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

Robert H. Moore1, Ellen E. Millman1, Estrella Alpizar-Foster2, Wenping Dai2 and Brian J. Knoll2,*

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

* Author for correspondence (e-mail: bknoll{at}uh.edu)

Accepted 19 February 2004


    Summary
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The pericentriolar recycling endosome (RE) may be an alternative compartment through which some ß2-adrenergic receptors 2ARs) recycle from early endosomes to the cell surface during prolonged exposure to agonist. For the transferrin receptor, CXCR2, and the M4-muscarinic acetylcholine receptor, trafficking through the RE and receptor recycling is regulated by the small GTPase rab11. The precise role of the RE and rab11 in regulating the cellular trafficking of the ß2AR is not understood. We therefore monitored trafficking of ß2ARs in HEK293 cells following the modulation of rab11 activity. Expression of a rab11 mutant deficient in GTP binding (as a fusion between enhanced green fluorescent protein (EGFP) and the rab11S25N mutant) significantly slowed receptor recycling to the cell surface from dispersed transferrin-positive peripheral vesicles following a brief exposure to agonist. The agonist was applied at a time when receptors have undergone only one or two rounds of endocytosis and recycling. In cells overexpressing wild-type rab11, ß2ARs localized to a rab11-positive compartment and the rate of ß2AR recycling to the cell surface was reduced, but only after prolonged exposure to agonist and multiple rounds of receptor endocytosis and recycling. This effect was associated with impaired ß2AR trafficking to lysosomes and receptor proteolysis, whereas the sorting of low-density lipoprotein from transferrin-positive vesicles to late endosomes and lysosomes was not affected. These data highlight a pivotal role for rab11 in regulating the traffic of a G protein-coupled receptor at the level of the RE, where modulation of rab11 activity dictates the balance between receptor recycling and downregulation during prolonged exposure to agonist.

Key words: ß2-adrenergic receptor, Rab11, Recycling endosome, Lysosome, Trafficking


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The intracellular trafficking of cell surface receptors plays a crucial role in the modulation of their activity. For ß2-adrenergic receptors (ß2ARs), members of the G protein-coupled receptor (GPCR) superfamily, their distribution during prolonged exposure to agonist is regulated by a complex array of intracellular trafficking steps. Initially, phosphorylated receptors undergo rapid internalization into early endosomes via clathrin-coated vesicles. In the early endosome, ß2ARs are likely to be exposed to protein phosphatases and dephosphorylated, following which most receptors recycle to the cell surface in a fully sensitized state (Krueger et al., 1997Go; Moore et al., 1995Go; Morrison et al., 1996Go; von Zastrow and Kobilka, 1992Go, 1994Go; Yu et al., 1993Go). With prolonged agonist exposure, receptors undergo continuous rounds of endocytosis and recycling (Morrison et al., 1996Go), but over time, some ß2ARs are diverted to lysosomes where receptors are degraded (Kallal et al., 1998Go; Moore et al., 1999bGo).

Some cell surface receptors, such as the G protein-coupled chemokine receptor CCR5, V2 vasopressin, endothelin A, and M4-muscarinic receptors, and the transferrin receptor, may utilize an indirect route in recycling from early endosomes to the cell surface (Bremnes et al., 2000Go; Innamorati et al., 2001Go; Ren et al., 1998Go; Ullrich et al., 1996Go; Volpicelli et al., 2002Go). This indirect route involves transit from early endosomes to the pericentriolar recycling endosome (RE), then to the plasma membrane. The effect of trafficking through the RE on receptor function is unknown, other than that it results in slowed receptor recycling (Innamorati et al., 2001Go; Ren et al., 1998Go; Ullrich et al., 1996Go). We have reported two observations raising the possibility that some ß2ARs also utilize this slow recycling pathway. First, during prolonged exposures to agonist, the rate of ß2AR recycling from intracellular compartments to the plasma membrane significantly decreases (Moore et al., 1999aGo). Additionally, in cells pretreated with bafilomycin A1, a proton pump inhibitor that raises endosome pH and inhibits traffic between early and late endosomes (Clague et al., 1994Go), the normal trafficking of ß2ARs to lysosomes is blocked and receptors are markedly localized with rab11, a marker for the RE (Moore et al., 1999bGo). We hypothesized, therefore, that the RE and slow recycling pathway is important in the regulation of ß2AR trafficking in a rab11-dependent manner during receptor downregulation.

Rab proteins are members of the small GTPase superfamily and as with all GTPases, they shuttle between two activity states determined by the phosphorylation status of a bound guanine nucleotide. Every rab protein has a characteristic subcellular distribution where it functions as an essential regulator of vectorial traffic (Novick and Zerial, 1997Go; Zerial and McBride, 2001Go). Rab11 localizes to and regulates traffic through the RE (Green et al., 1997Go; Ren et al., 1998Go; Ullrich et al., 1996Go) as well as between endosomes and the trans-Golgi network (TGN) (Chen et al., 1998Go; Wilcke et al., 2000Go). Studies of the transferrin receptor indicate that rab11 activity, and subsequently receptor traffic through the RE, can be modulated by overexpressing wild-type or mutant rab11 proteins (Ren et al., 1998Go; Ullrich et al., 1996Go; Wilcke et al., 2000Go). Overexpression of rab11S25N, which is deficient in GTP binding and functions as a dominant negative mutant, inhibits the delivery of transferrin to the RE and is believed to slow the recycling of receptors from early endosomes to the cell surface (Ren et al., 1998Go). Alternatively, overexpression of rab11Q70L, a mutation that confers constitutive activity for many rab proteins, causes the accumulation of transferrin in the RE (Ren et al., 1998Go; Ullrich et al., 1996Go). Among GPCRs, recycling of the M4-muscarinic receptor and CXCR2 are significantly slowed in cells expressing the rab11S25N mutant (Fan et al., 2003Go; Volpicelli et al., 2002Go).

In the present study, we modulated rab11 activity by overexpressing wild-type and mutant rab11 proteins in human embryonic kidney 293 (HEK293) cells expressing ß2ARs. The expression of rab11S25N significantly impaired ß2AR recycling from transferrin-positive but EEA1-negative vesicles to the cell surface after both brief and prolonged exposure to agonist. Conversely, the overexpression of both wild-type rab11 (rab11WT) and the Q70L mutant induced the redistribution of ß2ARs into rab11-positive compartments and inhibited the trafficking of ß2ARs to lysosome-associated membrane protein 2 (LAMP-2)-positive late endosomes and lysosomes. The morphologic changes following prolonged exposures to agonist in cells overexpressing rab11WT were associated with a reduced rate of receptor recycling and a marked inhibition of receptor degradation. These findings indicate a pivotal role for the RE and rab11 in regulating the endosome sorting of ß2ARs.


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell culture and reagents
12ß6 cells (a gift of B. Kobilka, Stanford, CA) were cultured in DME with 10% fetal bovine serum and 200 µg/ml Geneticin (Life Technologies, Gaithersburg, MD). This cell line was derived from HEK293 cells and expresses human ß2ARs with an N-terminal hemaglutinin (HA) epitope tag at a level of 300,000/cell (Moore et al., 1995Go; von Zastrow and Kobilka, 1992Go). Mouse monoclonal antibodies against the HA-tag (mHA.11) and LAMP-2 were obtained from Berkeley Antibody Co. (Berkeley, CA) and BD Pharmingen (San Diego, CA), respectively, and mouse monoclonal antibodies against rab11 and EEA1 were obtained from BD Transduction Laboratories (Franklin Lakes, NJ). Rabbit polyclonal antibodies against the terminal 15 amino acids of the ß2AR C-terminus and rab11 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Zymed Laboratories (South San Francisco, CA), respectively. Species-appropriate goat secondary antibodies and transferrin conjugated to Alexa 594 were purchased from Molecular Probes (Eugene, OR). Cy5-transferrin was purchased from Jackson ImmunoResearch Labs (West Grove, PA). FuGene 6 transfection reagent was obtained from Roche Molecular Biochemicals (Basel). The pEGFP (enhanced green fluorescent protein) expression vector was obtained from CLONTECH Laboratories (Palo Alto, CA) and rab11 cDNAs cloned into pEGFP were kindly provided by Dr Angela Wandinger-Ness (Albuquerque, NM). All other reagents were obtained from Sigma Chemical Co. (St Louis, MO) unless otherwise noted.

Immunofluorescence microscopy
12ß6 cells growing on poly-D-lysine coated cover slips in 6-well clusters were treated as indicated in the figure legends. Following treatment, the cells were washed with PBS containing 1.2% sucrose (PBSS), fixed with 4% paraformaldehyde in PBSS at 4°C for 10 minutes, then washed again with PBSS. The following steps were done at 25°C, with PBSS used for washes. The fixed cells were incubated in 0.34% L-lysine, 0.05% Na-m-periodate for 20 minutes, then washed and permeabilized with 0.2% Triton X-100. After a further wash, the cells were blocked with 10% normal goat serum for 15 minutes. Primary antibodies were diluted in PBSS with 0.2% normal goat serum and 0.05% Triton X-100, then added to the cells and left for 1-12 hours. The cells were washed four times before labeling with secondary antibodies using the same procedure as for primary antibodies. We used the following concentrations of antibodies: mHA.11, 5 µg/ml; polyclonal anti-ß2AR C-terminus 2 µg/ml; anti-rab11, 10 µg/ml; anti-LAMP-2, 5 µg/ml; anti-EEA1, 1:100 dilution; Alexa 488- and Alexa 647-anti-mouse IgG, 1:200 dilution; and Alexa 594 anti-rabbit IgG, 1:200 dilution. The cover slips were mounted in Mowiol and viewed using a DeltaVision deconvolution microscopy system (Applied Precision Inc., Issaquah, WA) equipped with a Zeiss Axiovert microscope. Imaging was performed using a Zeiss x100 oil immersion lens and sections were collected at an optical depth of 150 nm in the z-plane. The localization of vesicles was verified by the examination of multiple planes. Images were optimized using DeltaVision deconvolution software, pseudocolored using MetaMorph software (Universal Imaging Corporation, West Chester, PA), and transferred to Photoshop 6.0 (Adobe Systems, San Jose, CA) for the production of final figures.

To quantify ß2AR trafficking to late endosomes and lysosomes following prolonged exposures to agonist, the colocalization of receptors and LAMP-2 was determined using MetaMorph software. Quantification of overlap was performed following the construction of a binary image for each channel based on the merged image. The extent of colocalization was determined from four independent sections from each cell. The final values reflect the means±s.e.m. for 7-10 cells from 3 separate experiments.

Transient overexpression of EGFP-rab11 constructs in 12ß6 cells
12ß6 cells were plated onto poly-D-lysine-coated 22-mm-diameter glass coverslips at 50% confluence in 6-well clusters. After 24 hours and just prior to transfection, the media was removed and replaced with antibiotic-free media containing 3% fetal bovine serum. Cells were transfected with 2 µg per well of empty pEGFP vector as a control, pEGFP-rab11WT, or pEGFP-rab11S25N using FuGene 6 transfection reagent according to the manufacturer's instructions, with a DNA to FuGene 6 ratio of 2 µg/3 µl. The transfection mixture remained on cells for 48 hours, at which time the cells were treated as indicated in the figure legends. In control studies, FuGene 6 transfection reagent alone had no effect on cell morphology or the distribution of ß2ARs and organelle markers (data not shown).

To study the effects of rab11 overexpression on the distribution of ß2ARs following prolonged exposures to ISO (isoproterenol), leupeptin (100 µM) was added prior to ISO to allow the accumulation of receptors in lysosomes (Moore et al., 1999bGo). In some experiments cells were fed Alexa 594-transferrin (50-100 µg/ml) in serum-free media as described in the figure legends and receptors identified using mHA-11 antibody followed by anti-mouse Alexa 647 IgG. In specified experiments, cells were washed, fixed, and labeled for ß2ARs and either LAMP-2 or EEA1, and the secondary antibodies were anti-rabbit Alexa 594 IgG (1:200 dilution) and anti-mouse Alexa 647 IgG (1:100).

For morphologic studies of ß2AR recycling, cells were treated with ISO (5 µM) for 20 minutes or 6 hours, washed thoroughly, and placed in warm media containing 5 µM propranolol for varying times. In some experiments, cells were fed Alexa 594-transferrin as indicated in the figure legends to selectively label the endocytic pathway.

Inducible overexpression of rab11 proteins
EcR293 cells stably express the regulatory protein VgRXR, a chimeric steroid receptor that is activated by synthetic ecdysteroids such as ponasterone (No et al., 1996Go). Cells were initially transfected to stably express human ß2ARs with an N-terminus FLAG epitope using FuGene transfection reagent as previously described, with selection for G418 resistance. EGFP-rab11 constructs were cloned into the pIND-Hygro vector, which has a promoter element responsive to VgRXR, cloning sites for insertion of genes and a hygromycin resistance gene. pIND-Hygro/EGFP-rab11 and pIND-Hygro/EGFP rab11S25N were stably transfected into EcR293 cells using FuGene6, and hygromycin-resistant clones screened for ponasterone-inducible fluorescence. For induction of rab11 overexpression, cells were treated for 72 hours with 5 µM ponasterone, whereas control cells were treated with vehicle alone (0.125% ethanol).

Immunoblotting
Following treatment with ponasterone A (PON) or ethanol for 72 hours, transfected cells were washed with PBS, then dissolved in Laemmli sample buffer (Laemmli, 1970Go). Samples were electrophoresed through a 4-15% Tris-HCl SDS-polyacrylamide gradient gel (Ready Gel, BioRad, Hercules, CA) and transferred to Immobilon-P membranes (Millipore, Bedford, MA). The membranes were blocked in 5% dried milk in 0.2% Tween and probed with anti-rab11 mAb at a dilution of 1:2000, and bound antibodies were detected by chemiluminescence (ECL-Pierce Chemical Co., Rockford, IL). Immunoblots were quantified by densitometry using SigmaGel software (SPSS Science, Chicago, IL).

Measurement of ß2AR recycling kinetics in inducible cell lines
Cells growing on poly-D-lysine coated 24-well clusters were cultured in the presence of ponasterone or ethanol for 72 hours, then treated with ISO for 20 minutes or 6 hours. The cells were washed extensively and incubated in DME with 20 mM HEPES, pH 7.4 (DME-H) prewarmed to 37°C to allow recycling. At varying times up to 90 minutes, the cells were chilled and surface receptors measured by incubation in DME-H containing 6 nM [3H]CGP12177 at 0°C for 2 hours, followed by washing with cold DME-H. The monolayers were then lysed with 1% SDS and 1% NP-40, and the lysates counted by scintillation spectroscopy. Nonspecific binding was determined by parallel incubations with 5 µM propranolol and was less than 5% of total binding. For every experiment, binding at each time point was measured in triplicate. The fraction of internal receptors remaining was plotted versus time of recycling, and the curves fitted by nonlinear regression using GraphPad Prism software (version 3.0) and recycling rate constants determined as previously described (Morrison et al., 1996Go). Recycling rate constants are presented as the mean±s.e.m. of three separate experiments.

Rab11 proteins and LDL trafficking
Transient overexpression of rab11WT, rab11S25N, and empty vector was performed in 12ß6 cells as described above. After 48 hours, the media was removed and replaced with serum-free media containing DiI-LDL (20 µg/ml) for 5 minutes at 37°C. Cells were washed four times in chilled DME, then incubated for 45 minutes at 37°C in DME containing Cy5-transferrin (25 µg/ml). Following fixation in 4% paraformaldehyde and lysine/Na-m-periodate, cells were mounted using Mowiol and viewed and processed as described above. In other experiments, the cells were fed DiI-LDL for 5 minutes as above, washed, and then after 45 minutes fixed and labeled for LAMP-2, as previously described.

Measurement of ß2AR degradation
Transfected cells growing on poly-D-lysine coated 6-well clusters were cultured in the presence of either ethanol or ponasterone for 48 hours to induce EGFP-rab11 expression. The monolayers were then treated with EZ-link sulfo-NHS-biotin (0.5 mg/well) for 30 minutes at 25°C to biotinylate surface receptors. The biotinylated cells were washed with Dulbecco's phosphate buffered saline (DPBS) containing 0.9 mM CaCl2 and 0.5 mM MgCl2 to remove excess biotin and treated with ISO (5 µM) for the indicated times up to 24 hours. Following incubation with agonist, cells were washed and harvested in DPBS containing leupeptin (10 µg/ml), then pelleted and solubilized at 4°C in n-dodecyl-ß-D-maltoside (DDM) buffer (20 mM HEPES, pH 7.4, 300 mM NaCl, 5 mM EDTA, 0.8% DDM, and CompleteTM EDTA-free protease inhibitor at standard concentration). Lysates were centrifuged at 16,000 g to remove cellular debris. From each sample, 50 µg of protein was added to 50 µl streptavidin agarose beads (Sigma) at 4°C for 1 hour to bind biotinylated receptors, then the beads were pelleted by centrifugation and washed using DDM buffer followed by DPBS. The final pellets were resuspended in Laemmli buffer, heated to 65°C for 15 minutes, and centrifuged at 11,000 g to free biotinylated receptors from the streptavidin-agarose. A 10-µg aliquot of eluted protein from each sample was treated with 2 µl peptide N-glycosidase F (New England Biolabs, Beverly, MA) for 90 minutes at 37°C to remove N-terminal sugar moieties and improve the resolution of ß2ARs. The samples were electrophoresed and transferred to Immobilon-P membranes as described above. The membranes were probed with the anti-ß2AR C-terminus polyclonal antibody at a dilution of 1:1000 and detected using chemiluminescence. Bands were quantified by densitometry using SigmaGel software. Results are shown as the mean±s.d. of three separate experiments.


    Results
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Overexpressing rab11S25N inhibits ß2AR recycling from transferrin-positive vesicles
Previous data implicates roles for rab5 and rab4 in the endocytosis and rapid recycling of ß2ARs during brief exposures to agonist (Seachrist et al., 2000Go). We have reported that the rate of ß2AR recycling decreases with prolonged exposure to agonist (Moore et al., 1999aGo), suggesting that some receptors also utilize a slow recycling pathway. Furthermore, in cells pretreated with bafilomycin A1, ß2ARs accumulate in the RE where they localize with rab11 (Moore et al., 1999bGo). Thus, we predicted that ß2AR recycling should be altered in cells expressing wild-type (WT) or dominant-negative-mutant rab11 (S25N). We therefore tracked the recycling of ß2ARs in HEK293 cells stably expressing ß2ARs and transiently expressing EGFP-rab11 fusion proteins or empty pEGFP vector as a control following brief (20 minute) and prolonged (6 hour) exposures to agonist.

EGFP-rab11S25N localized to the cytosol and to a perinuclear compartment (Fig. 1A,b'), which we identified as trans-Golgi network by labeling with antibody against the TGN-specific adaptor protein AP1 (data not shown), consistent with the reported localization of rab11S25N mutant in BHK cells (Chen et al., 1998Go). EGFP-rab11WT was largely localized to perinuclear vesicles, consistent with REs (Fig. 1A,c').



View larger version (51K):
[in this window]
[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.

 

We determined the effects of rab11 on ß2AR recycling following a 20-minute exposure to agonist, a time point at which receptors have undergone only one or two rounds of endocytosis and recycling (Morrison et al., 1996Go). In control and rab11-transfected cells, there was a rapid redistribution of ß2ARs from the plasma membrane into punctate vesicles that were positive for EEA1 and transferrin (data not shown). Cells were then thoroughly washed and placed in warm media containing the antagonist propranolol to prevent any additional receptor internalization. In both control cells (Fig. 1A,a) and cells expressing EGFP-rab11WT (Fig. 1A,c), ß2ARs primarily localized to the plasma membrane within 15 minutes following the removal of agonist. The small fraction of receptors that failed to recycle were present in a perinuclear distribution in the case of control cells (Fig. 1A,a) or localized with EGFP-rab11WT (Fig. 1A,c). By 30 minutes after removal of agonist, recycling was virtually complete in control (Fig. 1B,a) and rab11WT-transfected cells (Fig. 1B,c). In cells overexpressing rab11S25N, some receptors remained localized to EEA1-positive endosomes after 15 minutes of recycling (Fig. 1A,b), although virtually all ß2ARs had exited these EEA1 endosomes after 30 minutes of recycling (Fig. 1B,b). However, many receptors failed to recycle even after 30 minutes and localized to dispersed vesicles devoid of EEA1. There was little or no colocalization of receptors with rab11S25N after 15 or 30 minutes of recycling, suggesting that ß2ARs do not transit through the TGN.

To characterize these EEA1-negative ß2AR-containing vesicles further, we treated cells transiently expressing EGFP-rab11S25N as above except during the recycling period Alexa 594-transferrin (50 µg/ml) was added to label the endocytic and recycling pathways. Nonrecycled ß2ARs were largely localized to transferrin-positive vesicles in cells expressing EGFP-rab11S25N after 15 minutes (data not shown), 30 minutes (Fig. 2A,b), and 60 minutes of recycling, (data not shown). To determine if these transferrin-positive compartments represent post-early endosome vesicles, we performed pulse-chase experiments in which Alexa 594-transferrin was added for 20 minutes during the exposure to ISO, washed, and allowed to chase for 15 minutes during ß2AR recycling. In both vector- and rab11S25N-transfected cells, transferrin was predominately localized to EEA1-positive early endosomes following the pulse and largely segregated from EEA1 after the chase (data not shown), but with strikingly different distributions. In control cells, transferrin and ß2ARs colocalized in a perinuclear compartment consistent with the RE (Fig. 2B,a), whereas in cells expressing rab11S25N they partially colocalized in vesicles dispersed throughout the cytoplasm (Fig. 2B,b). This finding suggests that rab11S25N inhibits ß2AR recycling from post-early endosome vesicles.



View larger version (33K):
[in this window]
[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.

 

To quantify the effects of modulating rab11 activity on ß2AR traffic, we created EcR293 (EcR293: ß2AR) cell lines stably expressing epitope-tagged ß2ARs in which wild-type or mutant rab11 could be inducibly overexpressed upon addition of the synthetic insect hormone ponasterone A. Using immunoblot analysis, we found that EGFP-rab11 proteins were significantly overexpressed following 72 hours of induction by ponasterone A, with the levels of expression of rab11WT and the rab11S25N mutant being approximately 4-fold and 2-fold, respectively, that of endogenous rab11 (Fig. 3). The overexpression of both rab11WT and rab11S25N to these levels had no effect on either the rate or extent of ß2AR internalization (Table 1 and data not shown).



View larger version (32K):
[in this window]
[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 this table:
[in this window]
[in a new window]
 
Table 1. ß2AR recycling-rate constants (min–1) in EcR293 cells

 



View larger version (13K):
[in this window]
[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 ({blacktriangleup}) or vehicle ({blacksquare}) 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., 1996Go). 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 (25K):
[in this window]
[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 ({blacktriangleup}) or vehicle ({blacksquare}) 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.

 
In transfected EcR293: ß2AR cells, we directly measured ß2AR recycling in cells overexpressing rab11 proteins and in vehicle control cells following brief (20-minute) exposures to agonist. As shown in Table 1, the first-order rate constants for receptor recycling (kr) in uninduced cells were similar to those for untransfected EcR293:ß2AR cells and to those previously measured in 12ß6 cells (Morrison et al., 1996Go), indicating that neither transfection with pIND-EGFP-rab11 proteins nor the slight leak in their expression significantly affected the rate of receptor recycling. In cells overexpressing the rab11S25N mutant, receptor recycling was significantly impaired following a brief exposure to agonist (Fig. 4A and Table 1). In contrast, the overexpression of rab11WT had no effect on recycling following a 20-minute exposure to agonist (Fig. 4B and Table 1), consistent with our morphologic data in transiently transfected cells.

Overexpressing wild-type rab11 slows ß2AR recycling through the recycling endosome
Although overexpressing wild-type rab11 did not overtly alter the trafficking of ß2ARs following a brief exposure to agonist, we considered the possibility that there may be an effect following prolonged exposures to agonist as we have previously shown that ß2AR recycling slows significantly during downregulation (Moore et al., 1999aGo). Cells transiently expressing EGFP-rab11WT or vector were exposed to agonist for 6 hours, then immediately fixed or washed to allow receptor recycling for 15 or 30 minutes, as described above. Under both conditions, some receptors were localized in EEA1-positive early endosomes following 6 hours of agonist treatment (Fig. 5A). In both control cells and cells expressing EGFP-rab11WT, receptors also were distributed to dense perinuclear compartments that contained either pulse-chased transferrin (Fig. 5B) or EGFP-rab11 (Fig. 5A,b), indicating that ß2ARs accumulate in REs. After removal of agonist, receptors largely recycled to the cell surface in control cells after 15 minutes (Fig. 6A,a) and 30 minutes (Fig. 6B,a). However, in cells expressing EGFP-rab11WT, ß2ARs had exited EEA1-positive early endosomes within 15 minutes (Fig. 6A,b) but some receptors remained in the RE even after 30 minutes (Fig. 6B, b), suggesting that rab11WT slows recycling from the RE but not from early endosomes. The effect of expressing EGFP-rab11S25N on ß2AR recycling following a prolonged exposure to agonist was similar to that observed after a 20-minute agonist treatment, with nonrecycled ß2ARs largely appearing in dispersed EEA1-negative but transferrin-positive vesicles (data not shown).



View larger version (32K):
[in this window]
[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.

 

We next quantified ß2AR recycling in transfected EcR293: ß2AR cells following a 6-hour exposure to agonist as described above. As expected, the expression of EGFP-rab11WT following ponasterone A treatment significantly decreased both the rate and extent of ß2AR recycling following a prolonged exposure to agonist (Fig. 6C and Table 1). Similar to its effect after a brief exposure to agonist, EGFP-rab11SN also significantly slowed receptor recycling following a 6-hour exposure to agonist (Table 1).

Overexpressing wild-type rab11 inhibits ß2AR trafficking to lysosomes and degradation
As the overexpression of rab11WT induced the accumulation of ß2ARs in the RE during prolonged exposures to agonist, we considered that overexpressing wild-type or mutant rab11 also may alter ß2AR trafficking to the degradative pathway. 12ß6 cells transiently overexpressing empty vector or rab11WT were treated with leupeptin (100 µM) to allow the accumulation of ß2ARs in lysosomes (Moore et al., 1999bGo) and exposed to ISO (5 µM) for 6 hours. In control cells, ß2ARs (Fig. 7A,a) had a punctate labeling pattern and extensively localized with the late endosome/lysosome marker LAMP-2, similar to our previous observations (Moore et al., 1999bGo). However, in cells overexpressing rab11WT (Fig. 7A,b) or rab11Q70L (data not shown), there was significantly less colocalization of ß2ARs and LAMP-2 (percent colocalization in control cells, 44.60±3.07 versus 11.51±1.65 for rab11WT cells, P<0.05). Instead, ß2AR extensively localized to rab11-positive structures and to peripheral endosomes.



View larger version (29K):
[in this window]
[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 ({blacksquare}) or ponasterone ({blacktriangleup}) 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.

 

To correlate this finding with a physiologic outcome, we directly quantified ß2AR degradation in the inducible EcR293:ß2AR:EGFP-rab11WT cell line by measuring the loss of surface-biotinylated receptors after varying exposures to agonist in cells pretreated with either ponasterone A or vehicle. ß2AR degradation in vehicle treated cells proceeded in a manner similar to untransfected cells (data not shown). However, in cells overexpressing rab11WT (Fig. 7B,C), receptor degradation was significantly inhibited following a 6-hour exposure to agonist, consistent with our morphologic data showing decreased receptor localization to lysosomes at this time point.

The finding that overexpressing rab11WT inhibited ß2AR trafficking to LAMP2-positive late endosomes and lysosomes suggests that rab11 regulates the trafficking of ß2ARs to lysosomes during prolonged exposure to agonists. We sought to determine if this effect resulted from a general disruption of trafficking directly from early endosomes to late endosomes. To address this question, we examined the effects of overexpressing rab11WT on the trafficking of an endocytic ligand, low-density lipoprotein (LDL). LDL internalizes with its receptor into early endosomes, where it localizes with transferrin, and then is sorted to late endosomes and lysosomes (Ghosh et al., 1994Go). If overexpressing rab11WT has a direct effect on early endosome to late endosome/lysosome traffic, we predicted that LDL also would be unable to transit to lysosomes but would remain in early endosomes and localize with transferrin. To test this prediction, we pulsed 12ß6 cells transiently overexpressing rab11 or vector with the fluorescent ligand DiI-LDL (20 µg/ml) for 5 minutes, washed, and allowed DiI-LDL to chase for 45 minutes in the presence of Cy5-transferrin (25 µg/ml) to label the endocytic pathway. As seen in Fig. 8, the overexpression of rab11WT had no effect on the distribution of DiI-LDL when compared to control cells expressing EGFP alone. In all cases, only a small amount of colocalization of DiI-LDL and Cy5-transferrin was observed (Fig. 8A), consistent with the normal transiting of LDL through early endosomes to the degradative pathway. To confirm this result, we performed similar pulse-chase experiments with DiI-LDL in control cells (data not shown) and in cells overexpressing rab11WT (Fig. 8B) and then labeled cells for LAMP-2. As expected and similar to control cells (data not shown), most DiI-LDL localized with LAMP-2 after a 45-minute chase, indicating that rab11WT does not inhibit the trafficking of LDL to late endosomes and lysosomes.



View larger version (24K):
[in this window]
[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.

 


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We and others have shown that during agonist treatment that ß2ARs undergo continuous rounds of endocytosis into and recycling from early endosomes, processes that are regulated by rab5 and rab4, respectively (Morrison et al., 1996Go; Seachrist et al., 2000Go; von Zastrow and Kobilka, 1994Go). Previous studies fail to demonstrate significant localization of ß2ARs with rab11 following 1 hour and 6 hours of agonist treatment, suggesting that these receptors do not transit through the RE (Innamorati et al., 2001Go; Moore et al., 1999bGo). In this study, we now present evidence that ß2ARs also recycle via the rab11-positive RE and that rab11 regulates multiple steps of agonist-induced ß2AR trafficking. First, in control cells, some ß2ARs localized to a perinuclear compartment that also contained pulse-chased transferrin following a prolonged exposure to agonist. Second, overexpressing rab11S25N slowed ß2AR recycling from transferrin-positive vesicles following both brief and prolonged exposures to agonist. Third, the overexpression of rab11WT inhibited ß2AR recycling from the RE following prolonged exposures to agonist. Fourth, the overexpression of rab11WT inhibited ß2AR trafficking to lysosomes and receptor degradation, whereas the sorting of DiI-LDL from transferrin-containing endosomes to late endosomes and lysosomes was not affected.

The effects of overexpressing rab11WT on ß2AR recycling following prolonged exposures to agonist are similar to those previously described for transferrin recycling (Ren et al., 1998Go; Ullrich et al., 1996Go). It is possible that overexpressing rab11WT slows receptor recycling by increasing ß2AR targeting to the RE, decreasing receptor efflux from this compartment, or both. However, studies of transferrin receptor trafficking in HeLa cells suggest that the main effect of rab11WT is in regulating the exit of receptors from the RE and not their delivery to that compartment (Wilcke et al., 2000Go). Our data showing that ß2ARs accumulated in the RE even after 30 minutes of recycling are consistent with this interpretation. Overexpressing rab11WT had no effect on either the rate or extent of ß2AR recycling following a brief exposure to agonist (Fig. 4B). At this time point most receptors are localized to early endosomes and receptors have undergone only one or two rounds of endocytosis and recycling. The trafficking of receptors from EEA1-positive early endosomes following prolonged exposure to agonist was also unimpaired (Fig. 6). These findings indicate that rab11WT does not regulate the direct, rapid recycling pathway but slows recycling through the RE. Further, these data suggest that the fraction of ß2ARs trafficking through the RE with each round of endocytosis and recycling must be relatively small compared with the rapid-recycling pathway.

Studies of transferrin receptor trafficking suggest that rab11S25N inhibits receptor recycling from early endosomes and prevents their delivery to the RE (Ren et al., 1998Go). In our studies, overexpressing rab11S25N induced a significant inhibition of ß2AR recycling following both short and prolonged exposures to agonist, with nonrecycled receptors largely being retained in dispersed transferrin-positive but primarily EEA1-negative vesicles 15 and 30 minutes after the removal of agonist (Figs 1, 2). This finding suggests that rab11S25N inhibits the recycling of ß2ARs from a post-early endosome recycling compartment and is consistent with recently published studies of M4-muscarininc receptor trafficking in PC12 cells (Volpicelli et al., 2002Go) and CXCR2 trafficking in HEK293 cells (Fan et al., 2003Go). However, the exact nature of these vesicles remains in question. Although it is possible that this compartment reflects dispersed recycling endosomes, in the case of M4 receptors these vesicles are believed to represent an intermediate compartment between early endosomes and REs (Volpicelli et al., 2002Go). M4-muscarinic receptors are known to largely accumulate in the RE, however this does not appear to be the case for ß2ARs (Innamorati et al., 2001Go). Instead, ß2ARs are believed to primarily recycle to the cell surface directly from early endosomes in a rab4-dependent manner (Seachrist et al., 2000Go). Thus, our results raise the possibility that these ß2AR-containing vesicles are located between early endosomes and the plasma membrane in the direct recycling pathway. Such a conclusion is consistent with published data suggesting that rab11S25N inhibits transferrin transit from sorting endosomes to both the RE and directly to the cell surface (Ren et al., 1998Go). Further, recent studies reveal the presence of recycling domains within endosomes that are occupied by both rab4 and rab11 (De Renzis et al., 2002Go; Sonnichsen et al., 2000Go), suggesting that these small GTPases may together coordinate proper sorting along the recycling pathway. A recently described 80 kDa protein, Rab Coupling Protein (RCP), serves as an effector for both rab4 and rab11 and is required for normal endosomal recycling (Lindsay et al., 2002Go). Thus, it is possible that rab11S25N impairs rapid recycling by binding RCP, or some other effector protein, thus preventing the functional interaction of effector with rab4.

We have shown that the overexpression of rab11WT inhibits the trafficking of ß2ARs to late endosomes and lysosomes and significantly delays receptor degradation (Fig. 7). However, our finding that LDL sorting from transferrin-containing endosomes to late endosomes/lysosomes was apparently not altered following the modulation of rab11 activity suggests that rab11 does not directly regulate early endosome to late endosome traffic (Fig. 8). This conclusion is consistent with published data showing that epidermal growth factor (EGF) receptors can travel to late endosomes and are normally degraded in HeLa cells overexpressing rab11Q70L (Wilcke et al., 2000Go). However, in contrast to EGF receptors, ß2ARs predominately recycle to the cell surface following endocytosis and only slowly travel to lysosomes (Moore et al., 1999aGo). Thus, it is likely that overexpression of rab11WT has an indirect effect on ß2AR trafficking to lysosomes by causing ß2ARs to become `trapped' in the slow recycling pathway, resulting in a diminished pool of receptors available for targeting to lysosomes. Supporting this explanation are our data showing significant colocalization of ß2ARs and rab11WT and slowed receptor recycling following prolonged exposure to agonist in cells overexpressing rab11WT. These data, along with our previous results in bafilomycin-A1-treated cells (Moore et al., 1999bGo), suggest that the slow recycling and degradative pathways are competitive and that receptor degradation can be decreased by inducing receptor accumulation in the RE.

Current data indicate that rab11 regulates the sorting of materials in the RE to at least two destinations, the plasma membrane and the TGN (Chen et al., 1998Go; Ren et al., 1998Go; Wilcke et al., 2000Go). Based on our results, we cannot exclude the possibility that there exists an additional rab11-regulated pathway from the RE to late endosomes or lysosomes. Recent data indicate that the delivery of cation-independent mannose-6-phosphate receptors to late endosomes is distal to their trafficking through the RE (Lin et al., 2004Go), although no such pathway has been described for any GPCR. It is conceivable that receptors, such as ß2ARs, that predominately recycle to the cell surface following endocytosis and only slowly move on to lysosomes, transit through the RE en route to the degradative pathway. Alternatively, rapidly degraded receptors, such as EGF receptors, are sorted directly from early endosomes to late endosomes, a process that is not dependent on rab11. Furthermore, a recent report suggests a role for rab4 in the regulation of both the recycling and degradative pathways (McCaffrey et al., 2001Go), indicating that a rab protein may direct multiple sorting events. Whatever the explanation, our results clearly show that although overexpressing rab11WT has no effect on the rapid recycling of ß2ARs, it inhibits their recycling from the RE and their trafficking to lysosomes and subsequent degradation.

In conclusion, these data indicate that ß2ARs are not only sorted in the early endosome to either the plasma membrane or to lysosomes, but also transit through the RE, a process that is regulated by rab11. It is unknown why some ß2ARs recycle via the RE and what targets receptors to this pathway. Studies of the V2 vasopressin receptor suggest that its accumulation in the RE is dependent upon the phosphorylation status of serine/threonine residues within a cluster in the cytoplasmic tail of the receptor (Innamorati et al., 1998Go; Innamorati et al., 1999Go; Innamorati et al., 2001Go). A similar serine cluster has been described for the ß2AR that is important in its rapid desensitization, but the role of these serines on receptor sorting is unknown (Seibold et al., 1998Go). Whatever the mechanism, our data clearly show that the trafficking of ß2ARs through the rab11-positive RE is associated with slowed rates of receptor recycling, diminished receptor delivery to lysosomes, and delayed receptor degradation. Although a recent report suggests that the RE is not essential for transferrin receptor recycling (Sheff et al., 2002Go), our findings raise the possibility that transit through the RE may serve dual roles in the modulation of ß2AR activity: contributing to receptor desensitization by slowing the recycling of dephosphorylated receptors to the cell surface as well as limiting receptor downregulation by protecting against receptor degradation.


    Acknowledgments
 
We wish to thank Angela Wandinger-Ness for providing the EGFP-rab11 constructs and Douglas Eikenburg, Keith Morrison, and Klaus-Peter Zimmer for their helpful comments. This work was supported by grants from the National Institutes of Health to R.H.M. (HL64934) and B.J.K. (HL57445 and HL50047).


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

Bremnes, T., Paasche, J. D., Mehlum, A., Sandberg, C., Bremnes, B. and Attramadal, H. (2000). Regulation and intracellular trafficking pathways of the endothelin receptors. J. Biol. Chem. 275, 17596-17604.[Abstract/Free Full Text]

Chen, W., Feng, Y., Chen, D. and Wandinger-Ness, A. (1998). Rab11 is required for trans-golgi network-to-plasma membrane transport and a preferential target for GDP dissociation inhibitor. Mol. Biol. Cell 9, 3241-3257.[Abstract/Free Full Text]

Clague, M. J., Urbe, S., Aniento, F. and Gruenberg, J. (1994). Vacuolar ATPase activity is required for endosomal carrier vesicle formation. J. Biol. Chem. 269, 21-24.[Abstract/Free Full Text]

De Renzis, S., Sonnichsen, B. and Zerial, M. (2002). Divalent Rab effectors regulate the sub-compartmental organization and sorting of early endosomes. Nat. Cell Biol. 4, 124-133.[CrossRef][Medline]

Fan, G. H., Lapierre, L. A., Goldenring, J. R. and Richmond, A. (2003). Differential regulation of CXCR2 trafficking by Rab GTPases. Blood 101, 2115-2124.[Abstract/Free Full Text]

Ghosh, R. N., Gelman, D. L. and Maxfield, F. R. (1994). Quantification of low density lipoprotein and transferrin endocytic sorting in HEp2 cells using confocal microscopy. J. Cell Sci. 107, 2177-2189.[Abstract/Free Full Text]

Green, E. G., Ramm, E., Riley, N. M., Spiro, D. J., Goldenring, J. R. and Wessling-Resnick, M. (1997). Rab11 is associated with transferrin-containing recycling compartments in K562 cells. Biochem. Biophys. Res. Commun. 239, 612-616.[CrossRef][Medline]

Innamorati, G., Sadeghi, H., Tran, N. T. and Birnbaumer, M. (1998). A serine cluster prevents recycling of the V2 vasopressin receptor. Proc. Natl. Acad. Sci. USA 95, 2222-2226.[Abstract/Free Full Text]

Innamorati, G., Sadeghi, H. and Birnbaumer, M. (1999). Phosphorylation and recycling kinetics of G protein-coupled receptors. J. Recept. Signal Transduct. Res. 19, 315-326.[Medline]

Innamorati, G., Le Gouill, C., Balamotis, M. and Birnbaumer, M. (2001). The long and the short cycle: alternative intracellular routes for G-protein coupled receptors trafficking. J. Biol. Chem. 276, 13096-13103.[Abstract/Free Full Text]

Kallal, L., Gagnon, A. W., Penn, R. B. and Benovic, J. L. (1998). Visualization of agonist-induced sequestration and down-regulation of a green fluorescent protein-tagged ß2-adrenergic receptor. J. Biol. Chem. 273, 322-328.[Abstract/Free Full Text]

Krueger, K. M., Daaka, Y., Pitcher, J. A. and Lefkowitz, R. J. (1997). The role of sequestration in G protein-coupled receptor resensitization. Regulation of ß2-adrenergic dephosphorylation by vesicular acidification. J. Biol. Chem. 272, 5-8.[Abstract/Free Full Text]

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]

Lin, S. X., Mallet, W. G., Huang, A. Y. and Maxfield, F. R. (2004). Endocytosed cation-independent mannose 6-phosphate receptor traffics via the endocytic recycling compartment en route to the trans-golgi network and a subpopulation of late endosomes. Mol. Biol. Cell 15, 721-733.[Abstract/Free Full Text]

Lindsay, A. J., Hendrick, A. G., Cantalupo, G., Senic-Matuglia, F., Goud, B., Bucci, C. and McCaffrey, M. W. (2002). Rab coupling protein (RCP), a novel Rab4 and Rab11 effector protein. J. Biol. Chem. 277, 12190-12199.[Abstract/Free Full Text]

McCaffrey, M. W., Bielli, A., Cantalupo, G., Mora, S., Roberti, V., Santillo, M., Drummond, F. and Bucci, C. (2001). Rab4 affects both recycling and degradative endosomal trafficking. FEBS Lett. 495, 21-30.[CrossRef][Medline]

Moore, R. H., Sadovnikoff, N., Hoffenberg, S., Liu, S., Woodford, P., Angelides, K., Trial, J., Carsrud, N. D. V., Dickey, B. F. and Knoll, B. J. (1995). Ligand-stimulated ß2-adrenergic receptor internalization via the constitutive endocytic pathway into rab5-containing endosomes. J. Cell Sci. 108, 2983-2991.[Abstract/Free Full Text]

Moore, R. H., Hall, H. S., Rosenfeld, J. L., Dai, W. and Knoll, B. J. (1999a). Specific changes in ß2-adrenoceptor trafficking kinetics and intracellular sorting during downregulation. Eur. J. Pharm. 369, 113-123.[CrossRef][Medline]

Moore, R. H., Tuffaha, A., Millman, E. E., Hall, H. S., Dai, W., Dickey, B. F. and Knoll, B. J. (1999b). Agonist-induced sorting of human ß2-adrenergic receptors to lysosomes during downregulation. J. Cell Sci. 112, 329-338.[Abstract/Free Full Text]

Morrison, K. J., Moore, R. H., Carsrud, N. D. V., Millman, E. E., Trial, J., Clark, R. B., Barber, R., Tuvim, M., Dickey, B. F. and Knoll, B. J. (1996). Repetitive endocytosis and recycling of the ß2-adrenergic receptor during agonist-induced steady-state redistribution. Mol. Pharmacol. 50, 692-699.[Abstract]

No, D., Yao, T. P. and Evans, R. M. (1996). Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc. Natl. Acad. Sci. USA 93, 3346-3351.[Abstract/Free Full Text]

Novick, P. and Zerial, M. (1997). The diversity of Rab proteins in vesicle transport. Curr. Opin. Cell Biol. 9, 496-504.[CrossRef][Medline]

Ren, M., Xu, G., Zeng, J., de Lemos-Chiarandini, C., Adesnik, M. and Sabatini, D. D. (1998). Hydrolysis of GTP on rab11 is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes. Proc. Natl. Acad. Sci. USA 95, 6187-6192.[Abstract/Free Full Text]

Seachrist, J. L., Anborgh, P. H. and Ferguson, S. S. (2000). Beta2-adrenergic receptor internalization, endosomal sorting, and plasma membrane recycling are regulated by rab GTPases. J. Biol. Chem. 275, 27221-27228.[Abstract/Free Full Text]

Seibold, A., January, B. G., Friedman, J., Hipkin, R. W. and Clark, R. B. (1998). Desensitization of ß2-adrenergic receptors with mutations of the proposed G protein coupled receptor kinase phosphorylation sites. J. Biol. Chem. 273, 7637-7642.[Abstract/Free Full Text]

Sheff, D., Pelletier, L., O'Connell, C. B., Warren, G. and Mellman, I. (2002). Transferrin receptor recycling in the absence of perinuclear recycling endosomes. J. Cell Biol. 156, 797-804.[Abstract/Free Full Text]

Sonnichsen, B., de Renzis, S., Nielsen, E., Rietdorf, J. and Zerial, M. (2000). Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11. J. Cell Biol. 149, 901-914.[Abstract/Free Full Text]

Ullrich, O., Reinsch, S., Urbe, S., Zerial, M. and Parton, R. G. (1996). Rab11 regulates recycling through the pericentriolar recycling endosome. J. Cell Biol. 135, 913-924.[Abstract]

Volpicelli, L. A., Lah, J. J., Fang, G., Goldenring, J. R. and Levey, A. I. (2002). Rab11a and myosin Vb regulate recycling of the M4 muscarinic acetylcholine receptor. J. Neurosci. 22, 9776-9784.[Abstract/Free Full Text]

von Zastrow, M. and Kobilka, B. K. (1992). Ligand-regulated internalization and recycling of human ß2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors. J. Biol. Chem. 267, 3530-3538.[Abstract/Free Full Text]

von Zastrow, M. and Kobilka, B. K. (1994). Antagonist-dependent and -independent steps in the mechanism of adrenergic receptor internalization. J. Biol. Chem. 269, 18448-18452.[Abstract/Free Full Text]

Wilcke, M., Johannes, L., Galli, T., Mayau, V., Goud, B. and Salamero, J. (2000). Rab11 regulates the compartmentalization of early endosomes required for efficient transport from early endosomes to the trans-golgi network. J. Cell Biol. 151, 1207-1220.[Abstract/Free Full Text]

Yu, S. S., Lefkowitz, R. J. and Hausdorff, W. P. (1993). ß-adrenergic receptor sequestration – a potential mechanism of receptor resensitization. J. Biol. Chem. 268, 337-341.[Abstract/Free Full Text]

Zerial, M. and McBride, H. (2001). Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2, 107-117.[CrossRef][Medline]