Expression of the porcine beta2-adrenergic receptor in Chinese hamster ovary cells.

Liang, Weida; Bidwell, Christopher A.; Collodi, Paul R.; Mills, S.E.

doi:10.2527/2000.7892329x

Cited by 14 publications

(13 citation statements)

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“…Values for the inhibition constant, K i , for the high-and low-affinity sites, respectively, were 1.6 and 2,630 nM with CGP 20712A as competitor, and 13.6 and 807 nM with bisoprolol as competitor. Expected values are based on data for each drug using the cloned β 1 AR and β 2 AR (Cao, 1998;Liang et al, 2000). Using β 1 AR selective antagonists, these data Values for K i are means ± SEM of three independent experiments with different pigs.…”

Section: Resultsmentioning

confidence: 99%

“…Stably transfected Chinese hamster ovary cell lines expressing the porcine β 1 AR or β 2 AR were grown in an atmosphere of 95% air and 5% (CO 2 at 37°C in a 1:1 F12:Dulbecco's modified Eagle's medium DMEM) media containing 100 U/mL of ampicillin, 200 U/mL of penicillin, 200 g/mL of streptomycin, 10 −8 M Se, and 1.2 mg/mL of NaHCO 3 in 1.5 mM HEPES, pH 7.4), plus 10% FBS and G418 (0.5 mg/mL; Liang et al, 2000). Confluent cells were washed twice with warm F12:DMEM and detached with 0.4% trypsin in F12:DMEM.…”

Section: Cell Linesmentioning

confidence: 99%

“…For species other than swine, ractopamine is suggested to have some selectivity for the β 1 AR (Smith et al, 1990;Moody et al, 2000). For the pig, the β 2 AR provides the best signal transduction (Mills et al, 2002), whereas binding affinity is similar for the β 1 AR and β 2 AR (Spurlock et al, 1993b;Liang et al, 2000;Mills et al, 2002). It was of interest, therefore, to determine which βAR mediates the activation of lipolysis in swine adipocytes.…”

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confidence: 99%

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β-Adrenergic receptor subtypes that mediate ractopaminestimulation of lipolysis1,2

2003

Journal of Animal Science

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Ractopamine HCl is an beta-adrenergic receptor (betaAR) ligand that was recently approved for use in swine to enhance carcass leanness. The RR stereoisomer of ractopamine is the most active of the four stereoisomers exhibiting the highest affinity and signaling response. The RR isomer exhibits selective activation of the porcine beta2AR, which might limit the lipolytic response to ractopamine because the betaAR is the predominant subtype in swine adipocytes and may mediate most of the lipolytic response. Therefore, we determined the betaAR subtypes that mediate the lipolytic response to ractopamine in swine adipocytes. In order to confirm the predominant role of the beta1AR in porcine adipocytes, isoproterenol-stimulated lipolysis was inhibited by increasing doses of subtype-selective antagonists. Inhibition curves were biphasic using beta1AR antagonists (CGP 20712A and bisoprolol) and curve analysis indicated that both beta1AR an beta2AR contributed to lipolysis with 50 to 60% of the response coming from the beta1AR. Inhibition with the beta2AR antagonist clenbuterol revealed only one class of betaAR that closely approximated the kinetics of the beta1AR. When the RR isomer of ractopamine was the lipolytic agent, similar results to isoproterenol were observed, except that the estimated contribution of the beta1AR was 38%. That beta2AR antagonists did not detect a contribution of the beta2AR to lipolysis may indicate that the beta1AR masked the response to the beta2AR. Dose titration with the RR isomer in the presence of a saturating concentration of beta1AR or beta2AR antagonists indicated that each subtype was present in sufficient quantities to stimulate lipolysis near maximally. Data indicate that both the beta1AR and beta2AR are functionally linked to lipolysis in swine adipocytes and that ractopamine activates each subtype. The RR isomer of ractopamine stimulated adenosine 3',5'-cyclic phosphate accumulation with equal efficacy to isoproterenol through the cloned porcine beta2AR, but was only 35% as efficacious through the cloned porcine beta1AR. These data confirm the beta2AR selectivity of the RR stereoisomer, but suggest the partial agonism through the beta1AR is sufficient to activate lipolysis through both subtypes in swine adipocytes.

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Section: Resultsmentioning

confidence: 99%

Section: Cell Linesmentioning

confidence: 99%

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confidence: 99%

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β-Adrenergic receptor subtypes that mediate ractopaminestimulation of lipolysis1,2

2003

Journal of Animal Science

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“…CHO cells stably transfected with a gene encoding the ␤ 2 -adrenergic receptor (␤ 2 AR) were provided by Dr. S. E. Mills (52,53). Before quantification of cAMP levels, untreated and filipintreated CHO-␤ 2 AR cells were incubated for 4 h with 100 ng/ml CT or 1 M ␤ 2 AR agonist isoproterenol.…”

Section: Methodsmentioning

confidence: 99%

Lipid Rafts Alter the Stability and Activity of the Cholera Toxin A1 Subunit

Ray

Taylor

Banerjee

et al. 2012

Journal of Biological Chemistry

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“…CHO cells stably expressing the M2 muscarinic receptor (10), M3 muscarinic receptor (11), and 5HT1A receptor (from Dr. D. Manning, University of Pennsylvania) have been described previously (12). CHO cells were grown in CHO IIIa medium (Invitrogen) containing dialyzed fetal bovine serum (M2-CHO, M3-CHO, CHO) or charcoal-stripped fetal bovine serum (5HT1A-CHO) (Atlanta Biologicals), methotrexate (M2-CHO, M3-CHO), penicillin, streptomycin, glutamine, and fungizone.…”

Section: Methodsmentioning

confidence: 99%

Receptor-mediated Reversible Translocation of the G Protein βγ Complex from the Plasma Membrane to the Golgi Complex

Akgöz¹,

Kalyanaraman²,

Gautam

2004

Journal of Biological Chemistry

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Heterotrimeric G proteins have been thought to function on the plasma membrane after activation by transmembrane receptors. Here we show that, after activation by receptors, the G protein ␤␥ complex selectively translocates to the Golgi. Receptor inactivation results in G␤␥ translocating back to the plasma membrane. Both translocation processes occur rapidly within seconds. The efficiency of translocation is influenced by the type of ␥ subunit present in the G protein. Distinctly different receptor types are capable of inducing the translocation. Receptor-mediated translocation of G␤␥ can spatially segregate G protein signaling activity.Heterotrimeric (␣␤␥) G proteins are localized to the plasma membrane of mammalian cells, facilitating interaction and activation by transmembrane G protein-coupled receptors (1-3). Extensive characterization of the effectors on which the G proteins act has suggested that the activated G protein ␣ and ␤␥ subunits function on the plasma membrane (3-6). It has been thought that the post-translational addition of a lipid moiety to the ␣ subunit and the ␥ subunit aids in the localization of G␣ and G␤␥ complex to the plasma membrane, where they act on the effector molecules (7). However, there is little information about the properties of these proteins or the signaling they mediate in intact live mammalian cells, because studies attempting to observe G protein function in living mammalian cells have been limited.To visualize the impact of receptor activation and inactivation on the spatial distribution of G protein subunits, we tagged G protein subunits with the yellow and cyan mutant forms of the green fluorescent protein, YFP 1 and CFP, respectively. The fusion proteins were expressed in mammalian cell lines and observed after activating overexpressed or endogenous receptors using fluorescence-based imaging methods. Although we have previously obtained evidence indicating the direct involvement of the G protein ␥ subunit in receptor interaction (8, 9), we examined the effect of receptor activation on ␣o-CFP, ␤1, and different ␥ subunit types tagged with YFP in Chinese hamster ovary (CHO) cells overexpressing M2 muscarinic receptors. We discovered that ␥-YFP translocated from the plasma membrane to the cell interior on receptor activation and translocated back to the plasma membrane on inactivation. The ␤ subunit co-translocated with the ␥ subunit. The rapidity of the translocation process and proportion of ␤␥ complex that translocated were dependent on the ␥ subunit type present in the expressed G protein. Experiments using a marker for the Golgi complex and a Golgi disrupting agent, brefeldin A, indicated that the ␤␥ complex translocates to the Golgi complex. The translocation was sensitive to G i/o -and G q -coupled receptor stimulation. Endogenous receptors also stimulated G␤␥ translocation. The translocation of the ␤␥ complex is selective, because ␣o-CFP or a chimeric ␣o-q-CFP that couples to G q -coupling receptors do not translocate from the plasma membrane in response to rece...

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Expression of the porcine beta2-adrenergic receptor in Chinese hamster ovary cells.

Cited by 14 publications

References 0 publications

β-Adrenergic receptor subtypes that mediate ractopaminestimulation of lipolysis1,2

β-Adrenergic receptor subtypes that mediate ractopaminestimulation of lipolysis1,2

Lipid Rafts Alter the Stability and Activity of the Cholera Toxin A1 Subunit

Receptor-mediated Reversible Translocation of the G Protein βγ Complex from the Plasma Membrane to the Golgi Complex

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