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First published online 9 June 2004
doi: 10.1242/jcs.01168

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Rab11 regulates the recycling and lysosome targeting of ß₂-adrenergic receptors

¹ Department of Pediatrics and Molecular Physiology and Biophysics, Baylor College of Medicine, 6621 Fannin, CCC 1040.00, Houston, TX 77030, USA
² Department of Pharmacological and Pharmaceutical Sciences, University of Houston, 521 Science and Research Building 2, Houston, TX 77204, USA

View larger version (51K): [in a new window]   Fig. 1. Overexpression of EGFP-rab11S25N inhibits ß2AR recycling from vesicles devoid of EEA1. 12ß6 cells transiently expressing empty EGFP vector (a,a'), EGFP-rab11S25N (b,b'), or EGFP-rab11WT (c,c') were treated with ISO for 20 minutes to induce ß2AR internalization. They were then washed and placed in media containing propranolol (5 µM) for 15 minutes (A) or 30 minutes (B) at 37°C to allow receptor recycling. ß2ARs were identified using an anti-C-terminal antibody and EEA1 was identified using a mouse monoclonal antibody. Panels a-c show the localization of ß2ARs (red) and EEA1 (green), with areas of colocalization appearing yellow, whereas a'-c' show the distribution of EGFP. Scale bar, 10 µm.

View larger version (33K): [in a new window]   Fig. 2. Overexpression of EGFP-rab11S25N inhibits ß2AR recycling from transferrin-positive endosomes. Cells transiently expressing vector (panels a,a'), EGFP-rab11S25N (panels b,b'), or EGFP-rab11WT (panels c,c') were treated with ISO for 20 minutes in serum free media, washed, and placed in serum-free media containing propranolol (5 µM) at 37°C to allow ß2AR recycling. (A) ß2ARs were allowed to recycle for 30 minutes in the presence of Alexa 594-transferrin (50 µg/ml). (B) Alexa 594-transferrin (100 µg/ml) was added to the media for 20 minutes during the incubation with ISO, washed, and both ß2ARs and transferrin were allowed to recycle for 15 minutes at 37°C. In both A and B, ß2ARs appear green and Alexa 594-transferrin appears red. Areas of colocalization of ß2ARs and transferrin appear yellow. Scale bar, 10 µm.

View larger version (32K): [in a new window]   Fig. 3. Inducible overexpression of EGFP:rab11 chimeras. EcR:EGFP-rab11 cells were grown for 72 hours in the presence of 0.125% vehicle (EtOH) or 5 µM ponasterone A (Pon A), then lysed and analysed by immunoblotting. Protein expression was determined using anti-rab11 antibody and ECL and then analyzed by densitometry. The upper bands (50 kDa) are EGFP-rab11 and the lower bands (25 kDa) are endogenous rab11. The levels of overexpression of EGFP-rab11WT and the S25N mutant were approximately fourfold and twofold, respectively, that of endogenous rab11. When observed by fluorescence microscopy, more than 80% of cells expressed EGFP-rab11 and their subcellular distributions were similar to those observed in transiently transfected cells (data not shown).

View larger version (13K): [in a new window]   Fig. 4. Inducible overexpression of EGFP-rab11S25N inhibits the rate of recycling of ß2ARs following a brief exposure to agonist. (A) EGFP-rab11S25N and (B) EGFP-rab11WT. Following treatment with ponasterone () or vehicle () for 72 hours, cells were treated with 5 µM ISO for 20 minutes, washed four times, and incubated in DME-H at 37°C for varying times up to 60 minutes to allow receptor recycling. Surface receptors were determined by incubation with 6 nM [3H]CGP12177 at 0°C and bound radioligand was quantified by scintillation spectroscopy. Each point represents the mean±s.e.m. of three independent experiments. The fraction of receptors that have recycled was plotted as a function of tim e following the removal of agonist and the curves fitted as previously described (Morrison et al., 1996). The zero point represents the 65-70% of receptors that internalized during agonist treatment and was not different in the two groups.

View larger version (32K): [in a new window]   Fig. 5. ß2ARs localize with EGFP-rab11WT and pulse-chased transferrin following a prolonged exposure to agonist. (A) 12ß6 cells transiently expressing empty EGFP vector (a,a') or EGFP-rab11WT (b,b') were treated with ISO for 6 hours, fixed immediately, and ß2ARs and EEA1 labeled as described. ß2ARs appear red and EEA1 appear green. The arrows in panel b indicates ß2ARs that localize with EGFP-rab11WT. (B) 12ß6 cells transiently expressing empty EGFP vector were treated with ISO for 6 hours. During the last 30 minutes of ISO treatment prior to fixation, cells were fed Alexa 594-transferrin for 15 minutes, washed, and the transferrin chased for 15 minutes in the continuous presence of ISO. ß2ARs are shown in green and Alexa 594-transferrin appears red. Areas of colocalization of receptors and transferrin appear yellow, as shown by the arrow. Scale bar, 10 µm.

View larger version (25K): [in a new window]   Fig. 6. Overexpression of EGFP-rab11WT inhibits ß2AR recycling from the RE. (A,B) 12ß6 cells transiently overexpressing empty vector (a,a') or EGFP-rab11WT (b,b') were treated with ISO (5 µM) for 6 hours, washed, and receptors were allowed to recycle in the presence of propranolol (5 µM) for 15 minutes (A) or 30 minutes (B) at 37°C. ß2ARs and EEA1 were labeled as described above and appear red and green, respectively. Arrows in panels b indicate areas of ß2AR and EGFP-rab11WT colocalization. Scale bars, 10 µm. (C) EcR293: ß2AR cells transfected with EGFP-rab11WT were treated with either ponasterone () or vehicle () for 72 hours to induce rab11 overexpression, then exposed to ISO for 6 hours. Cells were washed and incubated in DME-H at 37°C for varying times up to 90 minutes to allow receptor recycling and surface receptors were determined as described above. Each point represents the mean±s.e.m. of three independent experiments.

View larger version (29K): [in a new window]   Fig. 7. Overexpression of EGFP-rab11WT decreases ß2AR trafficking to lysosomes and inhibits receptor degradation. (A) 12ß6 cells transiently overexpressing empty vector (a,a') or EGFP-rab11WT (b,b') were treated with ISO for 6 hours in the presence of leupeptin. ß2ARs were identified using an anti-C terminal antibody and are visualized in red, whereas labeling with anti-LAMP-2 antibody is visualized in green. Areas of colocalization of ß2ARs and LAMP-2 appear yellow and are indicated by arrowheads in panel a. Arrows in panel b indicate areas of colocalization of ß2ARs and EGF-rab11WT. Scale bar, 10 µm. (B,C) EcR293: ß2AR cells transfected with EGFP-rab11WT were treated with either vehicle () or ponasterone () for 48 hours to induce rab11 overexpression, then surface receptors were biotinylated by exposure to EZ-link sulfo-NHS-biotin. Cells were washed and treated with ISO (5 µM) for the indicated times. Following incubation with agonist, the cells were lysed and receptors retrieved from the lysates for immunoblot analysis as described in Materials and Methods. Panel B, representative immunoblot of ß2ARs following indicated times of exposure to agonist, showing receptors migrating at 43 kDa. Panel C, quantification of ß2ARs remaining after varying times of exposure to agonist. Results are shown as the mean±s.d. of 3 separate experiments. * P<0.05 for the indicated time point.

View larger version (24K): [in a new window]   Fig. 8. Overexpression of EGFP-rab11WT or S25N mutant does not affect sorting of LDL. (A) Cells transiently expressing vector alone (a,a') or EGFP-rab11WT (b,b') were fed DiI-LDL (20 µg/ml) for 5 minutes, washed, and chased for 45 minutes in the presence of Cy5-transferrin (25 µg/ml). Panels a and b show the localization of transferrin (green) and DiI-LDL (red). (B) 12ß6 cells transiently expressing EGFP-rab11WT were fed DiI-LDL for 5 minutes, chased for 45 minutes, then immediately fixed and labeled for LAMP-2. Panel a shows the localization of DiI-LDL (red) and LAMP-2 (green), with areas of colocalization appearing yellow. Scale bar, 10 µm.