has shown that both the A1 and A2 adenosine receptors have three binding sites. The spatial relationship of these three binding sites has been defined. Adenosine orientation at A1 and A2 is different.     

病毒受体的研究方法

1概况 病毒受体可以定义为位于宿主细胞表面能够被病毒吸附蛋白识别并与之结合,从而引起病毒感染的分子复合物,其化学本质是糖蛋白、蛋白聚糖、脂类或糖脂,大多数属于蛋白质.病毒受体可以是单体也可以是多分子复合物,具有特异性、高亲和性、饱和性、结合位点及靶细胞部位的有限性以及独特的生物学活性等[1].病毒受体是公认的引发病毒感染宿主细胞的主要决定因素,也是影响病毒宿主特异性和组织亲嗜性的决定因素之一.     

病毒受体的研究方法

1概况病毒受体可以定义为位于宿主细胞表面能够被病毒吸附蛋白识别并与之结合,从而引起病毒感染的分子复合物,其化学本质是糖蛋白、蛋白聚糖、脂类或糖脂,大多数属于蛋白质。病毒受体可以是单体也可以是多分子复合物,具有特异性、高亲和性、饱和性、结合位点及靶细胞部位的有限性以及独特的生物学活性等[1]。病毒受体是公认的引发病毒感染宿主细胞的主要决定因素,也是影响病毒宿主特异性和组织亲嗜性的决定因素之一。研究病毒受体的特性及其功能对于从分子水平阐明病毒感染与免疫的机制,深刻理解病毒与宿主细胞的相互关系,研制更有效的病毒…     

Functional Neurochemical Evidence for the Presence of Presynaptic Nicotinic Acetylcholine Receptors at the Terminal Region of Myenteric Motoneurons: A Study with Epibatidine

The aim of this study was to verify the presence of presynaptic nicotinic acetylcholine receptors (nAChRs) at the terminals of myenteric motoneurons using a potent and highly selective nicotinic agonist, epibatidine. We examined contraction, and release of [³H]ACh on a guinea-pig longitudinal muscle strip preparation. First, we compared the ability of epibatidine and nicotine to induce isometric contraction and found epibatidine (EC₅₀ = 23.1 nM) to be 300-fold more potent than nicotine (EC₅₀ = 7.09 M). The release and contraction induced by 30 nM epibatidine were inhibited by the nicotinic antagonist mecamylamine (3 M) and the Na1-channel blocker TTX (1 M), indicating that the effects are mediated via nAChRs and are fully dependent on the propagation of action potentials. Atropine (0.1 M) significantly increased the [³H]ACh release but could not block contraction suggesting that a substantial part of the response develops via a noncholinergic mechanism. Epibatidine at a higher concentration (300 nM) induced contraction, which was only partly (45%) inhibited by TTX (1 M). The TTX-resistant contraction, however, was completely blocked by mecamylamine (3 M). Our data provide functional neurochemical evidence for the existence of presynaptic nAChRs at myenteric motoneuron terminals and suggest that these receptors can be activated only/by a higher concentration of agonists.     

兔肝细胞膜高密度脂蛋白受体酶联免疫检测法的研究

用辣根过氧化物酶标记羊抗人apoAI-IgG,建立了检测兔肝细胞膜HDL受体的酶联免疫吸附检测法.测定时, HDL结合量按抗体-配体-抗配体抗体酶交联物反应制作的标准曲线确定;膜蛋白非特异吸附则用与酶交联的抗体来源相同的同种动物血浆HDL平行抑制试验消除.实验测得正常家兔肝细胞膜HDL受体K_d值为7.17±1.18 mg/L,B_max值为(622.5±146.1)mg/g(n=7).     

Molecular Cloning and Functional Characterization of the Receptor for Clostridium perfringens Enterotoxin

A cDNA encoding the Clostridium perfringens enterotoxin receptor gene (CPE-R) was cloned from an expression library of enterotoxin-sensitive Vero cells. The nucleotide sequence of CPE-R showed that the enterotoxin receptor consists of 209 amino acids with a calculated molecular mass of 22,029 D. This receptor is highly hydrophobic, contains four putative transmembrane segments, and has significant similarity to the rat androgen withdrawal apoptosis protein RVP1 and the mouse oligodendrocyte specific protein, the functions of which are unknown. The expression of CPE-R was detected in the enterotoxin-sensitive Vero, Hep3B, and Intestine 407 cell lines, but not in the enterotoxin-insensitive K562 and JY cell lines. The CPE-R gene product expressed in enterotoxin-resistant L929 cells bound to enterotoxin specifically and directly and with high affinity and rendered the cells sensitive to the toxin, indicating that the cloned receptor is functional. Results showed that enterotoxin could not assemble into a complex with a defined structure unless it interacted with the receptor. From these results, it is proposed that the enterotoxin receptor is required for both target cell recognition and poreformation in the cell membrane.