Ligand regulation of green fluorescent protein-tagged forms of the human beta(1)- and beta(2)-adrenoceptors; comparisons with the unmodified receptors

Abstract

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.

Figure 1

Schematic diagram of the constructs which were stably expressed in HEK293 cells. cDNAs encoding the human β₁- and β₂-adrenoceptor (AR) were modified at their N-terminus to encode a FLAG^TM epitope. Following removal of the stop codon from the receptor sequences GFP was added in-frame.

Figure 2

Immunoblots of β₁-adrenoceptor and β₂-adrenoceptor constructs. Membranes expressing FLAG^TM β₂-adrenoceptor-GFP (1 and 4) or FLAG^TM β₁-adrenoceptor-GFP (2 and 3) were immunoblotted using either anti-β₂-adrenoceptor (1 and 2) or anti-β₁-adrenoceptor (3 and 4) antisera. Similar results were obtained in another set of immunoblots.

Figure 3

The β₁-adrenoceptor-GFP construct is internalized following addition of isoprenaline. A group of cells stably expressing the FLAG^TM β₁-adrenoceptor-GFP construct were exposed to isoprenaline (10 μM) for 0 (A), 10 (B), 20 (C) or 40 (D) min during visualization in the confocal microscope. Three separate experiments produced similar results.

Figure 4

Replacement of isoprenaline with betaxolol allows the β₁-adrenoceptor-GFP construct to recycle to the plasma membrane. Cells stably expressing the FLAG^TM β₁-adrenoceptor-GFP construct were exposed to isoprenaline (10 μM) for 0 (A) or 30 (B–D) min. Following washing in the presence of betaxolol (10 μM) the distribution of the receptor was visualized 10 (C) and 30 (D) min later. Three separate experiments produced similar results.

Figure 5

Internalization and recycling of β₂-adrenoceptor-GFP. Cells stably expressing the FLAG^TM β₂-adrenoceptor-GFP construct were exposed to isoprenaline (10 μM) for 0 (A) or 30 (B–D) min. Following washing with buffer containing alprenolol (10 μM) the distribution of the receptor was monitored 30 (C) and 40 (D) min later. Three separate experiments produced similar results.

Figure 6

Short-term treatment with isoprenaline internalizes but does not downregulate the FLAG^TM β₂-adrenoceptor-GFP construct. Cells stably expressing the FLAG^TM β₂-adrenoceptor-GFP construct were exposed to isoprenaline (10 μM) for varying times. Specific binding of a single concentration of either [³H]-DHA (2 nM) or [³H]-CGP-12177 (10 nM) to intact cells was then measured as described in Methods.

Figure 7

Comparison of the kinetics and extent of isoprenaline-induced internalization of the β₂-adrenoceptor and β₂-adrenoceptor-GFP. Cells stably expressing either the FLAG^TM β₂-adrenoceptor or the FLAG^TM β₂-adrenoceptor-GFP construct were exposed to isoprenaline (10 μM) for varying times. Specific binding of a single concentration of [³H]-CGP-12177 (10 nM) to intact cells was then measured as described in Methods. Results are presented as means±s.e.m. for five (FLAG^TM β₂-adrenoceptor) or four (FLAG^TM β₂-adrenoceptor-GFP) experiments.

Figure 8

Comparison of the kinetics and extent of isoprenaline-induced internalization of the β₁-adrenoceptor and β₁-adrenoceptor-GFP. Cells stably expressing either the FLAG^TM β₁-adrenoceptor or the FLAG^TM β₁-adrenoceptor-GFP construct were exposed to isoprenaline (10 μM) for varying times. Specific binding of a single concentration of [³H]-CGP-12177 (10 nM) to intact cells was then measured as described in Methods. Results are presented as means±s.e.m. for three (FLAG^TM β₁-adrenoceptor) or five (FLAG^TM β₁-adrenoceptor-GFP) experiments.

Figure 9

Long-term treatment with isoprenaline. Effects on distribution and downregulation of the β₂-adrenoceptor and β₂-adrenoceptor-GFP monitored by ligand binding studies. (A) Intact Cells. Cells stably expressing either the FLAG^TM β₂-adrenoceptor or the FLAG^TM β₂-adrenoceptor-GFP construct were either untreated or exposed to isoprenaline (10 μM) for 24 h. Specific binding of a single concentration of either [³H]-DHA (2 nM) or [³H]-CGP-12177 (10 nM) to intact cells was then measured as described in Methods. Results are presented as means±s.e.m. for three separate experiments. (B) Membranes. Cells stably expressing either the FLAG^TM β₂-adrenoceptor or the FLAG^TM β₂-adrenoceptor-GFP construct were either untreated or exposed to isoprenaline (10 μM) for 24 h. Membranes were prepared and the specific binding of a single concentration of [³H]-DHA (2 nM) was then measured as described in Methods. Results are presented as means±s.e.m. for three separate experiments.

Figure 10

Visualization of the location of β₂-adrenoceptor-GFP following long-term challenge with isoprenaline. Cells stably expressing the FLAG^TM β₂-adrenoceptor-GFP construct were either untreated (A) exposed to isoprenaline (10 μM) for 24 h (B). The distribution of the construct was then visualized. Two further experiments produced similar patterns.

Figure 11

Visualization of the location of β₁-adrenoceptor-GFP following long-term challenge with isoprenaline. Cells stably expressing the FLAG^TM β₁-adrenoceptor-GFP construct were either untreated (A) exposed to isoprenaline (10 μM) for 24 h (B) The distribution of the construct was then visualized. Two further experiments produced similar patterns.