Article |
Address correspondence to Tamas Balla, National Institutes of Health, Building 49, Rm 6A35, 49 Convent Drive, Bethesda, MD 20892-4510. Tel.: (301) 496-2136. Fax: (301) 480-8010. E-mail: tambal{at}box-t.nih.gov
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Abstract |
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Key Words: angiotensin II receptor recycling; endosomal sorting; G proteincoupled receptor; PI 3-kinase; Rab proteins
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Introduction |
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Agonist-induced endocytosis of most GPCRs occurs primarily via clathrin-coated vesicles (Ferguson, 2001). Previous studies suggested that internalization of the AT1R differs from that of ß-receptors in being dynamin- and ß-arrestinindependent (Zhang et al., 1996), but at physiological hormone concentrations, endocytosis of the AT1R occurs predominantly via a ß-arrestin and dynamin-dependent mechanism (Anborgh et al., 2000; Gáborik et al., 2001; Kohout et al., 2001; Qian et al., 2001). The functions of ß-arrestins, ß2-adaptin, and dynamin have all been found to involve binding to inositol lipids or inositol phosphates (Gaidarov et al., 1999), and phosphoinositides have been implicated at several steps in the endocytotic process (Achiriloaie et al., 1999; Bottomley et al., 1999; Gaidarov and Keen, 1999). Studies with muscarinic and ß-adrenergic receptors indicated that phosphorylation of lipids by phosphatidylinositol (PI) 4-kinase(s) is required for the internalization of GPCRs (Sorensen et al., 1998, 1999). However, recent studies have suggested that GPCR kinasemediated translocation of a PI 3-kinase to membranes may mediate this effect (Naga Prasad et al., 2001).
Although considerable progress has been made in defining the mechanisms involved in intracellular processing of internalized nutrient and growth factor receptors (Mellman, 1996), less information is available about the trafficking and fate of internalized GPCRs. Studies on numerous GPCRs have shown that most internalized receptors recycle to the cell surface and utilize a variety of trafficking pathways during this process. For example, the ß2-adrenergic receptor undergoes relatively rapid recycling to the surface via a route that requires a conformational change induced by acidification in the endosomes and subsequent dephosphorylation (Lefkowitz, 1998). Recent studies have demonstrated that GPCRs are also found in perinuclear recycling endosomes during their intracellular trafficking steps (Innamorati et al., 2001; Kreuzer et al., 2001; Schmidlin et al., 2001; Signoret et al., 2000).
Studies on the yeast mating factor receptor suggest that degradation of the internalized receptor via ubiquitination involves sorting into a lysosomal compartment that is also a part of the vacuolar sorting pathway from the Golgi apparatus (Katzmann et al., 2001; Simonsen et al., 2001). Both the sorting of molecules into this compartment and their exit therefrom depend on the function of Vps34p, the sole PI 3-kinase of yeast (Schu et al., 1993; Simonsen et al., 2001). The formation of PI 3-phosphate (PI[3]P) by Vps34p is required for membrane targeting of the early endosomal autoantigen 1 (EEA1), which promotes Rab5-mediated homotypic fusion of early endosomes (Simonsen et al., 2001). Moreover, further phosphorylation of PI(3)P by a 5-kinase, Fab1p, is necessary for the formation and processing of multivesicular bodies (MVBs) (Odorizzi et al., 1998). The functions of inositide kinases and phosphoinositides are also required for the endosomal processing of internalized growth factor and transferrin receptors (TfRs) (Corvera and Czech, 1998; Simonsen et al., 2001). However, little is known about the specific mechanisms by which phosphoinositides determine the endosomal processing of GPCRs in mammalian cells.
The aim of the present study was to characterize the intracellular compartments through which the internalized AT1R is processed, and to investigate the role of PI 3-kinases in the pathway(s) involved in AT1R trafficking. Our results indicate that PI 3-kinase inhibitors selectively impair a step in the processing of internalized AT1R by which the receptor rapidly recycles to the membrane from vesicles that resemble MVBs. We also demonstrate the presence of a slower process that occurs via recycling endosomes and is still operative in the presence of PI 3-kinase inhibitors. Based on these findings, we propose a model of the postendocytic trafficking of the AT1R that involves at least two major pathways of GPCR recycling.
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Results |
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Colocalization of fluorescent agonist with the AT1R during endocytosis
To follow the fate of the receptor and its ligand simultaneously in live cells, rhodamine-labeled fluorescent Ang II (RhodAng II) was used to stimulate cells expressing the AT1RGFP chimera. RhodAng II was rapidly bound to cell surface receptors (Fig. 2 A) and caused clustering of the receptors on the plasma membrane within a few minutes (Fig. 2 B). During the subsequent internalization of the hormonereceptor complex, both ligand and receptor were detectable in punctate intracellular structures (Fig. 2 C). Progressive internalization of the receptor, and its colocalization with the ligand, were evident for up to 30 min (Fig. 2 D). At this time, the receptor and its ligand began to appear in deeper compartments adjacent to the nucleus, in addition to the more peripheral vesicles (Fig. 2, D and E). At these later times, RhodAng II was also detectable in small punctate structures that did not contain the receptor (Fig. 2, D and E). Most cells showed extensive accumulation of the AT1RGFP chimera in colocalization with its fluorescent ligand in the juxtanuclear compartment after 1 h of incubation with RhodAng II (Fig. 2 E).
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When the GTP-bound mutant version of Rab5 (Rab5Q79L) was expressed in HEK 293 cells, large vesicles similar to those formed after WT treatment in control cells were observed (Fig. 7 A). However, a notable feature of the Rab5Q79L-induced large vesicles, as opposed to those of WT-treated cells, was the presence of large RhodAng II- and AT1R-containing clusters within their lumens (Fig. 7 A). Such clusters were not observed in cells incubated with RhodAng II after WT treatment and only the large vesicleassociated small red dots characteristic of WT-treated cells were present (Fig. 7 A). In contrast, expression of the GDP-bound mutant, Rab5S34N, prevented the formation of large vesicles after Ang II and WT treatment. The amount of internalized RhodAngII was greatly reduced in such cells after 30 min of incubation, although the initial internalization of AT1R was still observed (Fig. 7 B). Pretreatment of cells with the H+-ATPase inhibitor bafilomycin (100 nM) completely prevented the separation of AT1R and its ligand (Fig. 7 C, left) and abolished the formation of the large vesicles in WT-treated cells, leading to the accumulation of the RhodAng II and the AT1R in the juxtanuclear compartment (Fig. 7 C, right).
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Finally, the WT sensitivity of the recycling of 125I-Tf was examined by a similar approach to analyze one single cycle of prebound Tf, and in the conventional way by preloading cells with 125I-Tf for 30 min at 37°C and following the exit of internalized Tf in the presence or absence of WT. WT almost completely prevented the recycling of 125I-Tf in the one-cycle experiment (Fig. 9 A), but only partially inhibited the recycling of Tf that was loaded into the cells at 37°C during prolonged incubation (Fig. 9 B).
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Discussion |
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The processing of nutrient receptors from early endosomes has been well characterized and is known to involve the small GTP binding proteins Rab4, Rab5, and Rab11. Rab4 is believed to provide rapid recycling of receptors to the surface from Rab5-positive sorting endosomes, whereas Rab11 is believed to be involved in the recycling from the more slowly appearing juxtanuclear compartment often referred to as the recycling endosomes (Mellman, 1996). During maturation of the latter compartment, Rab4 and Rab11 proteins often appear together (Miaczynska and Zerial, 2002). The WT sensitivity of Tf recycling, found in experiments using tracer amounts of 125I-Tf and a "one-cycle" protocol, indicates that a large fraction of internalized TfRs recycle through a WT-sensitive mechanism. Only after longer incubations at 37°C does Tf reach a compartment from which Tf recycling occurs even in the presence of WT. These data are consistent with a model in which Rab4-dependent rapid cycling of TfR from sorting endosomes is more sensitive to PI 3-kinase inhibition than the route of Rab11-dependent recycling endosomes. The Tf receptors found in the latter compartment become loaded with Tf only after prolonged internalization or at high receptor occupancy (Trischler et al., 1999).
Our data on the colocalization of AT1R with Rab4- and Rab11-containing vesicles indicate that the AT1R also uses both of these mechanisms for receptor recycling. Processing of internalized AT1R from early endosomes showed several features that differ from those of the TfR. First, even in one-cycle experiments, only the most rapid fraction of the 125I-Ang II recycling was inhibited by WT and a significant fraction of the ligand was able to leave the cells. Similarly, only the rapid phase of AT1R recovery to the cell surface after termination of Ang II stimulation was sensitive to WT. These kinetic data were paralleled by morphological differences that became appreciable only after WT treatment. In the case of the AT1R, separation of a significant portion of the ligand from its receptor occurs by a WT-sensitive process that causes the appearance of both the receptor and its ligand in small vesicles within the larger WT-induced vesicles. Neither Tf nor its receptor is detected inside such enlarged vesicles, but they were occasionally found in the limiting membranes of the larger vesicles and in tiny vesicles associated with the latter. The vesicular structures observed after Ang II stimulation in the presence of WT were similar to the MVBs, in which inward membrane budding leads to the formation of small internal vesicles (Mellman, 1996). MVBs that are positive for Rab7 are known to process receptor tyrosine kinases, such as the EGF receptor, and appear to be the sorting organelles for proteins destined for lysosomal degradation (Felder et al., 1990; Mellman, 1996). However, kinase-deficient EGF receptors, unlike their wild-type counterpart, do not undergo lysosomal degradation and return to the plasma membrane (Felder et al., 1990). This suggests that receptors can recycle back to the cell surface from this organelle or from its precursor forms. A recent report also demonstrated the presence of the G proteincoupled m4 muscarinic receptor (but not of TfR) inside small vesicles within large endosomes formed in cells expressing Rab5Q79L, a structure considered by the authors to be the MVB (Volpicelli et al., 2001).
Several lines of evidence have indicated that protein sorting at the level of the MVB is dependent on PI 3-kinase products. In Saccharomyces cerevisiae, the formation of PI(3,5)P2 from PI(3)P by the Fab1p protein, a PI(3)P 5-kinase, is necessary for the inward invagination of membranes and formation of internal vesicles (Odorizzi et al., 1998). In mammalian cells, injection of an antimammalian Vps34p antibody inhibited internal vesicle formation in multivesicular endosomes (Futter et al., 2001). Other evidence indicates that PI 3-kinases are also involved during the formation of late endosomes and lysosomes (Fernandez-Borja et al., 1999). WT also induced morphological changes similar to those described in the present report during studies on the delivery of lysosomal enzymes mediated by the mannose 6-phosphate receptor (Brown et al., 1995). Our data on the heterogeneity of the large vesicles observed in WT-treated and Ang IIstimulated HEK cells are consistent with multiple defects in the processing of early endosomes as well as the MVB. These include the budding off of external vesicles (used by TfR and perhaps a fraction of the AT1R) as well as the formation of internal vesicles and the maturation toward late endosomes via MVBs. Inhibition of these processes by WT could contribute to the enlargement of internalized vesicles. Although the WT-sensitive component of AT1R recycling could represent the same Rab4-dependent pathway used by the TfR, it may also involve a process by which the receptor is retrieved from multivesicular compartments before maturation to late endosomes. It is likely that this route (even if not identical with the pathway of TfR recycling) also utilizes Rab4, because we clearly detected colocalization of RhodAng II and the HAAT1R with Rab4GFP. The G proteincoupled ß2 adrenergic receptor, which is known to use a rapid recycling route, has also been shown to recycle to the cell surface via a Rab4-dependent mechanism (Seachrist et al., 2000).
It is important to note that WT treatment had relatively minor effects on the slow component of AT1R recycling, and under these conditions both receptor and ligand were found in small juxtanuclear vesicles. This suggests that some small vesicles still can be formed, and that these probably represent the route through which receptor and ligand can recycle after WT treatment. Several observations suggest that this compartment is the Rab11-positive recycling endosome. The AT1R was found to colocalize with Alexa®Tf in the pericentriolar area of Ang IIstimulated cells even after WT treatment. Given the relative WT insensitivity of TfR recycling from the Rab11-positive recycling endosomal compartment, it is probable that AT1R recycling from the enlarged vesicles involves the same route. However, it is also possible that the slow recycling route observed in WT-treated cells is a distorted pathway that shares only some of the characteristics of the pathways occurring in normal cells. The preservation of the slow recovery phase of internalized receptors to the cell surface in WT-treated cells indicates that lysosomal degradation of the AT1R is not a major route in these cells, at least in the 2-h time frame of stimulation in the present experiments. We also could not detect the AT1R receptor (only the RhodAng II ligand) in Rab7-positive vesicles in WT-treated cells, consistent with the retrieval of the receptor before late endosomes.
The similarity of the large vesicular phenotype between WT treatment and Rab5Q79L expression, and the inability of added 3-phosphoinositides to reverse the large vesicular phenotype, has suggested that this effect of WT results from inhibition of Rab5 inactivation rather than PI 3-kinase activity (Chen and Wang, 2001a). However, although hydrolysis of Rab5-bound GTP may well be prevented by WT-sensitive mechanisms, our data suggest that the effect of WT is more complex than stabilization of Rab5GTP. The ability of Rab5S34N to prevent the formation of large vesicles after WT treatment indicates the need for continuous Rab5-assisted transfer of the receptorligand complex from internalized vesicles to early endosomes. However, not all of the enlarged vesicles contained wild-type Rab5GFP in WT-treated cells, suggesting that Rab5 must be released from at least some of the vesicles despite the presence of WT. Moreover, Rab5Q79L caused the accumulation of large clusters of RhodAng II and HAAT1R within the enlarged vesicles, a feature that was not seen in vesicles of WT-treated cells. Furthermore, although expression of Rab5Q79L was without effect on TfR recycling (Ceresa et al., 2001; Volpicelli et al., 2001), we observed that WT treatment inhibits early recycling of the AT1R. These data suggest that although the abnormal vesicles induced by WT or expression of Rab5Q79L share many similarities, they are not identical. Inhibition of H+-ATPase by bafilomycin prevented the separation of AT1R from its ligand, and also prevented the WT-induced enlargement of AT1R-containing vesicles. These findings are similar to those observed with major histocompatibility complex (MHC) class II molecules in melanoma cells (Fernandez-Borja et al., 1999), and indicate that acidification is a prerequisite for the maturation of endosomes as well as for the dissociation of the ligandreceptor complex.
Based on the present results and data available in the literature, a proposed model depicting the intracellular trafficking of AT1R and possibly other GPCRs is shown in Fig. 10. Agonist-induced internalization of the AT1R is rapidly followed by its appearance in early endosomes. Unlike the TfR, which leaves early endosomes and recycles via Rab4- and Rab11-positive recycling pathways, most of the AT1R initially follows a route leading to the formation of MVBs. Some of the AT1R and its ligand could bud off by a mechanism that is similar to that of the TfR. However, both can leave the vesicles by other means and may utilize mechanisms that are also involved in the formation of small internal vesicles. WT treatment causes multiple defects in early endosome maturation and processing. These include the inhibition of early recycling of TfR, probably via Rab4, with less impairment of the Rab11-positive slower recycling pathway. WT also inhibits the processing of AT1R toward the MVB, leading to the enlargement of this vesicular pool that contributes to the inhibition of recycling of receptor and ligand to the surface. The slow recycling of the receptor during WT treatment involves a distinct mechanism that proceeds via the Rab11 recycling pathway. Further studies are required to pinpoint the exact site(s) and enzyme(s) that are targeted by WT, and to validate the relevance of this model to the trafficking of other GPCRs.
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Materials and methods |
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Confocal analysis of receptor endocytosis
HEK 293 cells stably expressing the AT1RGFP chimera were plated onto polylysine-coated 25-mm-diameter glass coverslips at a density of 5 x 105 cells/dish and cultured for 3 d before analysis. HEK 293 cells stably expressing the wild type, HAAT1R were transfected with selected GFP constructs using the lipofectamine reagent for 24 h. Cells were washed twice with Dulbecco's PBS containing 0.1% BSA before analysis and treated with the indicated stimuli or inhibitors at 37°C. The coverslip was placed into a chamber mounted on a heated stage with the medium temperature kept at 33°C. Cells were incubated in 1 ml buffer and examined in an inverted microscope under a 40x oil immersion objective (Nikon) and a Bio-Rad Laboratories laser confocal microscope (MRC-1024). For double labeling experiments the 488- and 568-nm laser lines were used with 525 ± 20-nm and 580-nm-long pass filters for the detection of GFP and Alexa®, respectively.
Immunocytochemistry
HEK 293 cells grown on glass coverslips were transiently transfected with HAAT1R and the respective RabGFP constructs for 24 h. Cells were stimulated with Ang II for the times indicated and were washed twice with PBS before fixation with 4% paraformaldehyde in PBS (pH 7.4) for 20 min at room temperature. After treatment with sodium borohydride (1 mg/ml) for 15 min, cells were permeabilized with 0.1% Triton X-100 in PBS and incubated in blocking solution for 30 min (1% BSA in PBS), before incubation with anti-HA antibody (1:200 in blocking solution) for 1 h at room temperature. This was followed by two washes with TBST and incubation with rhodamine-conjugated antimouse antibody (1:200) for 1 h. The coverslips were mounted using Dako fluorescence mounting medium. Images were acquired with a ZEISS LSM 510 confocal laser scanning microscope in multi track mode, using the 488- and 543-nm laser lines with 500550-nm band pass and 560-nm long pass filters for the detection of GFP and rhodamine, respectively.
Recycling of the AT1R in HEK 293 cells
HEK 293 cells stably expressing AT1RGFP were cultured on 24-well plates and preincubated in medium 199 (Hank's salts) containing 20 mM Hepes (pH 7.4) and 1 g/liter BSA in the absence or presence of Ang II (1 µM) for 30 min at 37°C. Cells were then washed four times with 1 ml ice cold Dulbecco's PBS, and the incubation continued for the indicated times in medium 199 containing 20 mM Hepes (pH 7.4) and 1 g/liter BSA at 37°C. Inhibitors were added 10 min before the end of the preincubation and were readministered with medium 199 after washing the cells to remove Ang II. Incubations were terminated by placing the cells on ice and rapidly washing them twice with ice cold Dulbecco's PBS. The surface binding of the AT1R was then determined at equilibrium by incubating the cells in the presence of 125I-[Sar1,Ile8]Ang II (0.05 µCi/well) and unlabeled [Sar1,Ile8]Ang II (2 nM) at 4°C overnight. The cells were washed twice with 1 ml Dulbecco's PBS before solubilization with 0.5 M NaOH, 0.05% SDS for determination of cell-associated radioactivity.
Internalization kinetics of prelabeled AT1R or TfR
HEK 293 cells were cultured in four-well plates and were incubated in the presence of 125I-Ang II or 125I-Tf (0.05 µCi/well) in 0.5 ml medium 199 containing 20 mM Hepes (pH 7.4) and 1 g/liter BSA at 4°C for 4 h (Ang II) or 90 min (Tf) to permit binding to the cell surface receptors without internalization. The unbound tracer was removed by washing the cells twice with 1 ml ice cold Dulbecco's PBS. At this point, 0.5 ml warm (37°C) medium was added, and the cells were incubated for the indicated times at 37°C to allow receptor internalization and subsequent processing to proceed. In the case of TfR studies, 100 µM desferroxamine and 100 nM unlabeled Tf was added after 5 min to prevent possible reuptake of the recycled ligand. Incubations were terminated by placing the cells on ice, and the medium containing the radioligand released during the incubation was collected and replaced with 0.5 ml ice cold acid wash solution (150 mM NaCl and 50 mM acetic acid) to remove the surface-bound radioligand (Hunyady et al., 1994). After a 10-min incubation on ice, the extracellular (acid sensitive) tracer was collected. Cells were then lysed with 0.5 M NaOH and 0.05% SDS to determine the internalized (acid resistant) radioactivity. The total binding was calculated as the sum of the released (into the medium), extracellular (acid sensitive), and internalized (acid resistant) radioactivities. The internalized radioactivity was expressed as a percent of the total binding at each time point. Recycling of internalized TfR was also studied after loading cells with 125I-Tf for 30 min at 37°C and following the release of internalized 125I-Tf at 37°C after washing off the noninternalized tracer in the presence of desferroxamine.
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Footnotes |
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Acknowledgments |
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The work of L. Hunyady was supported by grants from the Hungarian Ministry of Education (FKFP-0318/1999, OMFB 02489/2000), the Hungarian Science Foundation (OTKA T-032179), and the Hungarian Ministry of Public Health (ETT 31/2000). The postdoctoral fellowship of R. Lodge was supported by the Canadian Medical Research Council/Canadian Institutes for Health Research.
Submitted: 6 November 2001
Revised: 3 April 2002
Accepted: 23 April 2002
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References |
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