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First published online 20 January 2004
doi: 10.1242/jcs.00878

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Differences in endosomal targeting of human ß₁- and ß₂-adrenergic receptors following clathrin-mediated endocytosis

Membrane Biochemistry Section, Laboratory of Molecular and Cellular Neurobiology, National Institute of Neurological Disorders and Stroke, The National Institutes of Health, Bethesda, MD 20892, USA

View larger version (35K): [in a new window]   Fig. 1. Comparison of agonist-mediated internalization of receptor subtypes. Cells stably expressing ß1AR () or ß2AR () were exposed to 1 µM ISO for the indicated times or 30 minutes, washed and assayed for [3H]CGP-12177 binding to surface receptors as described in Materials and Methods. (A) Time course of internalization in BHK-hß1 and -hß2 cells expressing 978±47 (n=6) and 1010±84 (n=5) fmol of ßAR/mg protein. (B) Effect of ßAR density on internalization. Different clonal lines of BHK-hß1 and -hß2 cells were assayed for the amount of internalization after 30 minutes of agonist treatment. Some were induced with zinc sulfate for 24 hours. Internalization is plotted as a function of initial surface receptor density. The slopes of the linear regression lines are 196 and 454 fmol/30 minutes/pmol, a difference of 2.3-fold. (C) Same as A except for HEK 293-hß1 and -hß2 cells expressing 1790±42 (n=3) and 1580±31 (n=4) fmol of ßAR/mg protein. (D) Same as A except for Arr2-BHK-hß1 and -hß2 expressing 1680±204 (n=6) and 1310±169 (n=5) fmol of ßAR/mg protein.

View larger version (36K): [in a new window]   Fig. 2. Effect of arrestins and dominant-negative mutants of arrestin-2 and dynamin on agonist-mediated internalization of ß-subtypes. Cells were transiently cotransfected with equal amounts of total plasmid DNA containing Zem228c-ß1AR or -ß2AR, and either pcDNA3 (con), pcDNA3-arrestin-2 (arr-2), -arrestin-3 (arr-3), -arrestin-2-(319-418) (DN-arr), or -dynamin-K44A (DN-dyn). After 48 hours, the cells were incubated for 30 minutes with or without 1 µM ISO and assayed for surface receptors. Results represent the means±s.e.m. of 3-6 experiments for each condition. Although ßAR expression levels varied, they were similar for both subtypes at each condition.

View larger version (47K): [in a new window]   Fig. 3. Differences in agonist-stimulated phosphorylation of ß1AR and ß2AR in BHK cells. Untransfected cells and cells stably expressing ß1AR or ß2AR were incubated for 3 hours with [32P]orthophosphate, incubated with or without 1 µM ISO for 15 minutes or the times indicated, washed and lysed. After solubilization, receptors were immunoprecipitated with antibodies to ß1AR (BHK and BHK-hß1 cells) or ß2AR (BHK-hß2 cells) and separated by SDS-PAGE. Receptor phosphorylation was detected on the dried gels and quantified by phosphor imaging as described in Materials and Methods. (A) A representative grayscale image of 32P-labeled receptors from control and ISO-treated cells. Immunoprecipitates of equal amounts of protein (0.5 mg), ß1AR (0.6 pmol) or ß2AR (0.45 pmol) were loaded on the gel. (B) Summary of the quantification of receptor phosphorylation. Results are expressed as fold stimulation by agonist and are the means±s.e.m. of three separate experiments. (C) Time course of agonist-stimulated phosphorylation of ß1AR () and ß2AR () in BHK cells. Results shown are from a single experiment.

View larger version (34K): [in a new window]   Fig. 4. Pharmacological and functional properties of wild-type ß1AR and ß1AR-GFP. BHK cells stably expressing ß1AR () and ß1ARGFP () at 0.7-1 pmol/mg protein were assayed for binding or cAMP accumulation as described in Materials and Methods. (A) Saturation binding of radioligand. Cell lysates were incubated for 3 hours at 30°C with increasing concentrations of 125ICYP with or without 10 µM propranolol. Kd values for ICYP were 18.2±3.0 and 20.4±1.9 pM (P>0.05). (B) Antagonist competition binding. Same as A except 30 pM 125ICYP and increasing concentrations of CGP-20712A were used. Ki values for CGP-2012A were 9.7±3.9 and 14.1±3.0 nM (P>0.05). (C) Agonist competition binding. Same as B except ISO plus 100 µM GTP were used. Kd values for ISO were 57.0±1.4 and 87.6±3.4 nM (P<0.002). (D) Cells were incubated with increasing concentrations of ISO or 100 µM forskolin for 10 minutes and assayed for cAMP. EC50 values for ISO were 0.80±0.1 and 1.26±0.3 nM (P>0.05). Vmax values for ISO as a percentage of forskolin stimulation were 74.7±1.3 and 50.0±3.6% (P<0.002). A representative experiment is shown in each panel. Values are the means±s.e.m. of 3 or 4 experiments.

View larger version (63K): [in a new window]   Fig. 5. Visualization of agonist-mediated internalization of ß1AR- and ß2AR-GFP. BHK cells stably expressing ß1AR-GFP (top row) or ß2AR-GFP (second row) or ß1AR-GFP and transiently co-expressing arrestin-2 (third row) or arrestin-3 (bottom row) were observed by confocal fluorescence microscopy during stimulation with 1 µM ISO for 0, 5, 10 and 20 minutes at 37°C as described in Materials and Methods. Whereas agonist treatment resulted in the redistribution of most of ß2AR-GFP from plasma membrane to large perinuclear vesicles, there was little redistribution of ß1AR-GFP until arrestin-2 or -3 was co-expressed. Under these conditions, most of ß1AR-GFP appeared in small, peripheral vesicles that were distinct from those containing ß2AR-GFP. Bar, 10 µm.

View larger version (115K): [in a new window]   Fig. 6. Colocalization of ß1AR-GFP with transferrin and with clathrin. (A-I) HEK cells transiently transfected with ß1AR-GFP without (D-F) or with (A-C,G-I) arrestin-2 were incubated at 4°C with Texas Red®-labeled transferrin (20 µg/ml) for 30 minutes and with 1 µM ISO at 37°C for another 20 minutes (D-I). The cells were washed and fixed as described in Materials and Methods. The distribution of ß1AR-GFP (green) and transferrin (red) was examined using a confocal microscope. Colocalization of transferrin with ß1AR-GFP (yellow) can be observed in the merged images. (J-R) Same as A-I except transferrin was omitted and the cells were permeabilized and stained for clathrin (red) with rabbit anti-clathrin followed by Cy5-conjugated anti-rabbit (color changed from blue). Colocalization of clathrin with ß1AR-GFP (yellow) can be observed in the merged images. Scale bar: 5 µm.

View larger version (92K): [in a new window]   Fig. 7. Agonist-mediated endocytosis of ß1AR and ß2AR co-expressed in HEK 293 cells. Three different approaches were used to show the distribution of ß1AR (left panels) and ß2AR (middle panels) and any colocalization (right panels) in control cells (-ISO) and cells stimulated with 1 µM ISO for 15 minutes (+ISO). (A-F) Cells stably expressing HA-ß1AR were transiently transfected with Flag-ß2AR and arrestin-2, incubated with and without agonist, washed and fixed. The cells were then processed for immunofluorescence as described in Materials and Methods. HA-ß1AR (red) and Flag-ß2AR (green) are shown separately and as merged images to show colocalization (yellow). (G-L) The same as described above except the cells were incubated at 4°C with fluorescent anti-HA and anti-Flag, washed, warmed up for 5 minutes, incubated with or without agonist and fixed. HA-ß1AR (green) and Flag-ß2AR (red) are shown separately and as merged images to show colocalization (yellow). (M-R) Cells stably expressing HA-ß2AR were transiently transfected with ß1AR and arrestin-2, incubated with or without agonist, washed, fixed and processed for immunofluorescence. ß1AR (green) and HA-ß2AR (red) are shown separately and as merged images to show colocalization (yellow).

View larger version (25K): [in a new window]   Fig. 8. Subcellular fractionation of control and agonist-treated cells by sucrose density gradient centrifugation. Arr2-BHK-hß1 (A,C) and -hß2 (B,D) cells were induced with zinc sulfate for 24 hours. Lysates prepared from control cells (open symbols) and cell treated with ISO for 15 minutes (closed symbols) were fractionated by sucrose density gradient centrifugation and the fractions assayed for binding with 125ICYP (,,,) and [3H]CGP-12177 (,,,) along with the sucrose concentration (,) as described in Materials and Methods. Distributions of total receptors between plasma membrane and endosomal fractions were for ß1AR: control, 82.7±0.6 and 17.3±0.6%; ISO-treated, 63.1±3.6 and 36.9±3.6% (n=4); and for ß2AR: control, 87.8±2.4 and 12.2±2.4%; ISO-treated, 49.0±1.1 and 51±1.1% (n=3).

View larger version (43K): [in a new window]   Fig. 9. Detection of agonist-mediated internalization of ß-subtypes by cell surface biotinylation. Arr2-BHK-hß1 and -hß2 cells were induced with zinc sulfate for 24 hours, incubated with or without 1 µM ISO for 15 minutes, and subjected to cell surface biotinylation for 30 minutes at 4°C, or biotinylated first and then exposed to agonist. The cells were lysed, extracted with detergent and clarified by centrifugation. The soluble receptors were immunoprecipitated with antibodies to ß1AR or ß2AR C-tail preabsorbed to protein A-agarose. The bound receptors were subjected to SDS-PAGE and blotting with HRP-streptavidin followed by chemiluminescence detection and densitometry. In addition, portions of the lysates were assayed for 125ICYP binding, and separately, intact cells were assayed for [3H]CGP-12177 binding, to quantify the amounts of total and internalized receptors. (A) Blots of biotin-labeled ß1AR (left) and ß2AR (right) from control cells (C), cells exposed to ISO and biotinylated (I/B) and cells biotinylated and treated with ISO (B/I). Equal amounts of receptor (10 fmol of total ßAR) were loaded on the gel. (B) Summary of the quantification of cell surface receptor internalization by [3H]CGP-12177 binding and biotinylation by densitometry). Values are expressed as percentage of control and represent the means±s.e.m. of three separate experiments, each assayed in triplicate.

View larger version (36K): [in a new window]   Fig. 10. ß1AR and ß2AR recycle with similar kinetics but differ in sensitivity to monensin. Cells stably expressing ß1AR and ß2AR were treated with 1 µM ISO for 15-30 minutes to induce maximum internalization, washed to remove the agonist and allowed to recycle for the indicated times. The cells were then assayed for surface receptors as described in Materials and Methods. Recycling of ß1AR () and ß2AR () in (A) BHK cells and (B) Arr2-BHK cells. (C) Recycling of ß1AR (,) and ß2AR (,) in control and monensin-treated HEK 293 cells. Cells were treated with 100 µM monensin (closed symbols) for 30 minutes before adding agonist, and monensin was present during the recycling period. (D) Summary of ßAR recycling in control and monensin-treated cells. Values are expressed as the percentage recycling of internalized receptors, and represent the means±s.e.m. of 3-5 independent experiments. Differences between control and monensin-treated ß1AR-expressing cells were not significant. *P<0.01; **P<0.001.

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