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Biochimica Et Biophysica Acta.... Sep 2017Even though there are hundreds of reports in the published literature supporting the hypothesis that G protein-coupled receptors (GPCR) form and function as dimers this...
Even though there are hundreds of reports in the published literature supporting the hypothesis that G protein-coupled receptors (GPCR) form and function as dimers this remains a highly controversial area of research and mechanisms governing homodimer formation are poorly understood. Crystal structures revealing homodimers have been reported for many different GPCR. For adrenergic receptors, a potential dimer interface involving transmembrane domain 1 (TMD1) and helix 8 (H8) was identified in crystal structures of the beta-adrenergic (β-AR) and β-AR. The purpose of this study was to investigate a potential role for TMD1 and H8 in dimerization and plasma membrane expression of functional β-AR. Charged residues at the base of TMD1 and in the distal portion of H8 were replaced, singly and in combination, with non-polar residues or residues of opposite charge. Wild type and mutant β-AR, tagged with YFP and expressed in HEK293 cells, were evaluated for plasma membrane expression and function. Homodimer formation was evaluated using bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and fluorescence correlation spectroscopy. Amino acid substitutions at the base of TMD1 and in the distal portion of H8 disrupted homodimer formation and caused receptors to be retained in the endoplasmic reticulum. Mutations in the proximal region of H8 did not disrupt dimerization but did interfere with plasma membrane expression. This study provides biophysical evidence linking a potential TMD1/H8 interface with ER export and the expression of functional β-AR on the plasma membrane. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.
Topics: Cell Membrane; Crystallography, X-Ray; Endoplasmic Reticulum; Gene Expression Regulation; HEK293 Cells; Humans; Mutation; Protein Conformation; Protein Domains; Protein Multimerization; Receptors, Adrenergic, beta-1; Receptors, Adrenergic, beta-2; Signal Transduction; Spectrometry, Fluorescence
PubMed: 27993566
DOI: 10.1016/j.bbamem.2016.12.007 -
The Journal of Biological Chemistry Oct 1984We have simultaneously quantitated alpha 1-adrenergic receptor occupation and agonist-elicited Ca2+ mobilization monitored as unidirectional 45Ca2+ efflux from intact...
We have simultaneously quantitated alpha 1-adrenergic receptor occupation and agonist-elicited Ca2+ mobilization monitored as unidirectional 45Ca2+ efflux from intact BC3H-1 muscle cells in order to examine the relationship between the number of surface receptors occupied and the functional response. [3H]Prazosin has been used to measure receptor number as well as the binding kinetics with surface receptors, and the observed equilibrium and kinetic constants are in close accord with values obtained previously in cellular homogenates. Since alpha 1-agonist-elicited 45Ca2+ efflux can be monitored over intervals of 3 min or less and prazosin dissociation from its receptor has a t 1/2 of 44 min, prazosin can be employed to produce a pseudoirreversible inactivation of receptors. A comparison of the remaining receptors and residual response reveals an inverse linear relationship between receptors inactivated by prazosin and 45Ca2+ efflux. A similar result is obtained following fractional receptor inactivation with the irreversible alkylating agent phenoxybenzamine. Parameters of receptor occupation and response also correlate well for the agonist phenylephrine and for the competitive antagonist phentolamine. The unitary relationship between sites available for occupation and response indicates that the alpha 1 receptor does not function as an oligomer where fewer bound antagonist molecules are required to block the receptor than sites of agonist occupation necessary for activation. Moreover, substantial evidence has accrued in intact smooth muscle for a receptor reserve or nonlinear coupling between alpha 1 receptor occupation and contraction in smooth muscle. Our findings demonstrate that such behavior does not exist for alpha 1 receptor-elicited mobilization of Ca2+ in the BC3H-1 muscle cell.
Topics: Animals; Binding, Competitive; Calcium; Cell Line; Cell Membrane; Kinetics; Mice; Mice, Inbred C3H; Muscles; Phenoxybenzamine; Phentolamine; Phenylephrine; Prazosin; Receptors, Adrenergic, alpha
PubMed: 6092338
DOI: No ID Found -
Journal of the American College of... Oct 1993Heart failure results in dramatic changes in certain neurotransmitter and hormone receptors. The majority of the changes occur in the heart and generally can be... (Comparative Study)
Comparative Study Review
Heart failure results in dramatic changes in certain neurotransmitter and hormone receptors. The majority of the changes occur in the heart and generally can be classified as regulatory phenomena that withdraw the failing heart from adrenergic stimulation. Of these, the most prominent is beta 1-receptor downregulation. Changes in vascular receptors are much less prominent and there is no direct evidence that any vascular receptor changes in heart failure. The changes that occur in myocardial receptors suggest that antiadrenergic therapy would be effective in the treatment of heart failure by removing adrenergic signaling transduced by the remaining components of the receptor pathways. Taken together, the receptor desensitization changes present in the failing heart provide a rationale for beta 1- plus beta 2-adrenergic blockade or even combined beta 1-, beta 2-alpha 1-adrenergic receptor blockade in heart failure.
Topics: Blood Vessels; Down-Regulation; GTP-Binding Proteins; Heart; Heart Failure; Humans; Receptors, Adrenergic, alpha; Receptors, Adrenergic, beta; Signal Transduction
PubMed: 8397233
DOI: 10.1016/0735-1097(93)90465-d -
Cellular Physiology and Biochemistry :... Sep 2020Trace amines (TA) are small organic compounds that have neuromodulator activity due to their interaction with some neuron-related receptors, such as trace amine...
BACKGROUND/AIMS
Trace amines (TA) are small organic compounds that have neuromodulator activity due to their interaction with some neuron-related receptors, such as trace amine associated receptors (TAARs), α2-adrenergic receptor (α2-AR) and ß-adrenergic receptor (ß-AR). However, there is little information on whether TA and dopamine (DOP) can interact with other adrenergic receptors (ARs) such as the mammalian α1-AR and the bacterial counterpart QseC, which is involved in quorum sensing of some Gram-negative pathogens. The aim of this study was to investigate the interaction of TA and DOP with α1-AR and QseC.
METHODS
We performed an in silico study using 3D structure from SWISS MODEL and analyzed the protein interaction via molecular docking using PyMol, PoseView and PyRX 8.0. For the in vitro study, we investigated the QseC kinase activity by measuring the remaining ATP in a reaction containing QseC-enriched membrane incubated together with purified QseB and EPI, TA, DOP, or PTL respectively. We also measured the intracellular Ca++ levels, which represents the α1-AR activation, in LNCAP (pancreatic cell line) cells treated with EPI, TA, DOP and PTL respectively using a fluorescence-based assay. The LNCAP cell proliferation was measured using an MTT-based assay.
RESULTS
Our in silico analysis revealed that TAs and DOP have high binding affinity to the human α1-AR and the bacterial adrenergic receptor (QseC), comparable to epinephrine (EPI). Both are membrane-bound kinases. Experimental studies with pancreatic cell line (LNCAP) showed that the TAs and DOP act as α1-AR antagonist by counteracting the effect of EPI. In the presence of EPI, TA and DOP trigger an increase of the intracellular Ca++ levels in the LNCAP cells leading to an inhibition of cell proliferation. Although in silico data suggest an interaction of TA and DOP with QseC, they do not inhibit the kinase activity of QseC, a histidine kinase receptor involved in quorum sensing which is also sensitive to EPI.
CONCLUSION
Our study showed that the TAs and DOP act as α1-AR antagonist but no effect was observed for QseC.
Topics: Amines; Animals; Computer Simulation; Dopamine; Escherichia coli Proteins; Humans; Molecular Docking Simulation; Phosphorylation; Receptors, Adrenergic, alpha-1; Signal Transduction; Trace Elements
PubMed: 32930525
DOI: 10.33594/000000276 -
Naunyn-Schmiedeberg's Archives of... Jun 2014Various naturally occurring polymorphic forms of human G protein-coupled receptors (GPCRs) have been identified and linked to diverse pathological diseases, including...
Various naturally occurring polymorphic forms of human G protein-coupled receptors (GPCRs) have been identified and linked to diverse pathological diseases, including receptors for vasopressin type 2 (nephrogenic diabetes insipidus) and gonadotropin releasing hormone (hypogonadotropic hypogonadism). In most cases, polymorphic amino acid mutations disrupt protein folding, altering receptor function as well as plasma membrane expression. Other pathological GPCR variants have been found that do not alter receptor function, but instead affect only plasma membrane trafficking (e.g., delta opiate and histamine type 1 receptors). Thus, altered membrane trafficking with retained receptor function may be another mechanism causing polymorphic GPCR dysfunction. Two common human α2A and α2C adrenergic receptor (AR) variants have been identified (α2A N251K and α2C Δ322-325 ARs), but pharmacological analysis of ligand binding and second messenger signaling has not consistently demonstrated altered receptor function. However, possible alterations in plasma membrane trafficking have not been investigated. We utilized a systematic approach previously developed for the study of GPCR trafficking motifs and accessory proteins to assess whether these α2 AR variants affected intracellular trafficking or plasma membrane expression. By combining immunofluorescent microscopy, glycosidic processing analysis, and quantitative fluorescent-activated cell sorting (FACS), we demonstrate that neither variant receptor had altered intracellular localization, glycosylation, nor plasma membrane expression compared to wild-type α2 ARs. Therefore, pathopharmacological properties of α2A N251K and α2C Δ322-325 ARs do not appear to be due to altered receptor pharmacology or plasma membrane trafficking, but may involve interactions with other intracellular signaling cascades or proteins.
Topics: Cell Membrane; Genetic Variation; HEK293 Cells; Humans; Intracellular Fluid; Polymorphism, Genetic; Protein Binding; Protein Transport; Receptors, Adrenergic, alpha-2
PubMed: 24643471
DOI: 10.1007/s00210-014-0972-6 -
Journal of Internal Medicine Jun 1999The genes causing obesity in rodent models have been characterized, but do not seem to be important for human obesity. Recently the putative association between obesity... (Review)
Review
The genes causing obesity in rodent models have been characterized, but do not seem to be important for human obesity. Recently the putative association between obesity and polymorphism in human beta-adrenergic receptor genes have been studied intensely in the light of the important role of these receptors in the regulation of energy mobilization and utilization. A polymorphism (Trp64Arg) in the beta3-adrenergic receptor gene is associated with obesity (relative risk approximately 2) in some but not all investigations on Caucasian and Japanese populations. When expressed in artificial cell systems, the polymorphism is associated with alterations of the beta -adrenoceptor. The genetic allele variance influences also the native receptor function when measured in isolated human fat cells. The human beta2-adrenoceptor gene shows a high degree of polymorphism. The role of beta2-receptor gene polymorphism for obesity has so far only been investigaed in women. A Gln27Glu variant is markedly associated with obesity with a relative risk for obesity of approximately 7 and odds ratio of approximately 10. Women who are homozygous for 27Glu have approximately 20 kg higher fat mass than controls. Thus, polymorphism in genes coding for different beta-adrenoceptor subtypes may be important for the development of human obesity.
Topics: Arginine; Humans; Mutation, Missense; Obesity; Polymorphism, Genetic; Receptors, Adrenergic, beta; Receptors, Adrenergic, beta-2; Receptors, Adrenergic, beta-3; Tryptophan
PubMed: 10395196
DOI: 10.1046/j.1365-2796.1999.00495.x -
Glia Sep 2022Norepinephrine exerts powerful influences on the metabolic, neuroprotective and immunoregulatory functions of astrocytes. Until recently, all effects of norepinephrine...
Norepinephrine exerts powerful influences on the metabolic, neuroprotective and immunoregulatory functions of astrocytes. Until recently, all effects of norepinephrine were believed to be mediated by receptors localized exclusively to the plasma membrane. However, recent studies in cardiomyocytes have identified adrenergic receptors localized to intracellular membranes, including Golgi and inner nuclear membranes, and have shown that norepinephrine can access these receptors via transporter-mediated uptake. We recently identified a high-capacity norepinephrine transporter, organic cation transporter 3 (OCT3), densely localized to outer nuclear membranes in astrocytes, suggesting that adrenergic signaling may also occur at the inner nuclear membrane in these cells. Here, we used immunofluorescence and western blot to show that β -adrenergic receptors are localized to astrocyte inner nuclear membranes; that key adrenergic signaling partners are present in astrocyte nuclei; and that OCT3 and other catecholamine transporters are localized to astrocyte plasma and nuclear membranes. To test the functionality of nuclear membrane β -adrenergic receptors, we monitored real-time protein kinase A (PKA) activity in astrocyte nuclei using a fluorescent biosensor. Treatment of astrocytes with norepinephrine induced rapid increases in PKA activity in the nuclear compartment. Pretreatment of astrocytes with inhibitors of catecholamine uptake blocked rapid norepinephrine-induced increases in nuclear PKA activity. These studies, the first to document functional adrenergic receptors at the nuclear membrane in any central nervous system cell, reveal a novel mechanism by which norepinephrine may directly influence nuclear processes. This mechanism may contribute to previously described neuroprotective, metabolic and immunoregulatory actions of norepinephrine.
Topics: Adrenergic Agents; Astrocytes; Catecholamines; Norepinephrine; Nuclear Envelope; Receptors, Adrenergic; Receptors, Adrenergic, beta; Receptors, Adrenergic, beta-1
PubMed: 35589612
DOI: 10.1002/glia.24219 -
The Journal of Pathology Jan 2023While multi-drug combinations and continuous treatment have become standard for multiple myeloma, the disease remains incurable. Repurposing drugs that are currently...
While multi-drug combinations and continuous treatment have become standard for multiple myeloma, the disease remains incurable. Repurposing drugs that are currently used for other indications could provide a novel approach to improve the therapeutic efficacy of standard multiple myeloma treatments. Here, we assessed the anti-tumor effects of cardiac drugs called β-blockers as a single agent and in combination with commonly used anti-myeloma therapies. Expression of the β -adrenergic receptor correlated with poor survival outcomes in patients with multiple myeloma. Targeting the β -adrenergic receptor (β AR) using either selective or non-selective β-blockers reduced multiple myeloma cell viability, and induced apoptosis and autophagy. Blockade of the β AR modulated cancer cell metabolism by reducing the mitochondrial respiration as well as the glycolytic activity. These effects were not observed by blockade of β -adrenergic receptors. Combining β AR blockade with the chemotherapy drug melphalan or the proteasome inhibitor bortezomib significantly increased apoptosis in multiple myeloma cells. These data identify the therapeutic potential of β AR-blockers as a complementary or additive approach in multiple myeloma treatment and support the future clinical evaluation of non-selective β-blockers in a randomized controlled trial. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Topics: Humans; Multiple Myeloma; Receptors, Adrenergic, beta-1; Signal Transduction; Bortezomib; Apoptosis
PubMed: 36245401
DOI: 10.1002/path.6020 -
Cancer Biomarkers : Section a of... 2013The importance of adrenergic pathways in cancer has long been suspected, but now there is mounting epidemiological, preclinical, and clinical evidence of its importance... (Review)
Review
The importance of adrenergic pathways in cancer has long been suspected, but now there is mounting epidemiological, preclinical, and clinical evidence of its importance in gynecologic cancers. To date, most of these effects are mediated primarily through the beta 2 adrenergic receptor activation of the tumor cell cyclic AMP-protein kinase A signaling pathway. This review will discuss the current knowledge about the neuroendocrine stress response in gynecologic tumor biology.
Topics: Animals; Female; Genital Neoplasms, Female; Humans; Receptors, Adrenergic; Signal Transduction
PubMed: 23912486
DOI: 10.3233/CBM-130324 -
The Journal of Biological Chemistry Mar 1986Human platelet alpha 2-adrenergic receptors have been purified approximately 80,000-fold to apparent homogeneity by a five-step chromatographic procedure. The overall...
Human platelet alpha 2-adrenergic receptors have been purified approximately 80,000-fold to apparent homogeneity by a five-step chromatographic procedure. The overall yield starting from the membranes is approximately 2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated protein from purified receptor preparations shows a single major band of Mr 64,000. The specific binding activity of the alpha 2-adrenergic receptor after four chromatographic steps is 14.5 nmol/mg protein. This value is consistent with the expected theoretical specific activity (15.6 nmol/mg) for a protein with a molecular mass of 64,000 daltons if it is assumed that there is one ligand-binding site/receptor molecule. The purified protein can be covalently labeled with the alkylating alpha-adrenergic ligand, [3H]phenoxybenzamine. This labeling is specific, and it shows that the Mr 64,000 protein contains the ligand binding site of the alpha 2-adrenergic receptor. In addition, the competitive binding of ligands to the purified receptor protein shows the proper alpha 2-adrenergic specificity. The alpha 2-adrenergic receptor contains an essential sulfhydryl residue. Thus, exposure of the purified receptor to the sulfhydryl-specific reagent, phenylmercuric chloride, resulted in an 80% loss of binding activity. This loss of binding activity was prevented when exposure to phenylmercuric chloride was done in the presence of alpha 2-adrenergic ligands, and it was reversed by subsequent exposure to dithiothreitol. Partial proteolysis of purified alpha 2-adrenergic receptors was obtained with Staphylococcus aureus V-8 protease, alpha-chymotrypsin, and papain. In a comparison with purified beta 2-adrenergic receptors, no common partial proteolytic products were found.
Topics: Blood Platelets; Chromatography, Affinity; Digitonin; Electrophoresis, Polyacrylamide Gel; Humans; Molecular Weight; Phenoxybenzamine; Phentolamine; Phenylmercury Compounds; Receptors, Adrenergic, alpha; Sepharose; Solubility; Sulfhydryl Compounds; Tritium
PubMed: 3005306
DOI: No ID Found