Article date: August 2000
By: Alison J McLean, Graeme Milligan in Volume 130, Issue 8, pages 1825-1832
Stable clones of HEK293 cells expressing either FLAGTM epitope‐tagged, wild type human β1‐ and β2‐adrenoceptors or C‐terminally green fluorescent protein (GFP)‐tagged forms of these receptors were established.
The binding affinity of [3H]‐dihydroalprenolol and other ligands was little affected by addition of GFP to the C‐terminal of either receptor.
Isoprenaline induced the internalisation of both β1‐adrenoceptor‐GFP and β2‐adrenoceptor‐GFP and following removal of the agonist both constructs were able to recycle to the cell surface.
The extent of internalisation of β2‐adrenoceptor‐GFP produced by isoprenaline was substantially greater than for β1‐adrenoceptor‐GFP.
C‐terminal addition of GFP slowed markedly the rate of internalization of both the β1‐adrenoceptor and the β2‐adrenoceptor in response to isoprenaline.
Sustained exposure to isoprenaline (24 h) produced substantially greater levels of downregulation of native β2‐adrenoceptor compared to β2‐adrenoceptor‐GFP although both were equally effectively removed from the plasma membrane.
Sustained exposure to isoprenaline resulted in a large fraction of β2‐adrenoceptor‐GFP becoming trapped in internal vesicles/lysosomes but not degraded.
Even after sustained exposure to isoprenaline a significant fraction of β1‐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.
Stable clones of HEK293 cells expressing either FLAGTM epitope‐tagged, wild type human β1‐ and β2‐adrenoceptors or C‐terminally green fluorescent protein (GFP)‐tagged forms of these receptors were established.
The binding affinity of [3H]‐dihydroalprenolol and other ligands was little affected by addition of GFP to the C‐terminal of either receptor.
Isoprenaline induced the internalisation of both β1‐adrenoceptor‐GFP and β2‐adrenoceptor‐GFP and following removal of the agonist both constructs were able to recycle to the cell surface.
The extent of internalisation of β2‐adrenoceptor‐GFP produced by isoprenaline was substantially greater than for β1‐adrenoceptor‐GFP.
C‐terminal addition of GFP slowed markedly the rate of internalization of both the β1‐adrenoceptor and the β2‐adrenoceptor in response to isoprenaline.
Sustained exposure to isoprenaline (24 h) produced substantially greater levels of downregulation of native β2‐adrenoceptor compared to β2‐adrenoceptor‐GFP although both were equally effectively removed from the plasma membrane.
Sustained exposure to isoprenaline resulted in a large fraction of β2‐adrenoceptor‐GFP becoming trapped in internal vesicles/lysosomes but not degraded.
Even after sustained exposure to isoprenaline a significant fraction of β1‐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
DOI: 10.1038/sj.bjp.0703506
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