Ligand regulation of green fluorescent protein‐tagged forms of the human β 1 ‐ and β 2 ‐adrenoceptors; comparisons with the unmodified receptors

• Published in: British journal of pharmacology Vol. 130; no. 8; pp. 1825 - 1832
• Main Authors: McLean, Alison J, Milligan, Graeme
• Format: Journal Article
• Language: English
• Published: 29.01.2009
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1038/sj.bjp.0703506

Stable clones of HEK293 cells expressing either FLAG TM epitope‐tagged, wild type human β 1 ‐ and β 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 β 1 ‐adrenoceptor‐GFP and β 2 ‐adrenoceptor‐GFP and following removal of the agonist both constructs were able to recycle to the cell surface. The extent of internalisation of β 2 ‐adrenoceptor‐GFP produced by isoprenaline was substantially greater than for β 1 ‐adrenoceptor‐GFP. C‐terminal addition of GFP slowed markedly the rate of internalization of both the β 1 ‐adrenoceptor and the β 2 ‐adrenoceptor in response to isoprenaline. Sustained exposure to isoprenaline (24 h) produced substantially greater levels of downregulation of native β 2 ‐adrenoceptor compared to β 2 ‐adrenoceptor‐GFP although both were equally effectively removed from the plasma membrane. Sustained exposure to isoprenaline resulted in a large fraction of β 2 ‐adrenoceptor‐GFP becoming trapped in internal vesicles/lysosomes but not degraded. Even after sustained exposure to isoprenaline a significant fraction of β 1 ‐adrenoceptor‐GFP remained at the cell surface. These results indicate that although GFP tagging of β‐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. British Journal of Pharmacology (2000) 130 , 1825–1832; doi: 10.1038/sj.bjp.0703506