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J. Biol. Chem., Vol. 275, Issue 29, 21969-21974, July 21, 2000

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Essential Role of Dynamin in Internalization of M₂ Muscarinic Acetylcholine and Angiotensin AT_1A Receptors^*

Yvonne Werbonat^§, Nina Kleutges^§, Karl H. Jakobs, and Chris J. van Koppen^¶

From the Institut für Pharmakologie, Universitätsklinikum Essen, D-45122 Essen, Germany

Received for publication, February 28, 2000, and in revised form, April 26, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Most G protein-coupled receptors (GPCRs), including the M₁ muscarinic acetylcholine receptor (mAChR), internalize in clathrin-coated vesicles, a process that requires dynamin GTPase. The observation that some GPCRs like the M₂ mAChR and the angiotensin AT_1A receptor (AT_1AR) internalize irrespective of expression of dominant-negative K44A dynamin has led to the proposal that internalization of these GPCRs is dynamin-independent. Here, we report that, contrary to what is postulated, internalization of M₂ mAChR and AT_1AR in HEK-293 cells is dynamin-dependent. Expression of N272 dynamin, which lacks the GTP-binding domain, or K535M dynamin, which is not stimulatable by phosphatidylinositol 4,5-bisphosphate, strongly inhibits internalization of M₁ and M₂ mAChRs and AT_1ARs. Expression of kinase-defective K298M c-Src or Y231F,Y597F dynamin (which cannot be phosphorylated by c-Src) reduces M₁ mAChR internalization. Similarly, c-Src inhibitor PP1 as well as the generic tyrosine kinase inhibitor genistein strongly inhibit M₁ mAChR internalization. In contrast, M₂ mAChR internalization is not (or is only slightly) reduced by expression of these constructs or treatment with PP1 or genistein. Thus, dynamin GTPases are not only essential for M₁ mAChR but also for M₂ mAChR and AT_1AR internalization in HEK-293 cells. Our findings also indicate that dynamin GTPases are differentially regulated by c-Src-mediated tyrosine phosphorylation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

For most G protein-coupled receptors (GPCRs),¹ receptor internalization is thought to be initiated by phosphorylation of the receptor by G protein-coupled receptor kinases and binding of the cytosolic protein -arrestin to the phosphorylated receptor (1). -Arrestin then sterically inhibits further interaction of the receptor with heterotrimeric G proteins and binds with high affinity to clathrin heavy chains (1). Through this interaction, GPCRs are believed to be targeted to clathrin-coated pits. Following transformation of the clathrin-coated pit into a clathrin-coated vesicle, the clathrin-coated vesicle pinches off from the plasma membrane. This process is catalyzed by the 100-kDa GTPase dynamin, which probably activates (as yet largely unknown) effectors of the fission machinery (2). Three closely related mammalian dynamin isoforms have been identified: neuronal dynamin-1, ubiquitously expressed dynamin-2, and dynamin-3, which is expressed in testes, neurons, and lung (3). Comparison of the primary sequence shows that all three dynamin isoforms contain three highly conserved GTP-binding motifs (i.e. elements I, II, and III). A Lys⁴⁴  Ala substitution in the first of the three putative GTP-binding motifs yields a dominant-negative dynamin mutant, which displays strongly impaired GTPase activity and is predicted to have a greatly reduced GTP binding affinity (4). The two other GTP-binding motifs in dynamin are likely to be involved in GTP binding as well. Mutation of the third GTP-binding motif (substitution Lys²⁰⁶  Asp in element III) or removal of all three GTP-binding motifs (amino acids 1-271 in dynamin-1) drastically reduces clathrin-coated vesicle-mediated internalization (4-6). A second important regulator of dynamin function is phosphatidylinositol 4,5-bisphosphate (PIP₂) (6-9). All three dynamin isoforms contain a pleckstrin homology domain that is able to bind PIP₂. Binding of PIP₂ to dynamin not only strongly increases the GTPase activity of dynamin but may also serve to target dynamin to the plasma membrane, allowing subsequent dynamin self-assembly at the neck of the clathrin-coated vesicle (6-9). Expression of the dynamin mutant K535M, which is not stimulatable by PIP₂, effectively blocks transferrin receptor internalization in clathrin-coated vesicles (6).

A large number of recent studies indicate that most GPCRs, including M₁, M₃, and M₄ muscarinic acetylcholine receptors (mAChRs) in HEK-293 cells, internalize in clathrin-coated vesicles in a dynamin-dependent manner. This evidence is primarily based on the inhibitory effect of the dominant-negative inhibitor of dynamin-mediated internalization, K44A dynamin (10-13). In contrast, M₂ mAChRs internalize in a clathrin-independent manner and irrespective of expression of K44A dynamin in HEK-293 cells (10, 12). Likewise, internalization of angiotensin AT_1A receptors (AT_1ARs) (13), dopamine D₂ receptors (14), and secretin receptors (15) is also insensitive to expression of K44A dynamin. This has led to the proposal that internalization of these GPCRs is dynamin-independent. However, in light of the notion that the binding of GTP to dynamin probably involves binding to all three GTP- binding motifs in the GTP-binding pocket, we reasoned that a dynamin mutant lacking all three GTP-binding motifs might be a more appropriate dominant negative dynamin mutant to determine whether internalization of a particular GPCR is dynamin-dependent. Indeed, we here demonstrate that internalization of M₂ mAChR and AT_1AR is strongly inhibited by expression of N272 dynamin, which lacks the complete GTP-binding domain. Also, expression of K535M dynamin, which lacks PIP₂-stimulated GTPase activity, significantly blocks internalization of these GPCR species.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- N-[³H]Methylscopolamine (82 Ci/mmol), [¹²⁵I]Tyr⁴-Sar¹-Ile⁸-angiotensin II (2200 Ci/mmol), and [-³²P]ATP (3000 Ci/mmol) were purchased from NEN Life Science Products. PP1 and genistein were purchased from Biomol and Calbiochem, respectively. The cDNA constructs encoding wild-type c-Src and K298M c-Src were gifts from Dr. J. T. Parsons. Rat wild-type dynamin-1aa and rat K535M dynamin-1aa cDNA in pCMV96-7 (6) were generously provided by Dr. J. P. Albanesi. Rat Y231F,Y597F dynamin-1aa in pCMV96-7 (16) was a gift from Dr. R. J. Lefkowitz. The mouse AT_1AR cDNA in pBC12BI was a gift from Dr. L. Hein. The cDNAs encoding HA-tagged wild-type and K44A human dynamin-1aa (4) in pRK5 were gifts from Dr. S. Schmid. Rat dynamin-1aa N272 was generated by digestion of rat wild-type dynamin-1aa in pCMV96-7 with BglII and EcoRV. The product was filled in with Klenow DNA polymerase and religated with T4 DNA ligase. The authenticity of the N272 dynamin mutant was confirmed by dideoxy DNA sequencing and Western blot analysis. Rabbit anti-c-Src polyclonal antibody (N-16), mouse anti-Tyr(P) antibody (PY20), and goat anti-dynamin-1 antibody (C-16) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse anti-Myc 9E10 antibody, mouse anti-dynamin antibody (clone 41), and peroxidase-conjugated goat anti-mouse antibody were obtained from Calbiochem, Transduction Laboratories, and Dianova, respectively. Peroxidase-conjugated rabbit anti-goat antibody, peroxidase-conjugated goat anti-rabbit antibody, and fluorescein isothiocyanate-labeled anti-mouse antibody were from Sigma.

Cell Culture, Plasmid Construction, and DNA Transfection-- HEK-293 tsA201 and HEK-293 cells were grown in Dulbecco's modified Eagle's medium (DMEM)/F-12 medium supplemented with 10% fetal calf serum, penicillin G (100 units/ml), and streptomycin (100 µg/ml) in an atmosphere of 5% CO₂. Cells plated on 150-mm plates were transiently transfected with mAChR/pCD-PS or AT_1AR/pBC12BI DNA, together with one of the vectors listed above as described before (10). For epitope tagging of M₂ mAChRs, the Myc sequence EQKLISEEDL was inserted after the initiator methionine in the extracellular amino terminus of the receptor by the polymerase chain reaction method with Pfu DNA polymerase (Stratagene). The complete receptor DNA sequence was verified by dideoxy DNA sequencing and subcloned in pcDNA3 (Invitrogen). Stably transfected HEK-293 cells expressing Myc-tagged M₂ mAChR were selected after culturing in medium containing 500 µg/ml G418 (Life Technologies, Inc.).

Immunoblot Analysis of c-Src and Dynamin Expression-- Forty-eight hours after DNA transfection, cells on 35-mm plates were washed twice with phosphate-buffered saline (150 mM NaCl, 6.5 mM Na₂HPO₄, 2.7 mM KCl, pH 7.4) and lysed by the addition of 0.5 ml of boiling lysis buffer (1% SDS, 10 mM Tris-HCl, pH 7.4). Lysate was transferred to a microcentrifuge tube and boiled for 5 min. After five passages through a 25-gauge needle, samples were centrifuged for 5 min to remove insoluble material and diluted to an equal amount of protein as measured by the BCA method (Pierce) with lysis buffer. Fifty microliters of electrophoresis sample buffer (250 mM Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromphenol blue, 2% 2-mercaptoethanol) were added to 50 µl of the diluted samples and boiled for another 5 min. After SDS-polyacrylamide gel electrophoresis on 10% polyacrylamide gels, protein was blotted onto nitrocellulose. Nitrocellulose was then blocked with 150 mM NaCl, 10 mM Tris-HCl, pH 7.5, containing 5% bovine serum albumin (BSA; fraction V; Sigma) (dynamin) or 5% skin milk (c-Src). After washing three times for 5 min in 150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% Tween 20, the blot was incubated with either anti-dynamin antibody (clone 41, 0.125 µg/ml; or C-16, 0.4 µg/ml) or anti-c-Src antibody N-16 (0.1 µg/ml) in blocking buffer for 1 h. Following four washes for 5 min in wash buffer and incubation in blotting buffer for 10 min, the blot was incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (0.2 µg/ml), horseradish peroxidase-conjugated rabbit anti-goat antibody (diluted 1:1000), or horseradish peroxidase-conjugated goat anti-rabbit antibody (diluted 1:1000) at room temperature. After 1 h, the blot was washed again, and immunoreactivity was visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech).

Immunocytochemical Localization of Myc-tagged M₂ mAChR-- HEK-293 cells stably expressing Myc-tagged M₂ mAChR at a density of 350 fmol/mg of protein and grown on poly-L-lysine-coated 18 × 18-mm glass coverslips were incubated in 25 mM HEPES-buffered DMEM/F-12 medium (pH 7.4) in the presence and absence of 1 mM carbachol for 60 min. Then cells were washed twice with phosphate-buffered saline, fixed, and permeabilized in methanol for 5 min at 4 °C. Cells were washed three times with phosphate-buffered saline for 5 min each and incubated in TBS (10 mM Tris-HCl, pH 7.4, 150 mM NaCl) containing 0.5% fatty acid-free BSA for 45 min at room temperature. After incubation with mouse anti-Myc 9E10 antibody (5 µg/ml) in TBS plus 0.5% BSA for 60 min at room temperature, cells were washed three times with TBS plus 0.5% BSA for 5 min each and subsequently incubated with fluorescein isothiocyanate-labeled anti-mouse antibody (10 µg/ml) in TBS plus 0.5% BSA for 60 min at room temperature in the dark. After three washes with TBS plus 0.5% BSA for 5 min each and once in TBS for 5 min, coverslips were mounted using Moviol (Calbiochem). Immunofluorescence was detected using a Zeiss Axiophot fluorescence microscope equipped with standard fluorescein filter. Immunofluorescence was marginal in nontransfected cells and cells expressing wild-type M₂ mAChRs (without Myc tag), as well as in Myc-tagged M₂ mAChR-expressing cells when second antibody without the first antibody was used.

Dynamin Immunoprecipitation-- HEK-293 cells on 150-mm plates stably expressing M₁ mAChRs (17) were serum-starved in DMEM/F-12 medium. After 16 h, cells were preincubated in 25 mM HEPES-buffered DMEM/F-12 medium for 30 min and then stimulated for 5 min with 100 µM carbachol. After rapid suction of medium from the plates, 1.5 ml of ice-cold radioimmune precipitation assay buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 500 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate, 20 mM NaF, 1 mM NaVO₄, 1 mM dithiothreitol, 2.5 µg/ml leupeptin, 2.5 µg/ml aprotinin, 100 µM phenylmethylsulfonyl fluoride) was added to the cell monolayer. After 10 min at 4 °C, cells were lysed by repeated aspiration through a 21-gauge and 25-gauge needle. The cell lysate was centrifuged at 14,000 × g for 10 min at 4 °C. The supernatant was incubated with mouse anti-dynamin monoclonal antibody (3.75 µg) for 1 h at 4 °C followed by incubation with 50 µl of Protein G Plus/Protein A-agarose beads (Calbiochem) for 2 h at 4 °C while rotating. Then immunoprecipitates were collected by centrifugation at 14,000 × g at 4 °C. The pellets were washed five times with ice-cold radioimmune precipitation buffer and once with ice-cold buffer A (110 mM NaCl, 3 mM KCl, 7 mM Na₂HPO₄, 2 mM KH₂PO₄, and 20 mM NaF, pH 7.4). The pellets were resuspended in 30 µl of 2× Laemmli sample buffer. After boiling for 5 min, samples were centrifuged, and protein in the supernatant was analyzed on a 10% SDS-polyacrylamide gel and Western blotting with anti-Tyr(P) antibody (0.2 µg/ml) and, after stripping, with anti-dynamin antibody (clone 41; 0.125 µg/ml). Immunoreactivity was visualized with peroxidase-conjugated goat anti-mouse antibody.

c-Src Immunoprecipitation and c-Src Kinase Activity Assay-- After serum depletion for 16 h, HEK-293 cells on 150-mm plates stably expressing M₁ or M₂ mAChRs (17) were stimulated for 5 min at 37 °C with 100 µM carbachol in 25 mM HEPES-buffered DMEM/F-12 medium. Then the medium was rapidly removed from the plates, and the cells were lysed in 1.5 ml of lysis buffer (10 mM Tris-HCl, pH 7.4, 500 mM NaCl, 10 mM EDTA, 20 mM NaF, 1 mM NaVO₄, 1.0% Nonidet P-40, 1 mM dithiothreitol, 2.5 µg/ml leupeptin, 2.5 µg/ml aprotinin, and 100 µM phenylmethylsulfonyl fluoride). From this lysate, c-Src was immunoprecipitated with anti-Src antibody N-16 (1.5 µg) with 50 µl of Protein A Plus/Protein G-agarose beads. Immunoprecipitates were washed five times with lysis buffer and once in c-Src kinase buffer (100 mM Tris-HCl, pH 7.4, 125 mM MgCl₂, 25 mM MnCl₂, 2 mM EGTA, 250 µM NaVO₄, 2 mM dithiothreitol), followed by resuspension in 85 µl of Src kinase buffer with prepared c-Src substrate peptide and [-³²P]ATP (125 µM, 10-20 µCi/vial) according to the manufacturer's instructions (Upstate Biotechnology, Inc., Lake Placid, NY). The mixture was incubated at 30 °C for 12 min while shaking, and the reaction was stopped by the addition of 40 µl of 40% trichloroacetic acid. Thirty-microliter aliquots of the reaction mixture were spotted on P81 phosphocellulose paper in duplicate, washed five times for 5 min each with 0.75% phosphoric acid and once with acetone for 3 min, followed by radioactivity counting.

Receptor Internalization Assays-- Internalization of mAChRs was determined 48 h after DNA transfection by [³H]NMS binding assays to intact cells in 25 mM HEPES-buffered saline, pH 7.4 (HBS), containing 113 mM NaCl, 6 mM glucose, 3 mM KCl, 3 mM MgCl₂, 2 mM MgSO₄, and 1 mM NaH₂PO₄ at 4 °C as described in detail previously (10). Expression levels of M₁ and M₂ mAChRs varied between 100 and 750 fmol/mg of protein. Where indicated, transfected HEK-293 tsA201 cells were serum-starved for an additional 16 h in DMEM/F-12 medium. AT_1AR internalization was measured following incubation in 25 mM HEPES-buffered DMEM/F-12 medium buffer containing 1 mg/ml BSA with 1 µM unlabeled human angiotensin II (Sigma) for 60 min at 37 °C. Cells were then washed twice with ice-cold HBS; twice with ice-cold 20 mM 2-morpholinoethanesulfonic acid, 300 mM NaCl (pH 5.0); and twice with ice-cold HBS buffer (3 min each) to remove angiotensin II from receptor. Thereafter, cells were incubated in HBS buffer (with 1 mg/ml BSA) at 4 °C with 4-5 pM [¹²⁵I]Tyr⁴-Sar¹-Ile⁸-angiotensin II with or without 10 µM angiotensin II to determine nonspecific and total binding, respectively. After 4 h, cells were washed three times with HBS buffer and solubilized in 0.1% Triton X-100, and radioactivity was counted. Depletion of [¹²⁵I]Tyr⁴-Sar¹-Ile⁸-angiotensin II was limited to maximally 20% of total added radioligand by the inclusion of 4 nM unlabeled angiotensin II. Receptor internalization is expressed as (1  quotient of cell surface receptors of agonist-treated and untreated cells) × 100%.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Subcellular Redistribution of M₂ mAChR in HEK-293 Cells in Response to Carbachol-- Internalization of M₂ mAChRs in HEK-293 cells was monitored by indirect immunofluorescence of M₂ mAChRs tagged with a c-Myc epitope at the extracellular amino terminus. For this, we used stably M₂ mAChR-expressing cells instead of transiently expressing HEK-293 tsA201 cells. In the latter cell type, there was a significant preexisting intracellular pool of M₂ mAChRs that did not permit unequivocal demonstration of receptor translocation from the plasma membrane into the cytosol upon carbachol treatment. Control [³H]NMS binding experiments demonstrated that the Myc-tagged M₂ mAChRs sequestered with similar characteristics as the wild-type M₂ mAChRs in either HEK-293 tsA201 or HEK-293 cells (data not shown). As shown in Fig. 1A, M₂ mAChRs in untreated cells were found predominantly at the cell surface. During 60 min of incubation with 1 mM carbachol, M₂ mAChRs translocated into the cytoplasm (Fig. 1B). These results indicate that M₂ mAChRs like M₁ mAChRs (18) internalize into the cell interior of HEK-293 cells.

View larger version (52K): [in this window] [in a new window]   Fig. 1.   Visualization of M2 mAChR internalization in HEK-293 cells by immunofluorescence microscopy. HEK-293 cells stably expressing Myc-tagged M2 mAChRs were incubated in the absence (A) and presence (B) of 1 mM carbachol for 60 min at 37 °C, fixed and permeabilized in methanol, and processed for immunofluorescence for the Myc-tagged M2 mAChR as described under "Experimental Procedures." Representative images of six experiments are shown.

Effect of N272 Dynamin on M₁-M₄ mAChR Internalization in HEK-293 Cells-- To investigate the role of dynamin in M₂ mAChR internalization, N-terminal deletion dynamin-1 mutant N272 was expressed with either M₁ or M₂ mAChR in HEK-293 tsA201 cells. Fig. 2 shows the overexpression of the various transfected dynamin constructs used in this study. Expression of all dynamin forms (with the exception of N272 dynamin) was determined with a dynamin antibody recognizing the N-terminal part of dynamin-1 and dynamin-2. N272 dynamin-1, which migrates with an apparent molecular mass of ~72 kDa instead of ~100 kDa, lacks the greater part of this antibody-binding epitope. Expression of N272 dynamin was therefore detected by a dynamin-1 antibody that specifically recognizes a C-terminal domain of dynamin-1. Fig. 3 shows the effect of expression of N272 dynamin on M₁ and M₂ mAChR internalization in HEK-293 tsA201 cells. Expression of N272 dynamin inhibited internalization of M₁ and M₂ mAChRs in response to receptor stimulation with 100 µM or 10 µM carbachol for 60 min by 68 and 55%, respectively. Also, sequestration of M₃ and M₄ mAChRs in response to 100 or 10 µM carbachol for 60 min was reduced from 20 ± 5 to 1 ± 1% and from 35 ± 3 to 12 ± 4%, respectively, by co-expression of N272 dynamin in HEK-293 tsA201 cells (n = 3 experiments; data not shown). In contrast, as reported earlier by us (10) and others (12), expression of K44A dynamin inhibited M₁ but not M₂ mAChR internalization (Fig. 3).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Expression of endogenous dynamin and transiently expressed dynamin proteins in HEK-293 tsA201 cells. HEK-293 tsA201 cells were transiently transfected with empty pRK5 (pRK5), pCMV96-7/N272 dynamin (N272 Dyn), pCMV96-7/wild-type dynamin (WT Dyn), pRK5/K44A dynamin (K44A Dyn), pCMV96-7/Y231F,Y597F dynamin (Y231,597F Dyn), or pCMV96-7/K535M dynamin (K535M Dyn). Equal amounts of total cell lysates (50 µg of protein/lane) were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting. Expression of endogenous dynamin (lane 1), wild-type dynamin, and the dynamin mutants K44A, K535M, and Y231F,Y597F was determined using a mouse anti-dynamin monoclonal antibody recognizing the N terminus of dynamin. N272 dynamin expression was determined using a rabbit anti-dynamin polyclonal antibody directed against the C terminus of dynamin-1. Control immunoblot experiments with the rabbit anti-dynamin polyclonal antibody showed that expression of N272 dynamin was similar to expression of the other transfected dynamin constructs (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Effects of expression of wild-type dynamin, N272 dynamin, and K44A dynamin on M1 and M2 mAChR internalization in HEK-293 tsA201 cells. HEK-293 tsA201 cells transiently transfected with pCD-PS/M1 or M2 mAChR together with empty pRK5 (pRK5), pCMV96-7/wild-type dynamin (WT Dyn), pCMV96-7/N272 dynamin (N272 Dyn), or pRK5/K44A dynamin (K44A Dyn) were incubated with 100 µM (M1 mAChR) or 10 µM (M2 mAChR) carbachol for 60 min. Internalization of receptors was determined by specific [3H]NMS binding to intact cells. Cell surface expression of M1 and M2 mAChRs was not affected by coexpression of either dynamin construct. Data are the mean ± S.E. of four (M1 mAChR) and six (M2 mAChR) experiments. *, p < 0.05 compared with response of empty pRK5 vector-transfected cells (two-tailed t test).

Role of c-Src in Dynamin-mediated mAChR Internalization in HEK-293 Cells-- The results presented above strongly suggested that dynamin is not only required for internalization of M₁, M₃, and M₄ mAChRs but also essential for M₂ mAChR internalization in HEK-293 tsA201 cells. We therefore set out to analyze whether dynamin function in the M₁ and M₂ mAChR internalization pathways is differentially regulated. Recently, it was reported that internalization of ₂-adrenergic receptors in HEK-293 cells requires c-Src-mediated phosphorylation of dynamin on two tyrosine residues (i.e. Tyr²³¹ and Tyr⁵⁹⁷) (16). c-Src is activated by -arrestin, which is bound to the agonist-occupied ₂-adrenergic receptor and targets the receptor to the clathrin-coated pit (16). As M₁ mAChRs in HEK-293 tsA201 cells internalize into clathrin-coated vesicles in a -arrestin-dependent manner (11), we first investigated the role of c-Src in M₁ mAChR internalization in HEK-293 cells. As shown in Fig. 4A, activation of M₁ mAChRs in HEK-293 cells with 100 µM carbachol for 5 min increased c-Src kinase activity by 208 ± 16%. Basal and receptor-stimulated c-Src activity was effectively blocked by treatment with the selective c-Src inhibitor PP1 (1 µM). Stimulation of M₁ mAChRs led to an 82 ± 29% increase in tyrosine phosphorylation of endogenously expressed dynamin (Fig. 4B) (mean ± S.E. of four independent experiments), an increase that is comparable with the increases observed in other experimental systems (16, 19). As shown in Fig. 5A, transfection of HEK-293 tsA201 cells with wild-type c-Src or K298M c-Scr cDNA led to a strong overexpression of the corresponding c-Src protein over endogenous c-Src. While the expression of wild-type c-Src had no effect, the expression of catalytically defective K298M c-Src reduced M₁ mAChR internalization by 52% (Fig. 5B). Also, expression of Y231F,Y597F dynamin, which cannot be tyrosine-phosphorylated by c-Src (16), inhibited M₁ mAChR internalization by 76% (Fig. 5B). In contrast, expression of a catalytically defective mutant of another tyrosine kinase, Pyk2 (K457A), did not affect M₁ mAChR internalization (data not shown). Like M₁ mAChRs, M₂ mAChRs stimulate c-Src activity in HEK-293 cells, albeit to a smaller extent than M₁ mAChRs (i.e. 78 ± 34%) (Fig. 4A). However, expression of K298M c-Src did not inhibit M₂ mAChR internalization, while expression of Y231F,Y597F dynamin slightly reduced M₂ mAChR internalization. These results are supported by the observation that M₂ mAChR internalization in HEK-293 tsA201 cells was not inhibited by pretreatment of the cells with PP1 (10 µM), while the generic tyrosine kinase inhibitor genistein (100 µM) inhibited M₂ mAChR internalization by 16% (Table I). In contrast, internalization of M₁ mAChRs was reduced by 41 and 48% following treatment of the cells with 10 µM PP1 or 100 µM genistein, respectively (Table I).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   mAChR-induced activation of c-Src and tyrosine phosphorylation of dynamin in HEK-293 cells. A, HEK-293 cells stably expressing M1 or M2 mAChRs and serum-starved overnight were stimulated without (Con) and with 100 µM carbachol (Car) for 5 min. Src kinase was immunoprecipitated from cell lysates, and c-Src kinase activity was determined with [-32P]ATP and Src substrate peptide. PP1 (1 µM) was included in the reaction mixture where indicated 15 min before the addition of [-32P]ATP. The presence of dimethyl sulfoxide as vehicle (final concentration of 0.01%) did not affect c-Src activity. Data are the mean ± S.E. of four (M1 mAChR) or six (M2 mAChR) experiments. B, HEK-293 cells stably expressing M1 mAChRs were stimulated without (Con) and with 100 µM carbachol (Car) for 5 min after serum starvation overnight. Dynamin immunoprecipitates (IP) were subjected to SDS-polyacrylamide gel electrophoresis, and tyrosine phosphorylation of dynamin was detected by immunoblotting (IB) with anti-phosphotyrosyl antibody (upper panel). The filter shown in the upper panel was stripped and blotted with anti-dynamin antibody to document equivalent dynamin loading (lower panel). Similar images were obtained in three other experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Effects of expression of wild-type c-Src, K298M c-Src, wild-type dynamin and Y231F,Y597F dynamin on M1 and M2 mAChR internalization in HEK-293 tsA201 cells. A, detection of c-Src expression in total lysates of control transfected HEK-293 tsA201 cells (pRK5) and cells overexpressing wild-type c-Src (WT Src) or K298M c-Src (K298M Src). Lane 1 (pRK5) shows expression level of endogenous c-Src. B, HEK-293 tsA201 cells transiently expressing M1 or M2 mAChR together with control vector (pRK5), wild-type Src (WT Src), K298M c-Src (K298M Src), wild-type dynamin (WT Dyn), or Y231F,Y597F dynamin (Y231,597F Dyn) were incubated with 100 µM (M1 mAChR) or 10 µM (M2 mAChR) carbachol for 60 min. Internalization of receptors was determined by specific [3H]NMS binding to intact cells. Data are the mean ± S.E. of five (M1 mAChRs) and six (M2 mAChRs) experiments each. *, p < 0.05 compared with response of empty pRK5 vector-transfected cells (two-tailed t test).

Effect of K535M Dynamin on M₁-M₄ mAChR Internalization in HEK-293 Cells-- Since PIP₂ has recently been implicated as an important regulator of dynamin function (6-9), we tested the role of PIP₂ binding in dynamin-mediated mAChR internalization by coexpression of K535M dynamin. Western blot analysis of K535M dynamin expression is shown in Fig. 2. As depicted in Fig. 6, expression of K535 dynamin inhibited internalization of M₁ and M₂ mAChRs by 64 and 73%, respectively. Carbachol-induced internalization of M₃ and M₄ mAChRs was inhibited to a similar extent by expression of K535M dynamin (n = 3 experiments; data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Effects of expression of wild-type dynamin and K535M dynamin on M1 and M2 mAChR internalization in HEK-293 tsA201 cells. HEK-293 tsA201 cells transiently transfected with pCD-PS/M1 mAChR or pCD-PS/M2 mAChR together with empty pRK5 (pRK5), pCMV96-7/wild-type dynamin (WT Dyn), or pCMV96-7/K535M dynamin (K535M Dyn) were incubated with 100 µM (M1 mAChR) or 10 µM (M2 mAChR) carbachol for 60 min. Internalization of receptors was determined by specific [3H]NMS binding to intact cells. Cell surface expression of M1 and M2 mAChRs was not affected by coexpression of either dynamin construct. Data are the mean ± S.E. of nine (M1 mAChRs) and 13 (M2 mAChRs) sets of experiments. *, p < 0.05 compared with response of empty pRK5 vector-transfected cells (two-tailed t test).

Effect of N272 and K535M Dynamin on AT_1AR Internalization in HEK-293 Cells-- Since internalization of AT_1ARs in HEK-293 cells has been previously reported to be insensitive to overexpression of K44A dynamin (13), we also determined whether N272 dynamin or K535M dynamin blocks AT_1AR internalization in HEK-293 tsA201 cells. As shown in Fig. 7, expression of N272 dynamin and K535M dynamin inhibited AT_1AR internalization by 63 and 71%, respectively. In accordance with the aforementioned study on AT_1AR internalization (13), expression of K44A dynamin did not affect AT_1AR internalization (Fig. 7).

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Effects of expression of wild-type dynamin and the dynamin mutants K44A, N272, and K535M on AT1AR internalization in HEK-293 tsA201 cells. HEK-293 tsA201 cells transiently transfected with AT1AR/pBC12BI together with empty pRK5 (pRK5), pCMV96-7/wild-type dynamin (WT Dyn), pRK5/K44A dynamin (K44A Dyn), pCMV96-7/N272 dynamin (N272 Dyn), or pCMV96-7/K535M dynamin (K535M Dyn) were incubated with 1 µM angiotensin II for 60 min. Internalization of receptors was determined by specific [125I]Tyr4-Sar1-Ile8-angiotensin II binding to intact cells as described under "Experimental Procedures." Total and nonspecific binding of [125I]Tyr4-Sar1-Ile8-angiotensin II varied between 1700 and 4000 and between 50 and 180 cpm/well of a 24-well plate, respectively. Data are the mean ± S.E. of four experiments. *, p < 0.05 compared with response of empty pRK5 vector-transfected cells (two-tailed t test).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the past few years, the question whether dynamin plays an essential role in the internalization of a particular GPCR has been mostly analyzed by using K44A dynamin as dominant-negative mutant. While internalization of most GPCRs is blocked by expression of K44A dynamin, some GPCRs like the M₂ mAChRs, D₂ dopamine receptors, secretin receptors, and AT_1ARs internalize irrespective of K44A dynamin expression, suggesting that internalization of these GPCRs is dynamin-independent (10, 12-15). We now report that, contrary to what is currently postulated, internalization of M₂ mAChR and AT_1AR is dynamin-dependent. Coexpression of the dominant-negative dynamin mutants N272 and K535M strongly inhibited M₂ mAChR and AT_1AR internalization in HEK-293 tsA201 cells. These findings imply that N272 and K535M dynamin are more appropriate dominant-negative dynamin mutants than K44A dynamin. In this context, it will be interesting to determine whether fluid-phase endocytosis (5, 20) and internalization of ricin (21) are affected by expression of N272 or K535M dynamin also, because these trafficking processes are not blocked by K44A dynamin and are thus considered to be dynamin-independent. It is intriguing that N272 dynamin, which lacks all three GTP-binding motifs, inhibits internalization of both M₁ and M₂ mAChR, while K44A dynamin, which lacks only the first GTP-binding motif, blocks only M₁ mAChR internalization. It is possible that K44A dynamin selectively sequesters away an essential component of the M₁ but not of the M₂ mAChR internalization pathway. Another potential explanation relates to the fact that K44A dynamin is able to coassemble with wild-type dynamin (22). Since dynamin assembly and interaction of dynamin with other proteins requires the C terminus of the dynamin, which varies among the dynamin isoforms (23), different internalization pathways may use different dynamin isoforms. As a result, different internalization pathways may display differential sensitivity toward interference of K44A dynamin. Perhaps assembled GTP-bound K44A dynamin is sufficiently active to catalyze the budding of M₂ mAChR- and AT_1AR-containing vesicles from the plasma membrane but is not able to support internalization of M₁ mAChRs in clathrin-coated vesicles.

In the present study, we observed that mAChR and AT_1AR internalization in HEK-293 cells is strongly inhibited by expression of K535M dynamin, a dynamin mutant, which lacks the putative PIP₂ binding site (6). At present, it is unknown at which stage of the vesicle budding process PIP₂ binding to dynamin is essential. It has been postulated that, after recruitment of dynamin to the clathrin-coated pit, dynamin's interaction with the plasma membrane is strengthened by the binding of dynamin's pleckstrin homology domain with PIP₂ in the plasma membrane (8). In addition, PIP₂ binding might promote self-assembly of the dynamin molecules at the neck of the clathrin-coated pit and stimulate dynamin's GTPase activity (8, 9). An alternative possibility is that lysine 535 in the pleckstrin homology domain of dynamin serves to promote interaction of dynamin with proteins rather than with PIP₂ (6). Regardless of the mechanism, our study clearly underscores the relevance of dynamin's lysine 535 residue in GPCR internalization. Identification of the binding partners of dynamin's pleckstrin homology domain will be important for understanding the mechanisms of GPCR internalization.

In analogy with recent studies on the regulation of internalization of ₂-adrenergic receptors in HEK-293 cells (16, 24), we show that internalization of M₁ mAChRs is strongly reduced by inhibition of c-Src activity and by overexpression of Y231F,Y597F dynamin, which cannot be phosphorylated by c-Src. Since M₁ mAChRs internalize in clathrin-coated vesicles in a -arrestin-dependent manner, we propose that, in analogy to ₂-adrenergic receptors, internalization of M₁ mAChRs involves -arrestin-mediated targeting of receptor in the clathrin-coated pit and activation of c-Src by -arrestin. c-Src then phosphorylates dynamin, a process that is required for M₁ mAChR internalization in HEK-293 cells. Whether tyrosine phosphorylation activates dynamin or allows activation of dynamin by other molecules remains to be determined. In contrast, dynamin-mediated internalization of M₂ mAChRs was found not to be inhibited by expression of kinase-defective K298M c-Src or treatment of the cells with the specific c-Src inhibitor PP1. Thus, c-Src does not play a role in M₂ mAChR internalization. These findings are supported by recent studies showing that M₂ mAChR internalization in HEK-293 cells is -arrestin-independent (11, 12). However, treatment of the cells with the generic tyrosine kinase, genistein, or coexpression of Y231F,Y597F dynamin did slightly reduce M₂ mAChR internalization. This suggests that M₂ mAChR internalization is regulated to a very limited extent by phosphorylation of dynamin by tyrosine kinases other than c-Src. On the basis of the present and previous findings (11), we propose that M₂ mAChR internalization in HEK-293 cells is catalyzed by a dynamin isoform that differs from the dynamin isoform involved in clathrin-mediated M₁ mAChR internalization. Much remains to be learned about the internalization pathway of M₂ mAChR (and AT_1AR) in HEK-293 cells. We have observed that pretreatment of HEK-293 cells with 0.45 M sucrose fully blocks M₂ (and M₁) mAChR internalization in HEK-293 cells.² Yet expression of a dominant-negative clathrin mutant or -arrestin V53D, which inhibits clathrin-mediated internalization of GPCRs, blocks M₁ but not M₂ mAChR internalization (11). Similarly, expression of the amphiphysin SH3 domain, which blocks the targeting of dynamin to clathrin-coated pits, inhibits M₁ but not M₂ mAChR internalization in HEK-293 cells.² On the basis of these findings, we conclude that M₂ mAChR internalization in HEK-293 cells is clathrin-independent and that the inhibitory effect of sucrose on receptor internalization is not specific for clathrin-mediated internalization. In this respect, it is important to note that hypertonic sucrose treatment of HEK-293 cells does not only block vesicle formation at the plasma membrane but may also induce other cellular responses including MAP kinase activation (25), which may inhibit receptor internalization indirectly (26). Interestingly, similar findings have been obtained recently with the secretin receptor. Internalization of secretin receptors in HEK-293 cells is unaffected by expression of dynamin K44A and -arrestin V53D but is sensitive to sucrose pretreatment (15). Presently, it is unknown through which vesicles AT_1AR internalizes in HEK-293 cells. In adrenal glomerulosa and Chinese hamster ovary cells, AT_1AR may internalize through clathrin-coated vesicles, although this has only been inferred from biochemical (and not morphological) experiments using hypertonic sucrose treatment or potassium depletion (27). In vascular smooth muscle cells, however, AT_1ARs have been found to internalize through noncoated pits, possibly caveolae, as well as coated pits (27, 28). Thus, the internalization of AT_1AR and other GPCRs (10) appears to differ among different cell types. The identification of the budding vesicles through which AT_1AR and M₂ mAChR internalize in HEK-293 cells and other cell types will provide important information on the molecular mechanisms of GPCR internalization.

	ACKNOWLEDGEMENTS

We thank Riccarda Krudewig and Barbara Langer for expert technical assistance. We are indebted to Drs. J. P. Albanesi, L. Hein, R. J. Lefkowitz, J. T. Parsons, and S. Schmid for their gift of the various DNA plasmids and Dr. G. Rijksen for providing anti-Src antibody.

	FOOTNOTES

^* This work was supported by a grant from the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by an IFORES predoctoral fellowship from the Universitätsklinikum Essen.

^§ These authors contributed equally to this work.

^¶ To whom correspondence should be addressed: Institut für Pharmakologie, Universitätsklinikum Essen, Hufelandstr. 55, D-45122 Essen, Germany. Tel.: 49-201-723-3462; Fax: 49-201-723-5968; E-mail: van_koppen@uni-essen.de.

Published, JBC Papers in Press, May 2, 2000, DOI 10.1074/jbc.M001736200

² Y. Werbonat, N. Kleutges, K. H. Jakobs, and C. J. van Koppen, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; AT_1AR, angiotensin AT_1A receptor; BSA, bovine serum albumin; mAChR, muscarinic acetylcholine receptor; NMS, N-methylscopolamine; PIP₂, phosphatidylinositol 4,5-bisphosphate; HBS, HEPES-buffered saline; DMEM, Dulbecco's modified Eagle's medium.

	REFERENCES

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