Ligand regulation of green fluorescent protein-tagged forms of the human beta(1)- and beta(2)-adrenoceptors; comparisons with the unmodified receptors.
Stable clones of HEK293 cells expressing either FLAG(TM) epitope-tagged, wild type human beta(1)- and beta(2)-adrenoceptors or C-terminally green fluorescent protein (GFP)-tagged forms of these receptors were established. The binding affinity of [(3)H]-dihydroalprenolol and other ligands was little affected by addition of GFP to the C-terminal of either receptor. Isoprenaline induced the internalisation of both beta(1)-adrenoceptor-GFP and beta(2)-adrenoceptor-GFP and following removal of the agonist both constructs were able to recycle to the cell surface. The extent of internalisation of beta(2)-adrenoceptor-GFP produced by isoprenaline was substantially greater than for beta(1)-adrenoceptor-GFP. C-terminal addition of GFP slowed markedly the rate of internalization of both the beta(1)-adrenoceptor and the beta(2)-adrenoceptor in response to isoprenaline. Sustained exposure to isoprenaline (24 h) produced substantially greater levels of downregulation of native beta(2)-adrenoceptor compared to beta(2)-adrenoceptor-GFP although both were equally effectively removed from the plasma membrane. Sustained exposure to isoprenaline resulted in a large fraction of beta(2)-adrenoceptor-GFP becoming trapped in internal vesicles/lysosomes but not degraded. Even after sustained exposure to isoprenaline a significant fraction of beta(1)-adrenoceptor-GFP remained at the cell surface. These results indicate that although GFP tagging of beta-adrenoceptors can provide qualitative visual patterns of agonist-induced receptor trafficking and regulation in HEK293 cells the quantitative details vary markedly from those obtained with the unmodified receptors.
McLean
A J
AJ
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ.
Milligan
G
G
D015784:Betaxolol / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D004082:Dihydroalprenolol / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D049452:Green Fluorescent Proteins; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics; D011412:Propanolamines / Q000378:metabolism; D011869:Radioligand Assay; D018342:Receptors, Adrenergic, beta-1 / Q000235:genetics / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014316:Tritium
D008024:Ligands; D008164:Luminescent Proteins; D009842:Oligopeptides; D010455:Peptides; D011412:Propanolamines; D018342:Receptors, Adrenergic, beta-1; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D014316:Tritium; D049452:Green Fluorescent Proteins; D004082:Dihydroalprenolol; C085706:FLAG peptide; D007545:Isoproterenol; D015784:Betaxolol; C033394:CGP 12177