Uneven cellular expression of recombinant α2A-adrenoceptors in transfected CHO cells results in loss of response in adenylyl cyclase inhibition

AbstractTwo populations of Chinese hamster ovary (CHO) cells expressing similar numbers of recombinant human alpha2A-adrenergic receptors (α2A-AR) showed different capacity to inhibit adenylyl cyclase (AC) activity. Cells transfected with an integrating vector exhibited agonist-dependent inhibition of forskolin-stimulated AC, whereas cells transfected with a non-integrating episomal vector showed no inhibition. Fluorescent microscopy and flow cytometry revealed a very uneven receptor distribution in the episomally transfected cell population. Monoclonal cell populations were expanded from this parent population. Most clones lacked significant amounts of receptors, while a few expressed receptors at high density; these exhibited efficient agonist-dependent inhibition of forskolin-stimulated AC activity. Thus, dense receptor expression in only a few cells is not sufficient to evoke a significant inhibitory response in a functional assay where AC is stimulated in all cells. Consequently, a false negative result was produced. Furthermore, the cell population transfected with an integrating vector showed loss of homogeneity with increasing passage number

Characterizing the Key Metabolic Pathways of the Neonatal Mouse Heart Using a Quantitative Combinatorial Omics Approach

The heart of a newborn mouse has an exceptional capacity to regenerate from myocardial injury that is lost within the first week of its life. In order to elucidate the molecular mechanisms taking place in the mouse heart during this critical period we applied an untargeted combinatory multiomics approach using large-scale mass spectrometry-based quantitative proteomics, metabolomics and mRNA sequencing on hearts from 1-day-old and 7-day-old mice. As a result, we quantified 1.937 proteins (366 differentially expressed), 612 metabolites (263 differentially regulated) and revealed 2.586 differentially expressed gene loci (2.175 annotated genes). The analyses pinpointed the fructose-induced glycolysis-pathway to be markedly active in 1-day-old neonatal mice. Integrated analysis of the data convincingly demonstrated cardiac metabolic reprogramming from glycolysis to oxidative phosphorylation in 7-days old mice, with increases of key enzymes and metabolites in fatty acid transport (acylcarnitines) and beta-oxidation. An upsurge in the formation of reactive oxygen species and an increase in oxidative stress markers, e.g., lipid peroxidation, altered sphingolipid and plasmalogen metabolism were also evident in 7-days mice. In vitro maintenance of physiological fetal hypoxic conditions retained the proliferative capacity of cardiomyocytes isolated from newborn mice hearts. In summary, we provide here a holistic, multiomics view toward early postnatal changes associated with loss of a tissue regenerative capacity in the neonatal mouse heart. These results may provide insight into mechanisms of human cardiac diseases associated with tissue regenerative incapacity at the molecular level, and offer a prospect to discovery of novel therapeutic targets.Peer reviewe

Cell Model Systems in Characterizing Alpha2-Adrenoceptors as Drug Targets.

The three alpha2-adrenoceptor (alpha2-AR) subtypes belong to the G protein-coupled receptor superfamily and represent potential drug targets. These receptors have many vital physiological functions, but their actions are complex and often oppose each other. Current research is therefore driven towards discovering drugs that selectively interact with a specific subtype. Cell model systems can be used to evaluate a chemical compound's activity in complex biological systems. The aim of this thesis was to optimize and validate cell-based model systems and assays to investigate alpha2-ARs as drug targets. The use of immortalized cell lines as model systems is firmly established but poses several problems, since the protein of interest is expressed in a foreign environment, and thus essential components of receptor regulation or signaling cascades might be missing. Careful cell model validation is thus required; this was exemplified by three different approaches. In cells heterologously expressing alpha2A-ARs, it was noted that the transfection technique affected the test outcome; false negative adenylyl cyclase test results were produced unless a cell population expressing receptors in a homogenous fashion was used. Recombinant alpha2C-ARs in non-neuronal cells were retained inside the cells, and not expressed in the cell membrane, complicating investigation of this receptor subtype. Receptor expression enhancing proteins (REEPs) were found to be neuronalspecific adapter proteins that regulate the processing of the alpha2C-AR, resulting in an increased level of total receptor expression. Current trends call for the use of primary cells endogenously expressing the receptor of interest; therefore, primary human vascular smooth muscle cells (SMC) expressing alpha2-ARs were tested in a functional assay monitoring contractility with a myosin light chain phosphorylation assay. However, these cells were not compatible with this assay due to the loss of differentiation. A rat aortic SMC cell line transfected to express the human alpha2B-AR was adapted for the assay, and it was found that the alpha2-AR agonist, dexmedetomidine, evoked myosin light chain phosphorylation in this model.Siirretty Doriast

Evaluation of Optogenetic Electrophysiology Tools in Human Stem Cell-Derived Cardiomyocytes

Current cardiac drug safety assessments focus on hERG channel block and QT prolongation for evaluating arrhythmic risks, whereas the optogenetic approach focuses on the action potential (AP) waveform generated by a monolayer of human cardiomyocytes beating synchronously, thus assessing the contribution of several ion channels on the overall drug effect. This novel tool provides arrhythmogenic sensitizing by light-induced pacing in combination with non-invasive, all-optical measurements of cardiomyocyte APs and will improve assessment of drug-induced electrophysiological aberrancies. With the help of patch clamp electrophysiology measurements, we aimed to investigate whether the optogenetic modifications alter human cardiomyocytes' electrophysiology and how well the optogenetic analyses perform against this gold standard. Patch clamp electrophysiology measurements of non-transduced stem cell-derived cardiomyocytes compared to cells expressing the commercially available optogenetic constructs Optopatch and CaViar revealed no significant changes in action potential duration (APD) parameters. Thus, inserting the optogenetic constructs into cardiomyocytes does not significantly affect the cardiomyocyte's electrophysiological properties. When comparing the two methods against each other (patch clamp vs. optogenetic imaging) we found no significant differences in APD parameters for the Optopatch transduced cells, whereas the CaViar transduced cells exhibited modest increases in APD-values measured with optogenetic imaging. Thus, to broaden the screen, we combined optogenetic measurements of membrane potential and calcium transients with contractile motion measured by video motion tracking. Furthermore, to assess how optogenetic measurements can predict changes in membrane potential, or early afterdepolarizations (EADs), cells were exposed to cumulating doses of E-4031, a hERG potassium channel blocker, and drug effects were measured at both spontaneous and paced beating rates (1, 2Hz). Cumulating doses of E-4031 produced prolonged APDs, followed by EADs and drug-induced quiescence. These observations were corroborated by patch clamp and contractility measurements. Similar responses, although more modest were seen with the I-Ks potassium channel blocker JNJ-303. In conclusion, optogenetic measurements of AP waveforms combined with optical pacing compare well with the patch clamp gold standard. Combined with video motion contractile measurements, optogenetic imaging provides an appealing alternative for electrophysiological screening of human cardiomyocyte responses in pharmacological efficacy and safety testings.Peer reviewe

REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity

<div><p>Receptor expression enhancing proteins (REEPs) were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs), specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein) family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi transport and processing/neuronal localization of cargo proteins. However, these other potential REEP functions and the mechanism by which they selectively enhance GPCR cell surface expression have not been clarified. By utilizing several REEP family members (REEP1, REEP2, and REEP6) and model GPCRs (α2A and α2C adrenergic receptors), we examined REEP regulation of GPCR plasma membrane expression, intracellular processing, and trafficking. Using a combination of immunolocalization and biochemical methods, we demonstrated that this REEP subset is localized primarily to ER, but not plasma membranes. Single cell analysis demonstrated that these REEPs do not specifically enhance surface expression of all GPCRs, but affect ER cargo capacity of specific GPCRs and thus their surface expression. REEP co-expression with α2 adrenergic receptors (ARs) revealed that this REEP subset interacts with and alter glycosidic processing of α2C, but not α2A ARs, demonstrating selective interaction with cargo proteins. Specifically, these REEPs enhanced expression of and interacted with minimally/non-glycosylated forms of α2C ARs. Most importantly, expression of a mutant REEP1 allele (hereditary spastic paraplegia SPG31) lacking the carboxyl terminus led to loss of this interaction. Thus specific REEP isoforms have additional intracellular functions besides altering ER structure, such as enhancing ER cargo capacity, regulating ER-Golgi processing, and interacting with select cargo proteins. Therefore, some REEPs can be further described as ER membrane shaping adapter proteins.</p> </div

REEP expression in non-permeabilized HEK293A, NRK, and Rat1 cells.

<p>HEK293A, NRK, and Rat1 cells were transfected with Flag-REEP1, -REEP2, or –REEP6. Forty-eight hrs later, cells were fixed with 4% PFA, but not permeabilized, for immunofluorescent labeling. Flag-REEPs were labeled with M2 (anti-Flag) antisera and identified with Alexa 594-conjugated goat anti-mouse secondary antibody. Note the similar strong peri-nuclear and intracellular reticular staining pattern seen in all three cell lines. Representative of three separate transfections. Scale bars: 10 µm.</p

Amino acid comparison of REEP Families.

<div><p>The REEP family can be subdivided into two subfamilies, REEP1-4, and REEP5-6. Yeast Yop1 is most similar to the latter subfamily and has been included in the alignment. Residues completely conserved in each subfamily are highlighted in yellow, whereas partially similar residues are highlighted in blue (consensus residue derived from a block of similar residues at a given position) or green (consensus residue derived from the occurrence of greater than 50% of a single residue at a given position). Hydrophobic segments are boxed in black. The conserved 14-3-3 binding site (RSXpS) found in REEP1-4 is boxed in red. Conserved positively charged residues postulated to be involved with microtubulin binding in REEP1-4 are demarcated (*), whereas conserved negatively charged residues in REEP5-6/Yop1 are also shown (#) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076366#B34" target="_blank">34</a>]. Alignment performed using Vector NTI v.11 (Invitrogen). GenBank protein accession numbers utilized were:</p> <p>mouse REEP1 (Yip2a): NP_848723 </p> <p>mouse REEP2 (Yip2d): NP_659114 </p> <p>mouse REEP3 (Yip2b): NP_848721 </p> <p>mouse REEP4 (Yip2c): NP_850919 </p> <p>mouse REEP5 (Yip2e): NP_031900 </p> <p>mouse REEP6 (Yip2f): NP_647453</p> <p>Yop1: NP_0153</p></div

Immunoprecipitation of α2 ARs by REEPs.

<p>HEK293A cells were co-transfected with HA-α2A or -α2C ARs and control vector, Flag-REEP1, -REEP2, or –REEP6. Eighteen hrs post-transfection, total cell lysates were isolated and analyzed by co-immunoprecipitation (co-IP) with M2 antibody (anti-Flag) and immunoblotting. Transferred proteins were probed with rabbit anti-HA Ab (A/C) or anti-M2 (B/D). Molecular weight markers (kDa) are shown to the left. Lanes representing total cell lysates (input), REEP co-IP, and α2 AR co-IP are labeled at the bottom of the blots. <b>A</b>. Note absence of α2A AR co-IP with any REEP tested (middle). <b>B</b>. Similar amounts of REEP1, REEP2, and REEP6 were present in REEP co-IP assays. <b>C</b>. REEP1, REEP2, and REEP6 could co-IP the REEP-enhanced, minimally glycosylated form of α2C AR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076366#pone-0076366-g009" target="_blank">Figures 9</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076366#pone-0076366-g010" target="_blank">10</a>). <b>D</b>. As seen with α2A AR/REEP co-IP assays, similar amounts of REEP1, REEP2, and REEP6 were present in REEP co-IP assays. Neither α2A nor α2C ARs could co-IP any REEP tested (B and D, right). IgG light chain artifacts are indicated by an arrow (far right).</p