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
* Author for correspondence (e-mail: craig{at}pharm.sunysb.edu)
Accepted 18 September 2003
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Summary |
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Key words: G-protein-coupled receptors, Internalization, Counterregulation, Insulin, Agonist-induced, Trafficking
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Introduction |
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ß2ARs are phosphorylated by insulin treatment of cells (Karoor and Malbon, 1998). Studies in vivo have shown that insulin stimulates the phosphorylation of two major tyrosine residues, Y350 and Y364; both residues are located in the C-terminal cytoplasmic domain of the ß2AR (Karoor et al., 1995
). The phosphorylation of the Y350 residue in response to insulin creates an SH2-binding site to which Grb2, the p85 catalytic domain of phosphatidylinositol 3-kinase, and the GTPase dynamin can dock (Shih and Malbon, 1998
). Purified insulin receptor and recombinant ß2ARs have been used to show that insulin stimulates the insulin receptor-catalyzed phosphorylation of these same residues (Baltensperger et al., 1996
; Doronin et al., 2000
). Phosphorylation of the ß2AR impairs its ability to signal to its cognate G-protein Gs, a blockade that requires Grb2 with an intact SH2 domain (Shih and Malbon, 1998
).
Insulin catalyzes a robust internalization of ß2AR. Insulin thereby suppresses ß-adrenergic signaling, precluding access of ß-agonist to the ß2AR. In spite of similarities in the ability of ß-agonists and insulin to stimulate sequestration of the ß2AR, important differences may exist in the character of the pathways by which these sequestrations occur (Karoor et al., 1998). For example, ß2AR internalization in response to insulin, but not ß-adrenergic agonist, can be blocked with inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), such as wortmannin or LY294002 (Wang et al., 2000
), as well as by inhibitors of Src activity (Shumay et al., 2002
). In the current work, we extend the studies on GPCR trafficking and probe the role of cytoskeletal elements in the trafficking of ß2AR by insulin and by ß-agonists. The results provide compelling evidence that insulin and ß-adrenergic agonists employ unique cytoskeletal elements in trafficking receptor from the cell surface. Furthermore, the results reveal that the internalization and recycling aspects of receptor trafficking in response to insulin have unique cytoskeletal requirements: sequestration requires an intact actin cytoskeleton, whereas recycling to the cell surface requires intact microtubules.
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Materials and Methods |
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Cell culture
Human epidermoid carcinoma cells (A431) were maintained in Dulbecco modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), penicillin (60 µg/ml) and streptomycin (100 µg/ml), and grown in a humidified atmosphere of 5% CO2 and 95% air at 37°C. A431 clones stably expressing the GFP-tagged human ß2AR were cultivated with the addition of G418. Chinese hamster ovary (CHO) cells were obtained from the ATCC collection, propagated and stably transfected with pCDNA3 harboring the GFP-tagged human ß2AR (Liu et al., 1999a; Liu et al., 1999b
).
Immunoprecipitation and immunoblotting
For most studies, A431 cells were serum starved overnight with the following treatment as indicated. Cells were harvested and lysed in a lysis buffer (1% Triton X-100, 0.5% Nonidet-40, 10 mM dithiothreitol, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 100 µg/ml bacitracin, 100 µg/ml benzamidine, 2 mM sodium orthovanadate, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 40 mM sodium pyrophosphate, 50 mM KH2PO4, 10 mM sodium molybdate and 20 mM Tris-HCl, pH 7.4) at 4°C for 20 minutes. After centrifugation of the cell debris at 14,000 g for 30 minutes, clarified lysates were subjected to immunoprecipitation for 2 hours with antibodies specific for the ß2AR (CM04) linked covalently to agarose beads. Immune complexes were washed three times with RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 10 mM DTT, 1% Triton X-100, pH 8.0) and separated on 10% SDS-acrylamide Laemmli gels. Immunoblotting and detection of the ß-tubulin or actin were performed with anti-ß-tubulin and anti-actin antibody (both from Sigma), respectively, as previously described (Fan et al., 2001a; Fan et al., 2001b
).
Confocal microscopy
For the confocal microscopy studies, cells expressing ß2-AR and grown in eight-well Nunc chamber slides were serum starved and treated as indicated. Objects were imbedded in SlowFade (Molecular Probes) anti-fade reagent. Images were acquired on the Nikon Eclipse E600 microscope (oil-immersion, 60· objective) using He and Ne lasers. The digital images were exported as TIFF files and analyzed in Adobe Photoshop 5.5.
For immunofluorescent staining, cells were fixed with 3% paraformaldehyde and 0.25% glutaraldehyde in PBS pH 7.4 containing 2% of sucrose, permeabilized with 0.1% of Triton X-100 and incubated with blocking buffer (1% of normal goat serum in PBSTriton X-100) for 1 hour at room temperature. Fixed cells were incubated with primary antibody for 30 minutes (37°C); this was followed by extensive washing and a 30 minute incubation with the Alexa 655 conjugated secondary antibody (Molecular Probes, Eugene, OR) and final wash steps.
Hormone stimulation and drug treatment studies
A431 cells and stably transfected clones were routinely stimulated with either isoproterenol (10 µM) or insulin (100 nM) for 30 minutes and the trafficking of the GFP-tagged receptor monitored by fluorescence microscopy. Cells were serum-deprived for 18 hours before stimulation to remove growth factors and catechols from the cell media. For studies of the role of cytoskeleton elements in receptor trafficking, drugs were added either 30 minutes (for treatment with latrunculin or taxol) or 15 minutes (for treatment with nocodazole) in advance of the challenge with hormones. For studies with nocodazole only, cells were pre-cooled on ice before addition of the drug, to insure that the microtubules were fully depolymerized. The concentrations at which the drugs were used are as follows: nocodazole, 10 µM; taxol, 1 µM; and latrunculin, 1 µM.
ß-Adrenergic antagonist binding and receptor sequestration
The expression of ß2ARs in stably transfected A431 and CHO clones was quantified using the radiolabeled, high-affinity ß-adrenergic antagonist [125I]iodocyanopindolol (ICYP) to bind to intact cells. Identical radioligand binding assays were performed using the radiolabeled, water-soluble, membrane-impermeant [3H]CGP-12177 to determine the amount of cell-surface ß2AR in the untreated cells, as well as in the cells treated with either insulin or isoproterenol in the absence or presence of the cytoskeletal inhibitors/stabilizers (Karoor and Malbon, 1998). The data are presented as mean±s.e.m., where the amount of cell-surface receptor in the untreated cells is set as 100%.
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Results |
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It has been known for many years that microtubules have a role in the trafficking of GPCRs, including the ß2AR (Limas and Limas, 1983). We examined what effects disruption of the microtubules with nocodazole would have on the trafficking of ß2AR, observing that this agent, which in vivo binds tubulin and depolymerizes microtubules, blocks isoproterenol-induced internalization of ß2ARs (Fig. 1, +Noc.). Remarkably, nocodazole did not influence the response to insulin; insulin stimulated a robust internalization of ß2AR even in the presence of this microtubule inhibitor. Thus, we gained the first insight that the trafficking of the ß2AR by two potent regulators of internalization had some fundamental differences in mechanism. The role of the other major cytoskeletal system, the microfilaments formed from F-actin, was examined using latrunculin A, which binds the actin monomer and blocks F-actin dynamics. Treating A431 cells with latrunculin A had little influence on the sequestration of ß2ARs in response to isoproterenol, but effectively blocked the ability of insulin to internalize the ß2AR (Fig. 1, +Latr.).
The effects of nocodazole were examined by analyzing the trafficking of the GFP-tagged receptor and by confocal analysis of cells stained with anti-ß-tubulin (Fig. 2). Microtubules stained prominently in these epidermoid carcinoma cells (Fig. 2A), the patterns of microtubules being somewhat sensitive to treatment of the cells with either 100 nM insulin (+Ins) or with the ß-adrenergic agonist isoproterenol (10 µM, +Iso). In untreated cells, the majority of the ß2ARs were localized to the cell membrane. Merging the two images shows the redistribution of microtubules and marked internalization of ß2ARs that occurs when the cells are treated with either insulin (i.e. counterregulation) or isoproterenol (i.e. agonist-induced sequestration). The ß-agonist-induced induction of the ß2ARs, by contrast, was not observed in the nocodazole-treated cells (Fig. 2B). Treatment with nocodazole provoked a profound destabilization of the microtubular network in the untreated and hormone-treated cells alike (Fig. 2B). Microtubules were markedly shortened in the nocodazole-treated cells. Remarkably, in spite of the loss of much of the cytoskeletal architecture by depolymerization of microtubules, the internalization of ß2ARs in response to insulin stimulation was essentially the same as noted in the control cells (Fig. 2A). Treating the cells with latrunculin A did not alter the localization of the ß2ARs at the cell membrane in the absence of hormones (Fig. 2C). Agonist-induced sequestration of ß2ARs in response to isoproterenol proceeded normally in cells treated with latrunculin A (Fig. 2C). The ability of insulin to counterregulate ß2ARs and provoke internalization, by contrast, was essentially blocked in the latrunculin A-treated cells.
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Taxol stabilizes microtubules by binding to a pocket of ß-tubulin on the inner surface of a microtubule. This negates the effects of GTP hydrolysis that drives the depolymerization occurring on the other side of the monomer (He et al., 2001). Taxol treatment of A431 cells had a counterintuitive effect on the trafficking of ß2ARs. Treatment with taxol stabilized the microtubules, while still permitting the internalization of receptor in response to stimulation of either insulin or isoproterenol (Fig. 3A). Internalization of ß2ARs in response to either insulin or isoproterenol was attenuated modestly by taxol. Well-defined, cell-membrane localization of ß2ARs was evident, although perinuclear accumulation of receptor in response to either hormone was also noted. The distribution of receptors appears to be more homogeneously partitioned between the cell membrane and the perinuclear sites of accumulation, with less receptor found elsewhere in the cytoplasmic compartment. The effects of taxol treatment on the microtubules were profound, as noted by epifluorescence analysis of the ß-tubulin (Fig. 3B). In taxol-treated cells, relocalization of the microtubular network at the cell membrane was prominent. Stimulating taxol-treated cells with either isoproterenol or insulin reduced the accumulation of microtubules at the cell membrane and the formation of microtubule arrays elsewhere in the cell (Fig. 3B).
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Components of the multivalent, signaling complexes associated with the ß2AR include AKAP250 (gravin), AKAP79, protein kinase A, protein kinase C, Src and protein phosphatase 2B (Shih and Malbon, 1994; Cong et al., 2001
; Lin et al., 2000
; Fan et al., 2001a
; Fan et al., 2001b
; Oliveria et al., 2003
). ß2AR signaling complexes were isolated by immunoprecipitation to ascertain whether or not actin or ß-tubulin could be detected in the complex (Fig. 4). Analysis of the immune precipitations with anti-ß2AR antibodies revealed the presence of both actin and ß-tubulin. Treatment with insulin or isoproterenol increased the amount of both cytoskeletal elements associated with the signaling complexes. AKAP250 (gravin), a scaffold protein for the ß2AR, displays an F-actin binding site (Gelman, 2002
). Other elements of GPCR signaling complexes have been reported to interact with microtubules (Roychowdhury and Rasenik, 1994
; Wang et al., 1990
). Immunoprecipitations performed with unrelated antibodies and with unmodified matrix failed to pull down cytoskeletal elements, as determined by staining of immunoblots of the precipitates with either anti-ß-tubulin or anti-F-actin antibodies (data not shown).
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Compared with what is known about agonist- and insulin-induced internalization of ß2ARs, little is known about the recycling of receptor back to the cell membrane and what role, if any, the cytoskeleton plays in this process. To address the role of the F-actin cytoskeleton and the microtubular network in ß2AR recycling, cells were first stimulated with either insulin or isoproterenol for 30 minutes to induce full receptor sequestration. The cells were then either washed free of insulin to induce recovery from insulin treatment or incubated with 10 µM propranolol a high-affinity, ß-adrenergic antagonist to block isoproterenol binding to the receptors, and allowed to recover for up to 90 minutes (Fig. 5A). After 30 minutes with either insulin or isoproterenol, the bulk of the receptor was sequestered to perinuclear areas away from the cell membrane. The time-course for recovery reveals a progressive recycling of the ß2ARs to the cell membrane that is largely complete within 60-90 minutes. To test the role of microtubules in the recovery phase and in recycling of ß2ARs, cells were treated with either insulin or isoproterenol for 30 minutes and then co-treated with nocodazole for 30 minutes and finally washed free of insulin (insulin-induced) or treated with propranolol (isoproterenol-induced) in buffer containing nocodazole (Fig. 5B). The disruption of microtubules led to the attenuation of the recycling of receptors from isoproterenol- or insulin-treated cells. Although microtubule dynamics were not important to the ability of insulin to stimulate internalization of the ß2ARs, microtubules appeared essential to the recovery phase and recycling of the receptors back to the cell membrane. For ß2ARs internalized in response to isoproterenol, the recycling also was impaired by nocodazole. Thus, the internalization of ß2ARs in response to ß-adrenergic agonist, as well as the recycling of the internalized ß2ARs back to the cell membrane, was blocked by disruption of microtubules.
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To test the role of F-actin cytoskeleton in the recovery phase and recycling of ß2ARs, cells were stimulated with either insulin or isoproterenol for 30 minutes and then co-treated with latrunculin A for 30 minutes and finally washed free of insulin (insulin-induced) or treated with propranolol (isoproterenol-induced) in buffer containing latrunculin A (Fig. 5C). Disruption of F-actin dynamics markedly attenuated the recycling of receptors in cells stimulated with isoproterenol. The effects of latrunculin-A treatment on the recycling of receptors internalized in response to insulin stimulation were, by contrast, more modest. Normal F-actin dynamics were important to the ability of insulin to internalize the ß2ARs, but were nonessential to the recovery phase and recycling of the receptors back to the cell membrane (Fig. 5C). For ß2ARs internalized in response to isoproterenol, the recycling was impaired by latrunculin A, whereas the internalization process in response to ß-agonist was essentially insensitive to the disruption of F-actin cytoskeleton.
We noted the ability of isoproterenol to stimulate a dramatic change in the organization of the microtubule network (Fig. 2) and wondered whether the change was a result of the receptor activation per se or the most probable downstream response to the ß-adrenergic agonist, i.e. increased accumulation of intracellular cyclic AMP. Treating the cells with the plant diterpene forskolin led to robust accumulation of intracellular cyclic AMP by activating the adenylyl cyclase and bypassing the receptor/G-proteins upstream. Treatment with forskolin alone produced a pattern of microtubules somewhat similar to that observed in response to isoproterenol, suggesting that the regulation is probably dependent on both cyclic AMP and protein kinase A (Fig. 6). Nocodazole disrupted the microtubule network, but one could still observe arrays of fine microtubules stimulated by isoproterenol or by forskolin treatment. The effects of taxol on the stabilization of microtubules were profound (Fig. 3B). Treatment with either isoproterenol or forskolin (and presumably elevated cyclic AMP levels), by contrast, led to the appearance of radiant microtubules that were not present in the cells treated with taxol alone (Fig. 6).
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Discussion |
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Our current work provides several new insights into the manner in which ß-adrenergic agonists differ from insulin in the trafficking of ß2ARs. Most notably, this study reveals differing roles of the microtubule versus F-actin networks in enabling the internalization of ß2ARs for agonist-induced regulation versus counterregulation by insulin. Agonist-induced internalization of ß2ARs follows desensitization and is a hallmark for virtually all GPCRs (Morris and Malbon, 1999). Agonist-induced sequestration of the ß2AR to perinuclear locales occurs within minutes, but requires 15-30 minutes to reach maximal internalization. Nocodazole in vivo binds tubulin monomer and induces the depolymerization of microtubules. We found that nocodazole effectively blocks internalization of ß2ARs in response to ß-agonist, but it had no influence on the ability of insulin to counterregulate ß2ARs through sequestration. Thus, we speculate that an intact microtubular network enables agonist-induced sequestration of GPCRs.
The F-actin cytoskeletal network is essential for many cellular functions. Latrunculin A binds the actin-monomer and acts to sequester actin, blocking F-actin dynamics. In the current study, treatment with latrunculin A showed no influence on agonist-induced internalization of ß2ARs, but rather was found to block insulin-induced sequestration of the ß2ARs. These effects of disrupting actin cytoskeleton on insulin action are not a reflection of an effect proximal to receptor, as insulin receptor autophosphorylation, tyrosine phosphorylation of IRS-1,2 and Cbl, and serine/threonine phosphorylation of Akt in response to insulin are unaffected by latrunculin (Kanzaki and Pessin, 2001). Actin microfilaments do enable the translocation of the GLUT4 glucose transporter to the cell surface in response to insulin, a process that has many similarities to the counterregulation of ß2ARs in response to insulin that operates in the reverse orientation (Shumay et al., 2002
). Taken together, these results reinforce the notion that insulin-induced internalization and insulin-induced export of GLUT4 to the cell membrane may constitute use of the same cellular network.
One neglected feature of the agonist-induced trafficking of GPCRs is how the internalized receptors recycle to the cell membrane. We explored whether the recovery from desensitization and from counterregulation by insulin in cells made use of different cytoskeletal elements to recycle the ß2ARs. ß2ARs internalized by either ß-agonist or insulin displayed similar time-courses for the recycling of receptor back to the cell membrane. Although insulin-stimulated internalization of ß2ARs was insensitive to the disruption of the microtubules, the recovery of ß2ARs to the cell membrane was effectively blocked by nocodazole. Surprisingly, although the internalization of ß2ARs by agonist was blocked by nocodazole, the recycling of ß2ARs back to the cell membrane was only partially influenced by treatment with nocodazole. Treatment with latrunculin, by contrast, was effective at blocking the recycling of the ß2ARs internalized in response to ß-agonist. Thus, counterregulation by insulin and agonist-induced internalization both provoke a massive sequestration of these GPCRs from the cell surface to perinuclear locales in cells, but make use of very different cytoskeletal elements both to internalize and to recycle the receptors back to the cell surface.
Analysis of microtubules and actin microfilaments in cells challenged with ß-adrenergic agonist or insulin indicated that some level of rearrangement occurs in response to stimulated cells with these agents. For ß-agonist, it seems that the elevation of cyclic AMP may be responsible for the changes in microtubule architecture. For the insulin-stimulated cells, the response may be more difficult to define, because the downstream signaling for insulin, unlike ß-adrenergic agonist (Morris and Malbon, 1999), is populated with the mitogen-activated protein kinase cascades and many protein kinases and phosphatases whose activities are regulated by insulin (Pessin and Saltiel, 2000
; Czech and Corvera, 1999
; Saltiel and Kahn, 2001
). The ß2AR is a member of a multivalent signaling complex composed of the receptor in combination with AKAP250 (gravin), protein kinases A and C, Src and protein phosphatase 2B, and perhaps transiently with other signaling elements such as the heterotrimeric G-protein Gs. It has been shown that AKAP250 possesses an F-actin binding motif (Gelman, 2002
) and that microtubules can bind to Gs (Wang et al., 1990
). As tantalizing as these speculations may be, much work will be required to elucidate the molecular details and partners involved in the trafficking of GPCRs in response to agonist, as well as to receptor tyrosine kinases.
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Acknowledgments |
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References |
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