Effects of the β-agonist, isoprenaline, on the down-regulation, functional responsiveness and trafficking of β2-adrenergic receptors with N-terminal polymorphisms

Authors

  • Yulia Koryakina,

    Corresponding author
    1. Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
    2. Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
    3. Arkansas Children's Hospital Research Institute, Little Rock, AR 72202, USA
      (email yk6u@virginia.edu)
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  • Stacie M. Jones,

    1. Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
    2. Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
    3. Arkansas Children's Hospital Research Institute, Little Rock, AR 72202, USA
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  • Lawrence E. Cornett,

    1. Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
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  • Kathryn Seely,

    1. Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
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  • Lisa Brents,

    1. Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
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  • Paul L. Prather,

    1. Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
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  • Alexander Kofman,

    1. Department of Microbiology, Immunology and Cancer Biology, School of Medicine, University of Virginia, Charlottesville, VA 22908, USA
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  • Richard C. Kurten

    1. Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
    2. Arkansas Children's Hospital Research Institute, Little Rock, AR 72202, USA
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(email yk6u@virginia.edu)

Abstract

The β2-AR (β2-adrenergic receptor) is an important target for respiratory and CVD (cardiovascular disease) medications. Clinical studies suggest that N-terminal polymorphisms of β2-AR may act as disease modifiers. We hypothesized that polymorphisms at amino acids 16 and 27 result in differential trafficking and down-regulation of β2-AR variants following β-agonist exposure. The functional consequences of the four possible combinations of these polymorphisms in the human β2-AR (designated β2-AR-RE, β2-AR-GE, β2-AR-RQ and β2-AR-GQ) were studied using site-directed mutagenesis and recombinant expression in HEK-293 cells (human embryonic kidney cells). Ligand-binding assays demonstrated that after 24 h exposure to 1 μM isoprenaline, isoforms with Arg162-AR-RE and β2-AR-RQ) underwent increased down-regulation compared with isoforms with Gly162-AR-GE and β2-AR-GQ). Consistent with these differences in down-regulation between isoforms, prolonged isoprenaline treatment resulted in diminished cAMP response to subsequent isoprenaline challenge in β2-AR-RE relative to β2-AR-GE. Confocal microscopy revealed that the receptor isoforms had similar co-localization with the early endosomal marker EEA1 following isoprenaline treatment, suggesting that they had similar patterns of internalization. None of the isoforms exhibited significant co-localization with the recycling endosome marker Rab11 in response to isoprenaline treatment. Furthermore, we found that prolonged isoprenaline treatment led to a higher degree of co-localization of β2-AR-RE with the lysosomal marker LAMP1 (lysosome-associated membrane protein 1) compared with that of β2-AR-GE. Taken together, these results indicate that a mechanism responsible for differential responses of these receptor isoforms to the β-agonist involves differences in the efficiency with which agonist-activated receptors are trafficked to the lysosomes for degradation, or differences in degradation in the lysosomes.

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