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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).

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