Ligand regulation of green fluorescent protein-tagged forms of the human β1- and β2-adrenoceptors;: comparisons with the unmodified receptors

1 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. 2 The binding affinity of [H-3]-dihydroalprenolol and other Ligands was little affected by addition of GFP to the C-terminal of either receptor. 3 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. 4 The extent of internalisation of beta(2)-adrenoceptor-GFP produced by isoprenaline was substantially greater than for beta(1)-adrenoceptor-GFP, 5 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. 6 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. 7 Sustained exposure to isoprenaline resulted in a large fraction of beta(2)-adrenoceptor-GFP becoming trapped in internal vesicles/lysosomes but not degraded. 8 Even after sustained exposure to isoprenaline a significant fraction of beta(1)-adrenoceptor-GFP remained at the cell surface. 9 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.