Common ADRB2 Haplotypes Derived from 26 Polymorphic Sites Direct β2-Adrenergic Receptor Expression and Regulation Phenotypes

Background: The β2-adrenergic receptor (β2AR) is expressed on numerous cell-types including airway smooth muscle cells and cardiomyocytes. Drugs (agonists or antagonists) acting at these receptors for treatment of asthma, chronic obstructive pulmonary disease, and heart failure show substantial interindividual variability in response. The ADRB2 gene is polymorphic in noncoding and coding regions, but virtually all ADRB2 association studies have utilized the two common nonsynonymous coding SNPs, often reaching discrepant conclusions. Methodology/Principal Findings: We constructed the 8 common ADRB2 haplotypes derived from 26 polymorphisms in the promoter, 5′UTR, coding, and 3′UTR of the intronless ADRB2 gene. These were cloned into an expression construct lacking a vector-based promoter, so that β2AR expression was driven by its promoter, and steady state expression could be modified by polymorphisms throughout ADRB2 within a haplotype. “Whole-gene” transfections were performed with COS-7 cells and revealed 4 haplotypes with increased cell surface β2AR protein expression compared to the others. Agonist-promoted downregulation of β2AR protein expression was also haplotype-dependent, and was found to be increased for 2 haplotypes. A phylogenetic tree of the haplotypes was derived and annotated by cellular phenotypes, revealing a pattern potentially driven by expression. Conclusions/Significance: Thus for obstructive lung disease, the initial bronchodilator response from intermittent administration of β-agonist may be influenced by certain β2AR haplotypes (expression phenotypes), while other haplotypes may influence tachyphylaxis during the response to chronic therapy (downregulation phenotypes). An ideal clinical outcome of high expression and less downregulation was found for two haplotypes. Haplotypes may also affect heart failure antagonist therapy, where β2AR increase inotropy and are anti-apoptotic. The haplotype-specific expression and regulation phenotypes found in this transfection-based system suggest that the density of genetic information in the form of these haplotypes, or haplotype-clusters with similar phenotypes can potentially provide greater discrimination of phenotype in human disease and pharmacogenomic association studies

Common ADRB2 Haplotypes Derived from 26 Polymorphic Sites Direct β2-Adrenergic Receptor Expression and Regulation Phenotypes

The beta2-adrenergic receptor (beta2AR) is expressed on numerous cell-types including airway smooth muscle cells and cardiomyocytes. Drugs (agonists or antagonists) acting at these receptors for treatment of asthma, chronic obstructive pulmonary disease, and heart failure show substantial interindividual variability in response. The ADRB2 gene is polymorphic in noncoding and coding regions, but virtually all ADRB2 association studies have utilized the two common nonsynonymous coding SNPs, often reaching discrepant conclusions.We constructed the 8 common ADRB2 haplotypes derived from 26 polymorphisms in the promoter, 5'UTR, coding, and 3'UTR of the intronless ADRB2 gene. These were cloned into an expression construct lacking a vector-based promoter, so that beta2AR expression was driven by its promoter, and steady state expression could be modified by polymorphisms throughout ADRB2 within a haplotype. "Whole-gene" transfections were performed with COS-7 cells and revealed 4 haplotypes with increased cell surface beta2AR protein expression compared to the others. Agonist-promoted downregulation of beta2AR protein expression was also haplotype-dependent, and was found to be increased for 2 haplotypes. A phylogenetic tree of the haplotypes was derived and annotated by cellular phenotypes, revealing a pattern potentially driven by expression.Thus for obstructive lung disease, the initial bronchodilator response from intermittent administration of beta-agonist may be influenced by certain beta2AR haplotypes (expression phenotypes), while other haplotypes may influence tachyphylaxis during the response to chronic therapy (downregulation phenotypes). An ideal clinical outcome of high expression and less downregulation was found for two haplotypes. Haplotypes may also affect heart failure antagonist therapy, where beta2AR increase inotropy and are anti-apoptotic. The haplotype-specific expression and regulation phenotypes found in this transfection-based system suggest that the density of genetic information in the form of these haplotypes, or haplotype-clusters with similar phenotypes can potentially provide greater discrimination of phenotype in human disease and pharmacogenomic association studies

One probe, two-channel imaging of nuclear and cytosolic compartments with orange and red emissive dyes

Several new DNA-targeting probes that exhibit binding-induced 'turn on' fluorescence are presented. Two of the dyes, orange emissive 1, (E)-4-(4(-4-methylpiperazin-1-yl)phenyl)6-(4-(4-methylpi-perazin-1-yl)styryl)pyrimidin-2-ol), and red emissive 2, (E)-4-(4(-4-methyl-piperazin-1-yl)-phenyl)6-(4-(4-methylpiperazin-1-yl)styryl)-1,3-propanedionato-κO,κO']difluoroborane), are brightly fluorescent when bound to DNA, but are virtually non-fluorescent in aqueous solutions. Confocal fluorescence microscopy of live BT474, MCF7 and HEK293 cells demonstrates that both probes are cell permeable and rapidly accumulated intracellularly into cell nuclei and the cytosol. Taking advantage of their environmental sensitivity, these two pools of fluorophores are readily resolved into separate channels, and thus, a single dye allows two-color imaging of the nuclear and cytosolic compartments

Two-Photon Spectroscopy as a New Sensitive Method for Determining the DNA Binding Mode of Fluorescent Nuclear Dyes

A new optical strategy to determine the binding modes (intercalation vs groove binding) of small fluorescent organic molecules with calf thymus DNA was developed using two-photon absorption (TPA) spectroscopy. Two-photon excited emission was utilized to investigate a series of fluorescent nuclear dyes. The results show that TPA cross-sections are able to differentiate the fine details between the DNA binding modes. Groove binding molecules exhibit an enhanced TPA cross-section due to the DNA electric field induced enhancement of the transition dipole moment, while intercalative binding molecules exhibit a decrease in the TPA cross-section. Remarkably, the TPA cross-section of 4,6-bis(4-(4-methylpiperazin-1-yl)phenyl) pyrimidine is significantly enhanced (13.6-fold) upon binding with DNA. The sensitivity of our TPA methodology is compared to circular dichroism spectroscopy. TPA demonstrates superior sensitivity by more than an order of magnitude at low DNA concentrations. This methodology can be utilized to probe DNA interactions with other external molecules such as proteins, enzymes, and drugs

A New Design Strategy and Diagnostic to Tailor the DNA-Binding Mechanism of Small Organic Molecules and Drugs

The classical model for DNA groove binding states that groove binding molecules should adopt a crescent shape that closely matches the helical groove of DNA. Here, we present a new design strategy that does not obey this classical model. The DNA-binding mechanism of small organic molecules was investigated by synthesizing and examining a series of novel compounds that bind with DNA. This study has led to the emergence of structure–property relationships for DNA-binding molecules and/or drugs, which reveals that the structure can be designed to either intercalate or groove bind with calf thymus dsDNA by modifying the electron acceptor properties of the central heterocyclic core. This suggests that the electron accepting abilities of the central core play a key role in the DNA-binding mechanism. These small molecules were characterized by steady-state and ultrafast nonlinear spectroscopies. Bioimaging experiments were performed in live cells to evaluate cellular uptake and localization of the novel small molecules. This report paves a new route for the design and development of small organic molecules, such as therapeutics, targeted at DNA as their performance and specificity is dependent on the DNA-binding mechanism

Two-Photon Spectroscopy as a New Sensitive Method for Determining the DNA Binding Mode of Fluorescent Nuclear Dyes

A new optical strategy to determine the binding modes (intercalation vs groove binding) of small fluorescent organic molecules with calf thymus DNA was developed using two-photon absorption (TPA) spectroscopy. Two-photon excited emission was utilized to investigate a series of fluorescent nuclear dyes. The results show that TPA cross-sections are able to differentiate the fine details between the DNA binding modes. Groove binding molecules exhibit an enhanced TPA cross-section due to the DNA electric field induced enhancement of the transition dipole moment, while intercalative binding molecules exhibit a decrease in the TPA cross-section. Remarkably, the TPA cross-section of 4,6-bis­(4-(4-methylpiperazin-1-yl)­phenyl) pyrimidine is significantly enhanced (13.6-fold) upon binding with DNA. The sensitivity of our TPA methodology is compared to circular dichroism spectroscopy. TPA demonstrates superior sensitivity by more than an order of magnitude at low DNA concentrations. This methodology can be utilized to probe DNA interactions with other external molecules such as proteins, enzymes, and drugs

Turn-On, Fluorescent Nuclear Stains with Live Cell Compatibility

DNA-binding, green and yellow fluorescent probes with excellent brightness and high on/off ratios are reported. The probes are membrane permeable, live-cell compatible, and optimally matched to 405 nm and 514 nm laser lines, making them attractive alternatives to UV-excited and blue emissive Hoechst 33342 and DAPI nuclear stains. Their electronic structure was investigated by optical spectroscopy supported by TD-DFT calculations. DNA binding is accompanied by 27- to 75-fold emission enhancements, and linear dichroism demonstrates that one dye is a groove binder while the other intercalates into DNA

Lack of a relationship between baseline β₂AR expression and the % downregulation.

<p>The agonist-promoted downregulation, expressed as a percentage, was plotted against baseline β₂AR protein expression. There was no relationship (r² = 0.001, p>0.9) between the initial expression of β₂AR and the extent of downregulation. Results are from the 7 experiments of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011819#pone-0011819-g004" target="_blank">Figure 4</a>.</p

Phylogenetic tree of β₂AR haplotypes with phenotypic annotation.

<p>Shown is a phylogenetic tree constructed as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011819#s3" target="_blank">Methods</a> with the 8 β₂AR haplotype sequences. Shown are the expression phenotypes (I, increased; R, reference) and the dual phenotypes that incorporate expression and downregulation (denoted A, B, C from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011819#pone-0011819-g006" target="_blank">Figure 6</a>). L1 and L2 represent the two ancestral lineages, while L2(1) and L2(2) are lineages from L2.</p