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Grafting protein ligand monolayers onto the surface of microparticles for probing the accessibility of cell surface receptors

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BIOCONJUGATE CHEMISTRY 1999年第4期10卷 562-571页

作者： Frey, A Meckelein, B Schmidt, MA Univ Munster Inst Infektiol Zentrum Mol Biol Entzundung D-48129 Munster Germany

Coupling of a specific ligand to vaccines or drugs can be a powerful aid to route these compounds to a certain target cell population. However, if the targeted receptor is buried in a glycocalyx, binding of the ligand may be sterically hindered or even abolished, especially when the ligand is attached to bulky payloads. The antigen-transporting hi cells that cover the gut-associated lymphoid tissue have a less pronounced glycocalyx than neighboring enterocytes. Such architectural differences might provide a possibility for targeting micro- or nanoparticulate vaccines to the mucosal immune system. To investigate the influence of the glycocalyx on the accessibility of cell surface receptors, we developed a system where a monolayer of ligand molecules is coupled in spatially aligned manner onto the surface of microparticles. On the basis of fluorescent carboxylate-modified particles of 1 mu m diameter, different synthetic strategies were tested. Particles were first modified to display aldehyde functions on their surface, then protein ligands were coupled via Schiff base formation. The performance of the particles was tested on cultured mouse fibroblasts using the B subunit of cholera toxin as ligand and the plasma membrane glycolipid ganglioside G(M1) as receptor. Cholera toxin B subunit-coated microparticles generated by one of our synthetic pathways exhibited specific binding to fibroblasts which could be blocked with soluble cholera toxin B subunit. As particles as small as 50 nm and any proteinaceous ligand may be used, this system provides a versatile means for monitoring receptor accessibilities in vitro and in vivo.

关键词： 3T3细胞醛类/化学抗生物素蛋白/代谢结合部位羧酸类/化学细胞系成纤维细胞荧光染料氢离子浓度配体小鼠近交BALBC 微球体蛋白质类/化学蛋白质类/诊断应用受体细胞表面/药物作用动物小鼠

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Tight association of the human Mel_1a-melatonin receptor and G_i:: Precoupling and constitutive activity

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MOLECULAR PHARMACOLOGY 1999年第5期56卷 1014-1024页

作者： Roka, F Brydon, L Waldhoer, M Strosberg, AD Freissmuth, M Jockers, R Nanoff, C Univ Vienna Inst Pharmacol A-1090 Vienna Austria Inst Cochin Genet Mol F-75014 Paris France

If stably expressed in human embryonic kidney (HEK)293 cells, the human Mel(1a)-melatonin receptor activates G(i)-dependent, pertussis toxin-sensitive signaling pathways, i.e., inhibition of adenylyl cyclase and stimulation of phospholipase C beta;the latter on condition that G(q) is coactivated. The antagonist luzindole blocks the effects of melatonin and acts as an inverse agonist at the Mel(1a) receptor in both intact cells and isolated membranes. This suggests that the Mel(1a) receptor is endowed with constitutive activity, a finding confirmed on reconstitution of the Mel(1a) receptor with G(i). Because the receptor density is in the physiological range, constitutive activity is not an artifact arising from overexpression of the receptor. In addition, the following findings indicate that the Mel(1a) receptor forms a very tight complex with G(i) which can be observed both in the presence and absence of an agonist. 1) In intact cells and in membranes, high-affinity agonist binding is resistant to the destabilizing effect of guanine nucleotides. 2) The ability to bind an agonist with high affinity is preserved even after exposure of the cells to pertussis toxin, because a fraction of G(i) is inaccessible to the toxin in cells expressing Mel(1a) receptors (but not the A(1)-adenosine receptor, another G(i)-coupled receptor). 3) An antiserum directed against the Mel(1a) receptor coprecipitates G(i) even in the absence of an agonist. We therefore conclude that the Mel(1a) receptor is tightly precoupled and that its constitutive activity may play a role in pacing the biological clock, an action known to involve the melatonin receptors in the suprachiasmatic nucleus.

关键词：腺苷酸环化酶毒素结合竞争性细胞培养的 GTP结合蛋白质α亚单位 Gi-Go/代谢碘放射性同位素配体褪黑激素/类似物和衍生物褪黑激素/代谢百白咳毒素表型受体细胞表面/药物作用受体细胞表面/代谢受体胞质和核/药物作用受体胞质和核/代谢受体褪黑激素信号传导毒力因子鲍特菌属/药理学人类

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Activation of surfactant protein-B transcription: Signaling through the SP-A receptor utilizing the PI3 kinase pathway

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JOURNAL OF CELLULAR PHYSIOLOGY 2000年第2期184卷 229-238页

作者： Strayer, DS Korutla, L Thomas Jefferson Univ Jefferson Med Coll Dept Pathol & Cell Biol Philadelphia PA 19107 USA

This study describes receptor-activated signaling initiated by surfactant protein-A (SP-A), and the means by which it activates transcription of surfactant protein-B. Pulmonary surfactant is a mixture of lipids and associated proteins produced by type II pneumocytes. Interaction of SP-A with its cognate receptor (SPAR) on type II cells is involved in regulating surfactant secretion. This interaction also increases transcription of surfactant proteins and several other genes. To study SP-A cytokine activity, we used as a model surfactant-protein (SP-B) transcription, the activators of which have been characterized. HNF-3 and TTF-1 transcription factors are known to stimulate SP-B transcription. SP-A caused increased phosphorylation and nuclear localization of both. Corresponding increases in protein binding to the SP-B promoter were demonstrated by gel shift analysis. SP-A increased protein binding to HNF-3 and TTF-1 consensus recognition elements. Footprinting analysis indicated that SP-A-induced protein binding to SP-B promoter was greater in amount, but not different in location, from that seen in control cells, which normally transcribe SP-B. SP-A caused transient increases in PI3 kinase localization at the plasma membrane, and SP-A signaling to elicit increased SP-B transcription was blocked by LY294002, an inhibitor of PI3 kinase. Therefore, SP-A signals through PI3 kinase to increase SP-B transcription in type II pneumocytes by enhancing TTF-1 and HNF-3 activation of the SP-B promoter. SP-A activation of this signaling pathway, which affects many cellular functions and has not previously been implicated in type II cell transcriptional activity, has profound import for understanding type II cell biology. J. Cell. Physiol. 184:229-238, 2000. (C) 2000 Wiley-Liss, Inc.

关键词： 1-磷脂酰肌醇3-激酶/药物作用 1-磷脂酰肌醇3-激酶/生理学细胞培养的 DNA结合蛋白质类/药物作用 DNA结合蛋白质类/生理学糖蛋白类/药理学肝细胞核因子3α 肺/细胞学肺/药物作用肺/生理学核蛋白质类/药物作用核蛋白质类/生理学启动区(遗传学) 蛋白质亚型/药物作用蛋白脂质类/药物作用蛋白脂质类/遗传学蛋白脂质类/药理学蛋白脂质类/生理学肺表面活性物质相关蛋白质A 肺表面活性物质相关蛋白质类肺表面活性剂/药物作用肺表面活性剂/遗传学肺表面活性剂/药理学肺表面活性剂/生理学受体细胞表面/药物作用受体细胞表面/生理学转录因子/药物作用转录因子/生理学转录遗传/药物作用转录遗传/生理学动物女(雌)性大鼠

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Microtubule-dependent regulation of α_2B adrenergic receptors in polarized MDCKII cells requires the third intracellular loop but not G protein coupling

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MOLECULAR PHARMACOLOGY 2000年第1期57卷 44-52页

作者： Saunders, C Limbird, LE Vanderbilt Univ Med Ctr Dept Pharmacol Nashville TN 37232 USA

Previous studies in cultured, polarized Madin-Darby canine kidney II (MDCKII) renal epithelial cells have demonstrated that the apical steady-state localization and delivery of the A(1) adenosine receptor is modified by disruption of the microtubule network with colchicine, whereas the basolateral localization and trafficking of the alpha(2)-adrenergic receptors (alpha(2)AR) are not;instead, the binding capacity of the alpha(2B)AR, but not alpha(2A)AR or alpha(2C)AR subtypes, is increased in a time-dependent fashion. The present studies explore the molecular basis for this alpha(2B)AR subtype-selective phenomenon. Colchicine selectively increased alpha(2B)AR density at the cell surface, as determined by confocal microscopy, receptor binding, and surface biotinylation studies. The colchicine-induced increase in the functional density of the alpha(2B)AR requires the third intracellular loop because the alpha(2B)AR loop deletion (alpha(2B)AR Delta i3) mutant did not show an increased receptor density after colchicine treatment. Furthermore, the colchicine-mediated increase in alpha(2B)AR density is manifest only in polarized cells because colchicine treatment of nonpolarized MDCKII renal epithelial cells as well as simian kidney COSM6 and human embryonic kidney HEK293 cells did not effect an increase in alpha(2B)AR density. Colchicine-dependent increases in alpha(2B)AR density did not depend on functional coupling to G proteins, however, because pretreatment with pertussis toxin did not eliminate the effect of colchicine. These data indicate that microtubule-dependent regulation of alpha(2B)AR density at the basolateral surface of polarized MDCKII cells requires the third intracellular loop of alpha(2B)AR but not functional alpha(2B)AR-G protein coupling.

关键词： COS细胞细胞培养的秋水仙碱/药理学 GTP结合蛋白质类/化学 GTP结合蛋白质类/代谢微管/代谢蛋白质构象受体肾上腺素能α2/化学受体肾上腺素能α2/药物作用受体肾上腺素能α2/代谢受体细胞表面/药物作用受体细胞表面/代谢动物狗人类

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Lysophosphatidic acid-induced Ca²⁺ mobilization requires intracellular sphingosine 1-phosphate production -: Potential involvement of endogenous Edg-4 receptors

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第49期275卷 38532-38539页

作者： Young, KW Bootman, MD Channing, DR Lipp, P Maycox, PR Meakin, J Challiss, RAJ Nahorski, SR Univ Leicester Dept Cell Physiol & Pharmacol Leicester LE1 9HN Leics England Babraham Inst Mol Signalling Lab Cambridge CB2 4AT England SmithKline Beecham Pharmaceut Harlow CM19 5AW Essex England

Lysophosphatidic acid (LPA)-mediated Ca2+ mobilization in human SH-SY5Y neuroblastoma cells does not involve either inositol 1,4,5-trisphosphate (Ins(1,4,5)P-3)- or ryanodine-receptor pathways, but is sensitive to inhibitors of sphingosine kinase. This present study identifies Edg-4 as the receptor subtype involved and investigates the presence of a Ca2+ signaling cascade based upon the lipid second messenger molecule, sphingosine I-phosphate. Both LPA and direct G-protein activation increase [H-3]sphingosine l-phosphate levels in SH-SY5Y cells. Measurements of Ca-45(2+) release in premeabilized SH-SY5Y cells indicates that sphingosine I-phosphate, sphingosine, and sphingosylphosphorylcholine, but not N-acetylsphingosine are capable of mobilizing intracellular Ca2+. Furthermore, the effect of sphingosine was attenuated by the sphingosine kinase inhibitor dimethylsphingosine, or removal of ATP. Confocal microscopy demonstrated that LPA stimulated intracellular Ca2+ "puffs," which resulted from an interaction between the sphingolipid Ca2+ release pathway and Ins(1,4,5)P-3 receptors. Down-regulation of Ins(1,4,5)P-3 receptors uncovered a Ca2+ response to LPA, which was manifest as a progressive increase in global cellular Ca2+ with no discernible foci. We suggest that activation of an LPA-sensitive Edg-4 receptor solely utilizes the production of intracellular sphingosine l-phosphate to stimulate Ca2+ mobilization in SB-SY5Y cells. Unlike traditional Ca2+ release processes, this novel pathway does not require the progressive recruitment of elementary Ca2+ events.

关键词：咖啡因/药理学钙/代谢钙通道/生理学钙信号鸟苷5'-O-(3-硫代三磷酸)/代谢异源三聚体GTP结合蛋白质类/代谢肌醇1 4 5-三磷酸/代谢肌醇1 4 5-三磷酸受体动力学溶血磷脂素类/药理学神经母细胞瘤受体细胞表面/药物作用受体细胞表面/遗传学受体细胞表面/生理学受体胞质和核/生理学受体 G-蛋白偶联受体溶血磷脂酸逆转录聚合酶链反应鞘氨醇/类似物和衍生物鞘氨醇/代谢鞘氨醇/药理学肿瘤细胞培养的人类

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New role for Shc in activation of the phosphatidylinositol 3-kinase/Akt pathway

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MOLECULAR AND CELLULAR BIOLOGY 2000年第19期20卷 7109-7120页

作者： Gu, HH Maeda, H Moon, JJ Lord, JD Yoakim, M Nelson, BH Neel, BG Beth Israel Deaconess Med Ctr Canc Biol Program Dept Med Div Hematol & Oncol Boston MA 02115 USA Harvard Univ Sch Med Boston MA 02115 USA Univ Washington Virginia Mason Res Ctr Seattle WA 98195 USA Univ Washington Dept Immunol Seattle WA 98195 USA

Most, if not all, cytokines activate phosphatidylinositol 3-kinase (PI-3K). Although many cytokine receptors have direct binding sites for the p85 subunit of PI-3K, others, such as the interleukin-3 (IL-3) receptor beta common chain (beta c) and the IL-2 receptor beta chain (IL-2R beta), lack such sites, leaving the mechanism by which they activate PI-3K unclear. Here, we show that the protooncoprotein Shc, which promotes Ras activation by recruiting the Grb2-Sos complex in response to stimulation of cytokine stimulation, also signals to the PI-3K/Akt pathway. Analysis of Y--> F and "add-back" mutants of beta c show's that Y577, the Shc binding site, is the major site required for Gab2 phosphorylation in response to cytokine stimulation. When fused directly to a mutant form of IL-2R beta that lacks other cytoplasmic tyrosines. Shc can promote Gab2 tyrosyl phosphorylation. Mutation of the three tyrosyl phosphorylation sites of Shc, which bind Grb2, blocks the ability of the Shc chimera to evoke Gab2 tyrosyl phosphorylation. Overexpression of mutants of Grb2 with inactive SH2 or SH3 domains also blocks cytokine-stimulated Gab2 phosphorylation. The majority of cytokine-stimulated PI-3K activity associates with Gab2, and inducible expression of a Gab2 mutant unable to bind PI-3K markedly impairs IL-3-induced Akt activation and cell growth. Experiments with the chimeric receptors indicate that Shc also signals to the PI-3K/Akt pathway in response to IL-2, Our results suggest that cytokine receptors lacking direct PI-3K binding sites activate Akt via a Shc/Grb2/Gab2/PI-3K pathway, thereby regulating cell survival and/or proliferation.

关键词：衔接蛋白质类信号转导衔接蛋白质类膜泡运输氨基酸取代结合部位细胞因子类/药理学酶激活 GRB2衔接蛋白质白细胞介素2/药理学白细胞介素3/药理学肿瘤蛋白质类/生理学磷酸肌醇3-激酶类/生理学磷蛋白类/化学磷蛋白类/代谢磷酰化前体B细胞淋巴母细胞白血病淋巴瘤/病理学蛋白质加工转译后蛋白质丝氨酸苏氨酸激酶蛋白质类/生理学原癌基因蛋白质类/生理学原癌基因蛋白质c-akt 受体细胞表面/化学受体细胞表面/药物作用受体细胞表面/遗传学重组融合蛋白质类/药物作用重组融合蛋白质类/生理学 Shc信号衔接蛋白质类信号传导/生理学转染肿瘤细胞培养的 src同源域动物小鼠

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MECHANISM OF VASCULAR RELAXATION BY THE CALCITONIN GENE RELATED PEPTIDE

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ANNALS OF THE NEW YORK ACADEMY OF SCIENCES-SERIES 1992年第1期657卷 204-215页

作者： MARSHALL, I Department of Pharmacology University College London London WC1E 6BT England

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ACTIVATION OF OLIVOCEREBELLAR FIBERS INDUCES AN INCREASE IN CGRP CEREBELLAR BINDING-SITES

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ANNALS OF THE NEW YORK ACADEMY OF SCIENCES-SERIES 1992年第1期657卷 432-434页

作者： ROSINA, A MORARA, S PROVINI, L FORLONI, G UNIV MILAN FAC FARM IST FISIOL GEN CHIM BIOL I-20122 MILAN ITALY IST MARIO NEGRI MILAN ITALY

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CHARACTERIZATION OF CGRP RECEPTOR-SITES IN RAT AIRWAYS

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ANNALS OF THE NEW YORK ACADEMY OF SCIENCES-SERIES 1992年第1期657卷 441-443页

作者： LANOUE, C FOURNIER, A STPIERRE, S CADIEUX, A UNIV SHERBROOKE FAC MED DEPT PHARMACOL SHERBROOKE J1H 5N4 QUEBEC CANADA UNIV QUEBEC INST NATL RECH SCI SANTE POINTE CLAIRE H9R 1G6 QUEBEC CANADA

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L-Amino acid sensing by the extracellular Ca²⁺-sensing receptor

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2000年第9期97卷 4814-4819页

作者： Conigrave, AD Quinn, SJ Brown, EM Univ Sydney Dept Biochem Sydney NSW 2006 Australia Brigham & Womens Hosp Dept Med Div Endocrine Hypertens Boston MA 02115 USA Harvard Univ Sch Med Boston MA 02115 USA

The extracellular calcium (Ca-o(2+))-sensing receptor (CaR) recognizes and responds to (i.e., "senses") Ca-o(2+) as its principal physiological ligand, In the present studies, we document that the CaR is activated not only by extracellular calcium ions but also by amino acids, establishing its capacity to sense nutrients of two totally different classes, L-Amino acids, especially aromatic amino acids, including L-phenylalanine and L-tryptophan, stereoselectively mobilized Ca2+ ions in the presence of the CaR agonists, Ca-o(2+), gadolinium (Gd-o(3+)), and spermine in fura-2-loaded human embryonic kidney (HEK-293) cells stably transfected with the human CaR. L-amino acid-dependent effects were observed above, but not below, a threshold level of Ca-o(2+) of approximately 1.0 mM. L-Amino acids, particularly aromatic amino acids, also stereoselectively enhanced the sensitivity of the CaR to its agonists, Ca-o(2+) and spermine. Branched-chain amino acids were almost inactive, and charged amino acids, including arginine and lysine, were much less effective than aromatic and other amino acids. L-amino acid mixtures emulating the amino acid composition of fasting human plasma reproduced the effects of high concentrations of individual L-amino acids on Ca2+ mobilization and enhanced the sensitivity of the CaR to Ca-o(2+). The data presented herein identify the CaR as a molecular target for aromatic and other L-amino acids. Thus, the CaR can integrate signals arising from distinct classes of nutrients: mineral ions and amino acids. The actions of L-amino acids on the CaR may provide explanations for several long recognized but poorly understood actions of dietary protein on calcium metabolism.

关键词：氨基酸类/药理学氨基酸类/生理学钙/药理学钙/生理学细胞系克隆分子 Fura-2 钆/药理学肾动力学苯丙氨酸/药理学受体钙敏感受体细胞表面/药物作用受体细胞表面/遗传学受体细胞表面/生理学重组蛋白质类/药物作用重组蛋白质类/代谢精胺/药理学色氨酸/药理学人类

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