title: Effects of the β-agonist, isoprenaline, on the down-regulation, functional responsiveness and trafficking of β2-adrenergic receptors with N-terminal polymorphisms.
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 Arg16 (β2-AR-RE and β2-AR-RQ) underwent increased down-regulation compared with isoforms with Gly16 (β2-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.
authors.affiliation: Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. yk6u@virginia.edu
authors.forename: Yulia
authors.initials: Y
authors.lastname: Koryakina
authors.forename: Stacie M
authors.initials: SM
authors.lastname: Jones
authors.forename: Lawrence E
authors.initials: LE
authors.lastname: Cornett
authors.forename: Kathryn
authors.initials: K
authors.lastname: Seely
authors.forename: Lisa
authors.initials: L
authors.lastname: Brents
authors.forename: Paul L
authors.initials: PL
authors.lastname: Prather
authors.forename: Alexander
authors.initials: A
authors.lastname: Kofman
authors.forename: Richard C
authors.initials: RC
authors.lastname: Kurten
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects*; D057809:HEK293 Cells; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology*; D052118:Lysosomal-Associated Membrane Protein 1 / Q000032:analysis; D011110:Polymorphism, Genetic; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects*; D018343:Receptors, Adrenergic, beta-2 / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
chemical_list: D000318:Adrenergic beta-Agonists; D000242:Cyclic AMP; D007545:Isoproterenol; D052118:Lysosomal-Associated Membrane Protein 1; D020033:Protein Isoforms; D018343:Receptors, Adrenergic, beta-2; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins