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First published online 6 January 2004
doi: 10.1242/jcs.00890

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Trafficking of ß₂-adrenergic receptors: insulin and ß-agonists regulate internalization by distinct cytoskeletal pathways

¹ Departments of Pharmacology, Diabetes & Metabolic Diseases Research Center-HSC, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA
² Physiology & Biophysics, Diabetes & Metabolic Diseases Research Center-HSC, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA

View larger version (160K): [in a new window]   Fig. 1. Distinct elements of cytoskeleton are essential for sequestration of ß2AR induced by stimulation of cells with insulin (+Ins) or isoproterenol (+Iso). A431 cells stably expressing ß2AR tagged with enhanced GFP were used to monitor receptor internalization (by confocal microscopy) following stimulation with ß-adrenergic agonist (10 µM isoproterenol) or 100 nM insulin. Control cells, without any pretreatment or cells treated with cytoskeletal disrupting drugs nocodazole (10 µM) or latrunculin (1 µM) were subsequently stimulated with either insulin (100 nM, +Ins) or isoproterenol (10 µM, +Iso) for 30 minutes. After the fixation and embedding in SlowFade, cells were analyzed by confocal microscopy. In the control experiment (top panel), receptor relocated from cell periphery (area of plasma membrane, marked with yellow arrows) to nuclear proximity (white arrowheads). In the presence of nocodazole (+Noc.), stimulation with insulin (+Ins) induced sequestration of receptor from plasma membrane, whereas in cells stimulated with isoproterenol (+Iso), receptor remained on cell periphery (yellow arrows). Pretreatment of cells with latrunculin (+Latr.) suspended ß2AR relocation in response to stimulation with insulin. Bars, 10 µm.

View larger version (119K): [in a new window]   Fig. 2. Microtubule integrity is required for the ß2-AR trafficking induced by stimulation with isoproterenol: effect of nocodazole. A431 cells stably expressing GFP-tagged ß2AR were stimulated with either 100 nM insulin (+Ins) or 10 µM isoproterenol (+Iso) for 30 minutes (A). Fixed (2% paraformaldehyde in PBS, pH 7.2) cells were immunostained with monoclonal antibodies against -tubulin conjugated with Cy3 and examined by confocal microscopy (`microtubules'). The distribution of the GFP-tagged ß2AR and microtubules was analyzed simultaneously by confocal microscopy ((`beta2-AR'). Merging the results from these two analyzes (GFP-receptor in green, microtubules in red; (`merge(') provides a detailed image of the receptor localization. Treatment of the cells with nocodazole (B) induced tubulin depolymerization and blocked the sequestration of the ß2ARs in response to isoproterenol, but not to insulin. Treatment of the cells with latrunculin A (C) blocked sequestration of ß2AR in response to insulin, but not to isoproterenol. Bars, 10 µm.

View larger version (109K): [in a new window]   Fig. 3. Taxol-induced polymerization of microtubules does not prevent receptor sequestration in response to stimulation. A431 cells stably expressing GFP-tagged ß2AR (A) or wild-type (B) were stimulated with either insulin or isoproterenol for 30 minutes. Taxol (10 µM, (`+Taxol(') was added to cells for 30 minutes in advance. (A) GFP-tagged ß2AR internalization following stimulation was fixed and analyzed by confocal microscopy (control, upper panel). Pretreatment with taxol (lower panel) does not significantly affect receptor internalization in stimulated cells. (B) The same experiment performed on wild-type A431 cells: fixed (2% paraformaldehyde in PBS, pH 7.2) wild-type cells were immunostained with monoclonal antibodies against -tubulin coupled with FITC. Images from confocal microscopy showed the typical pattern of microtubules cytoskeleton in A431 cells (control experiment, upper panel). Treatment with taxol (lower panel) induced its polymerization and redistribution. Polymerized microtubules formed bulky rigid structures, localized in the cell periphery, in a parallel manner to the plasma membrane. Additional stimulation with isoproterenol ((`+Iso(') counteracts the effects of taxol and restores the arrangement of microtubules, radiating from nuclear vicinity with microtubules forming prominent arrays. Bars, 10 µm. ß2ARs localized either to the cell membrane (yellow arrows) or to the intracellular space (white arrowheads).

View larger version (25K): [in a new window]   Fig. 4. Stimulation with both insulin and isoproterenol results in an increased association of ß2AR with actin and tubulin. (A) Wild-type A431 cells were stimulated with either insulin or isoproterenol for 30 minutes. Cell lysates were immunoprecipitated using ß2AR-specific antibodies. Immunoprecipitated proteins were subjected to SDS-PAGE and immunoblotted with anti-tubulin, anti-actin or anti-ß2AR antibodies. Western blots show the increased density of bands corresponding to actin and tubulin. Bands, detected by ß2AR-antibodies, confirmed the equal loading of the samples. (B) Quantification of actin and tubulin detection in precipitates: data from western blots (as in A) were quantified by measuring area density (Adobe PhotoShop). The graphs show that the association of ß2AR with actin and tubulin is much greater in stimulated cells than that in unstimulated cells.

View larger version (73K): [in a new window]   Fig. 5. The integrity of the cytoskeleton is required for the recycling of ß2AR back to the plasma membrane. (A) Clones expressing ß2AR tagged GFP were stimulated with 100 nM insulin (+Ins, upper row) or with 10 µM isoproterenol (+Iso, bottom row) for 30 minutes, then removed from stimulation by either washout alone (for insulin) or washout and addition of the high-affinity ß-adrenergic antagonist propranolol (10 µM) (for isoproterenol-treated cells). The recovery process was monitored over a time period of 180 minutes, using confocal microscopy. In both cases, 180 minutes after washout the receptors are found to be relocated from the cytoplasm (white arrowheads) back to plasma membrane (yellow arrows). (B) Perturbation of microtubule cytoskeleton with nocodazole prevents the recycling of ß2ARs. Nocodazole was added to cultures after the stimulation by hormones and simultaneously with the termination of stimuli. Subsequent monitoring of receptor recovery revealed that after sequestration in response to stimulation either by insulin or by isoproterenol, recycling of ß2ARs back to the plasma membrane was impaired, i.e. a large pool of GFP-tagged receptor can be observed in the cytoplasm (white arrowheads) rather than in the cell membrane (yellow arrows). (C) Actin depolymerization blocks the recycling of ß2AR after its internalization induced by stimulation with isoproterenol. Clones were treated as above, except with latrunculin, rather than nocodazole. Scale bars: 10 µm. ß2ARs localized either to the cell membrane (yellow arrows) or to the intracellular space (white arrowheads).

View larger version (156K): [in a new window]   Fig. 6. Stimulation of cells with either isoproterenol or forskolin provokes microtubule reorganization. Untreated A431 cells (control, -), or cells pretreated with either nocodazole or taxol (see legends to Figs 1 and 3) were stimulated with either isoproterenol (10 µM, +Iso) for 30 minutes or with the plant diterpene forskolin (100 µM, +Forskolin) for 15 minutes. After the stimulation, the cells were washed, fixed and immunostained to visualize microtubules (see legend to Fig. 3). Stimulation by isoproterenol induces changes in the organization of microtubules, i.e. the radiance of microtubules from the central regions to the periphery of the cell was more prominent and multiple nucleation centers (note in the forskolin-treated cells) appeared. Treatment either with nocodazole or taxol perturbs these patterns of microtubule organization, i.e. radiating microtubule arrays were absent. Scale bar, 10 µm.