Receptor Subtype-specific Regulation of Muscarinic Acetylcholine Receptor Sequestration by Dynamin
DISTINCT SEQUESTRATION OF m2 RECEPTORS*

Oliver Vögler, Galina S. BogatkewitschDagger , Claudia Wriske, Patrick Krummenerl, Karl H. Jakobs, and Chris J. van Koppen§

From the Institut für Pharmakologie, Universität Gesamthochschule Essen, D-45122 Essen, Germany

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Sustained stimulation of muscarinic acetylcholine receptors (mAChRs) and other G protein-coupled receptors usually leads to a loss of receptor binding sites from the plasma membrane, referred to as receptor sequestration. Receptor sequestration can occur via endocytosis of clathrin-coated vesicles that bud from the plasma membrane into the cell but may also be accomplished by other, as yet ill-defined, mechanisms. Previous work has indicated that the monomeric GTPase dynamin controls the endocytosis of plasma membrane receptors via clathrin-coated vesicles. To investigate whether mAChRs sequester in a receptor subtype-specific manner via dynamin-dependent clathrin-coated vesicles, we tested the effect of overexpressing the dominant-negative dynamin mutant K44A on m1, m2, m3, and m4 mAChR sequestration in HEK-293 cells. The m1, m2, m3, and m4 mAChRs sequestered rapidly in HEK-293 cells following agonist exposure but displayed dissimilar sequestration pathways. Overexpression of dynamin K44A mutant fully blocked m1 and m3 mAChR sequestration, whereas m2 mAChR sequestration was not affected. Also, m4 mAChRs, which like m2 mAChRs preferentially couple to pertussis toxin-sensitive G proteins, sequestered in a completely dynamin-dependent manner. Following agonist removal, sequestered m1 mAChRs fully reappeared on the cell surface, whereas sequestered m2 mAChRs did not. The distinct sequestration of m2 mAChRs was also apparent in COS-7 and Chinese hamster ovary cells. We conclude that the m2 mAChR displays unique subtype-specific sequestration that distinguishes this receptor from the m1, m3, and m4 subtypes. These results are the first to demonstrate that receptor sequestration represents a new type of receptor subtype-specific regulation within the family of mAChRs.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Muscarinic acetylcholine receptors (mAChRs)1 belong to the superfamily of plasma membrane receptors that regulate a large number of signal transduction pathways via activation of heterotrimeric GTP-binding proteins (G proteins). The mAChR family consists of five subtypes of cloned mAChRs and can be subdivided into two functional groups: the m1, m3, and m5 subtypes, which preferentially couple to the Gq family of G proteins, and the m2 and m4 subtypes, which effectively activate the Gi family of G proteins. The m1, m2, m3, and m4 receptors are widely expressed in the central nervous system and peripheral tissues, whereas m5 receptors are present in only minute amounts in hippocampus and other areas of the brain (1). Prolonged exposure of mAChRs and other G protein-coupled receptors to agonists usually results in the attenuation of the cellular response. One molecular mechanism of attenuation involves the phosphorylation of the receptors by G protein-coupled receptor kinases (GRKs) and increased binding of the inhibitory protein beta -arrestin to the phosphorylated receptors, thereby inhibiting the coupling with G proteins (2). Another regulatory mechanism is the sequestration of receptors by which G protein-coupled receptors become inaccessible for hydrophilic membrane-impermeable ligands, including agonists. The sequestration of receptors may represent endocytosis of receptors via clathrin-coated vesicles (3, 4), caveolae (5, 6), or noncoated vesicles (7), or may be associated with conformational changes of the receptor in the plasma membrane that make the receptor inaccessible to agonists (8). For some G protein-coupled receptors like the beta 2-adrenergic receptors, sequestration is required to allow resensitization of desensitized receptors in a presumably subcellular compartment from which they can recycle back to the plasma membrane (9, 10). For other G protein-coupled receptors, including mAChRs and secretin receptors, receptor sequestration is a desensitization mechanism (11-13). For example, sequestration of m4 mAChRs in CHO cells delays receptor resensitization by more than 2 h, whereas nonsequestered receptors acquire full responsiveness within 10 min after agonist removal (12).

A number of monomeric GTPases has been implicated as key regulators of various stages of endocytosis, including the 100-kDa GTPase dynamin. Dynamin is an essential, early acting component of the endocytic pathway that operates via clathrin-coated vesicles. This GTPase controls the formation of clathrin-coated vesicles by regulating the constriction and budding of clathrin-coated pits from the plasma membrane (14, 15). Overexpression of a dominant-negative dynamin mutant (dynamin K44A), which displays reduced GTP binding affinity and hydrolysis, potently inhibits the sequestration of several plasma membrane receptors, including receptors for transferrin and epidermal growth factor. Recently, Zhang et al. (16) showed that functional dynamin is required for the internalization of beta 2-adrenergic receptors in HEK-293 cells via clathrin-coated vesicles. In contrast, angiotensin II AT type 1A receptors, which are structurally and functionally distinct from beta 2-adrenergic receptors, sequester in a dynamin-independent manner (16). Thus, G protein-coupled receptors are able to sequester via dynamin-dependent and dynamin-independent sequestration pathways.

Previous radioligand binding studies have suggested that mAChRs may sequester in a receptor subtype-specific manner. For example, whereas both m1 and m2 mAChRs rapidly sequester in Y1 adrenal cells following agonist exposure, only m1 mAChRs sequester upon addition of phorbol ester (17). Likewise, in JEG-3 cells, agonist-treatment induces m2 but not m1 mAChR sequestration (18). Similarly, immunocytochemical studies on the localization of alpha 2-adrenergic receptor subtypes in HEK-293 cells have shown that following agonist binding, alpha 2A-adrenergic receptors do not sequester, whereas alpha 2B-adrenergic receptors are able to undergo agonist-induced sequestration (19). Thus, within a subfamily of structurally related G protein-coupled receptors, receptors can also differ in their capacity to sequester. The goal of the present work was to analyze the functional role of the GTPase dynamin in mAChR sequestration, and to investigate whether sequestration of the m1, m2, m3, and m4 mAChR subtypes is differentially regulated by dynamin. Here we demonstrate that whereas m1, m3, and m4 mAChR sequestration is regulated by dynamin, m2 mAChRs sequester in a dynamin-independent manner. These results provide strong evidence that receptor sequestration represents a novel type of receptor subtype-specific regulation of mAChRs.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- [3H]Quinuclidinyl benzilate ([3H]QNB, specific activity 43 Ci/mmol) and N-[3H]methylscopolamine ([3H]NMS, specific activity 84 Ci/mmol) were purchased from New England Nuclear. Mouse anti-dynamin monoclonal antibody and peroxidase-conjugated goat anti-mouse antibody were obtained from Transduction Laboratories and Dianova, respectively.

Plasmid Construction-- All recombinant DNA procedures were carried out following standard protocols. DNA encoding mouse m1 mAChR (20), porcine m2 mAChR (21), human m3 mAChR (22), and mouse m4 mAChR (23) were subcloned into pCD-PS expression vector. The cDNAs encoding hemagglutinin-tagged dynamin wild-type and K44A (14) were subcloned into pRK5 expression vector.

Cell Culture and Transfection-- HEK-293 tsA201 cells stably expressing simian virus 40 large T antigen (24) and COS-7 cells (American Type Culture Collection) were grown in Dulbecco's modified Eagle's/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. CHO cells (American Type Culture Collection) were grown in alpha  medium containing 10% fetal calf serum, penicillin G (100 units/ml), and streptomycin (100 µg/ml). Cell media were from Life Technologies, Inc. Cells on 150-mm plates were transfected with either 12.5 or 25 µg pCD-PS containing m1 or m2 mAChR DNA, respectively, together with either (unless indicated otherwise) 50 µg of pRK5 dynamin wild-type, pRK5 dynamin K44A, or empty pRK5, using the calcium phosphate method (25). Transfection efficiency of HEK-293 tsA201 cells was determined by in situ staining for beta -galactosidase activity of the cells cotransfected with the constitutively active pSVbeta -gal (Promega).

Immunoblot Analysis of Dynamin Expression-- Cells on 150-mm plates were washed twice with phosphate-buffered saline (150 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 6.5 mM Na2HPO4, pH 7.4) and lysed by the addition of 1.0 ml of lysis buffer (1% SDS, 10 mM Tris-HCl, pH 7.4). Lysate was transferred to a microcentrifuge tube and boiled for 5 min. After 5 passages through a 25-gauge needle, samples were centrifuged for 5 min to remove insoluble material and diluted with lysis buffer to an equal amount of protein as measured by the BCA method (Pierce). 100 µl 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 100 µl of the diluted samples and boiled for another 5 min. After SDS-polyacrylamide gel electrophoresis on 7.5% polyacrylamide gels, protein was blotted onto nitrocellulose. Nitrocellulose was then blocked with 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20, and 5% bovine serum albumin (Fraction V, Sigma). After washing three times for 5 min in 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20, the blot was incubated with mouse anti-dynamin monoclonal antibody (diluted 1:1000; 0.25 µg/ml) in blocking buffer for 1 h. Following three washes for 5 min, the blot was incubated with peroxidase-conjugated goat anti-mouse antibody (diluted 1:5000; 0.16 µg/ml) at room temperature. After 1 h, the blot was washed again and immunoreactivity was visualized by enhanced chemiluminescence (Amersham). Densitometric analysis of the immunoblots was performed using a Shimadzu CS-9000 dual wavelength spot scanner.

mAChR Sequestration Assay-- Briefly, 24 h after transfection, cells from 150-mm plates were replated on poly-L-lysine-coated 24-well plates and allow to reattach and grow for another 24 h. The cells were then incubated with or without carbachol for 0-60 min in 25 mM HEPES-buffered Dulbecco's modified Eagles's/F-12 medium. For each manipulation, 6 wells of cells were taken. After washing with ice-cold phosphate-buffered saline, 4 wells were incubated with 2 nM [3H]NMS in 500 µl of ice-cold phosphate-buffered saline, and 2 wells received atropine also (final concentration of 3 µM) to measure nonspecific binding amounting to maximally 10% of total binding. After 4 h of incubation at 4 °C, cells were washed with ice-cold phosphate-buffered saline, solubilized in 1% Triton X-100, scraped, and transferred into scintillation vials, which received 3.5 ml of scintillation fluid prior to radioactivity counting. Sequestration is expressed as (1 - quotient of cell surface receptors of carbachol-treated and untreated cells) × 100%. Total receptor number was determined in crude cell homogenates by binding of the membrane-permeable muscarinic radioligand [3H]QNB at 37 °C as described previously (25). Untransfected HEK-293 tsA201, COS-7, and CHO cells do not express detectable levels of mAChR as determined by [3H]QNB binding to total cell homogenates.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Effect of Overexpression of Dynamin Wild-type and K44A Mutant on m1 mAChR Sequestration-- To determine whether mAChR sequestration is regulated in a receptor subtype-selective manner, we first examined the effect of overexpressing dynamin wild-type and K44A mutant on m1 and m2 mAChR sequestration in HEK-293 tsA201 cells. Transfection of HEK-293 tsA201 cells with 50 µg of DNA encoding the dynamin wild-type and K44A mutant resulted in 15-30-fold overexpression of the corresponding protein over the endogenous dynamin expression level as measured by laser densitometry and corrected for an average transfection efficiency of 10-20% (Fig. 1A). Stimulation of m1 mAChR-expressing HEK-293 tsA201 cells with a receptor-saturating concentration of 1 mM carbachol for a period of 60 min resulted in a 47 ± 5% loss in cell surface receptor number, as measured with the membrane-impermeable muscarinic antagonist [3H]NMS (Fig. 1B). There was no detectable change in total mAChR number as measured by binding of the membrane-permeable muscarinic antagonist [3H]QNB to homogenates prepared from transfected HEK-293 cells (data not shown). Overexpression of dynamin K44A inhibited m1 mAChR sequestration in a dose-dependent manner. Transfection of cells with 5 µg of pRK5 dynamin K44A per 150-mm tissue culture plate inhibited m1 mAChR sequestration by about 60%, whereas sequestration was blocked by about 80 and 90% following transfection with 50 and 150 µg of pRK5 dynamin K44A DNA, respectively (Fig. 1B). In subsequent experiments, cells were transfected with 50 µg of pRK5 dynamin wild-type or K44A mutant per 150-mm tissue culture plate. The inhibition of receptor sequestration was due to a decrease in the extent rather than the rate of mAChR sequestration (Fig. 2A). Sequestration in both control cells and cells overexpressing dynamin K44A was essentially complete within 10-30 min of exposure to 1 mM carbachol. Inhibition of m1 mAChR sequestration was apparent at low concentrations of carbachol as well (Fig. 2B). Whereas incubation with 10-5 M carbachol reduced cell surface number on control cells by 22 ± 3%, receptor sequestration in dynamin K44A-overexpressing cells was only 7 ± 3%. In contrast to dynamin K44A, overexpression of dynamin wild-type did not change the time course of m1 mAChR sequestration or the potency of carbachol to induce receptor sequestration, indicating that endogenous expression levels of dynamin are not rate-limiting (Fig. 2, A and B). Taken together, our data provide strong evidence that m1 mAChR sequestration in HEK-293 tsA201 cells is regulated by dynamin.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.   Overexpression of dynamin wild-type and K44A mutant in HEK-293 tsA201 cells. Influence of dynamin K44A expression on mAChR sequestration is shown. A, detection of dynamin in total lysates of HEK-293 tsA201 cells grown on 150-mm plates and transiently transfected with 50 µg of empty pRK5 DNA (pRK5), 50 µg of pRK5 dynamin wild-type (WT), or K44A mutant (K44A) by immunoblotting. Lane 1 (pRK5) shows expression level of endogenous dynamin. Equal amounts of cell lysates (50 µg of protein/lane) were resolved on polyacrylamide gels, transferred to nitrocellulose, and immunoblotted using anti-dynamin monoclonal antibody. B, HEK-293 tsA201 cells were transfected with m1 mAChR DNA in pCD-PS together with empty pRK5 vector (open bar), or 0.5-150 µg of pRK5 dynamin K44A DNA (solid bars) per 150-mm plate. Total µg DNA per plate was kept constant. Cells grown on 24-well plates were treated with or without 1 mM carbachol for 1 h at 37 °C, and the cell surface receptor number was determined by specific [3H]NMS binding. Specific [3H]NMS binding to untreated cells transfected with 150 µg of empty pRK5 vector and pRK5 dynamin K44A was 385 ± 14 and 392 ± 24 fmol/mg protein, respectively. The data represent the mean ± S.E. of three independent experiments.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   Time course and carbachol concentration dependence of m1 mAChR sequestration in HEK-293 tsA201 cells. The effect of overexpressing dynamin wild-type and dynamin K44A is shown. HEK-293 tsA201 cells transiently transfected with m1 mAChR in pCD-PS together with pRK5 dynamin wild-type (WT), K44A (K44A), or empty pRK5 (pRK5) were incubated in the absence and presence of 1 mM carbachol for 10, 30, or 60 min (A), or with 10-7, 10-5, or 10-3 M carbachol for 60 min (B) at 37 °C. Sequestration was assessed by [3H]NMS binding to intact cells at 4 °C. Data are the mean ± S.E. of three sets of experiments each. Specific [3H]NMS binding to untreated cells transfected with pRK5, pRK5 dynamin wild-type, and pRK5 dynamin K44A was 220 ± 61, 183 ± 24, and 264 ± 73 fmol/mg protein, respectively.

Sequestration of m2 mAChRs in HEK-293 tsA201 Cells-- We next determined whether m2 mAChR sequestration in HEK-293 cells is regulated by dynamin as well. As shown in Fig. 3A, m2 mAChRs rapidly sequestered, and cell surface receptor number was reduced by 60-80% within 10-30 min of incubation with 1 mM carbachol at 37 °C. The loss of cell surface receptor number took place without a measurable change in total receptor number as determined by [3H]QNB binding to cell homogenates (data not shown). Overexpression of dynamin wild-type or dynamin K44A did not alter m2 mAChR sequestration in HEK-293 tsA201 cells. In all three cell systems, m2 mAChRs sequestered with a similar time course, and the potency of carbachol in inducing receptor sequestration was indistinguishable (Fig. 3B). These results strongly suggest that in HEK-293 tsA201 cells m2 mAChRs sequester by a dynamin-independent pathway.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3.   Sequestration of m2 mAChRs in HEK-293 tsA201 cells. HEK-293 tsA201 cells transiently transfected with 25 µg of m2 mAChR in pCD-PS together with 50 µg of pRK5 dynamin wild-type (WT), dynamin K44A (K44A), or empty pRK5 (pRK5) per 150-mm tissue culture plate, were incubated in the absence or presence of 1 mM carbachol for 10, 30, or 60 min (A), or with 10-7, 10-5, or 10-3 M carbachol for 60 min (B) at 37 °C. Sequestration was assessed by [3H]NMS binding to intact cells. Data are the mean ± S.E. from 6 sets of experiments each. Specific [3H]NMS binding to untreated cells transfected with pRK5, pRK5 dynamin wild-type, and pRK5 dynamin K44A was 43 ± 27, 54 ± 15, and 36 ± 6 fmol/mg protein, respectively. Transfection with 150 µg instead of 50 µg of pRK5 dynamin K44A did not inhibit m2 mAChR sequestration in HEK-293 tsA201 cells either.

Reappearance of Sequestered m1 and m2 mAChRs in HEK-293 tsA201 Cells-- The dissimilar sequestration characteristics of m1 and m2 mAChRs in HEK-293 tsA201 cells prompted us to investigate whether m1 and m2 mAChRs also display different reappearance kinetics following agonist-induced receptor sequestration. For this, m1 and m2 mAChR-expressing cells were incubated with 1 mM carbachol for 60 min at 37 °C, and then washed to remove ligand. As shown in Fig. 4, cell surface mAChR numbers were reduced by 48 ± 5% and 62 ± 5% in carbachol-treated m1 and m2 mAChR-expressing cells. Within 1-3 h, almost all sequestered [3H]NMS binding sites on m1 mAChR-expressing HEK-293 cells reappeared. In contrast, reappearance of sequestered [3H]NMS binding sites on m2 mAChR-expressing cells was only very small. 3 h after carbachol removal, only 10-20% of initial binding had reappeared, and in the next 3 h, there was no further increase. These data are consistent with the observation that m1 and m2 mAChRs utilize distinct pathways of sequestration in HEK-293 cells.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Reappearance of sequestered m1 and m2 mAChRs in HEK-293 tsA201 cells. HEK-293 tsA201 cells transiently expressing m1 or m2 mAChRs were incubated with or without 1 mM carbachol for 60 min at 37 °C. Cell surface receptor number was determined by specific [3H]NMS binding to intact cells following incubation in the absence of agonist for the time periods indicated. Data are the mean ± S.E. from three sets of experiments. Specific [3H]NMS binding to untreated cells expressing m1 and m2 mAChRs was 256 ± 151 and 214 ± 37 fmol/mg protein, respectively. Cell surface m1 and m2 mAChR number in untreated cells did not change significantly during the experimental time periods.

Sequestration of mAChR Subtypes in HEK-293 tsA201, COS-7, and CHO Cells-- To test whether the alternative sequestration pathway of m2 mAChRs in HEK-293 cells is a property unique to m2 mAChRs, we investigated whether other members of the mAChR family share this ability. To do this, HEK-293 tsA201 cells were cotransfected with either m3 or m4 mAChR DNA along with dynamin K44A DNA. As shown in Table I, overexpression of dynamin K44A completely blocked the sequestration of m3 mAChRs in HEK-293 tsA201 cells, whereas sequestration of m4 mAChRs was inhibited by 80%. This suggests that the dynamin-independent sequestration of m2 mAChRs is not correlated with a preferential coupling profile to pertussis toxin-sensitive G proteins. This finding was confirmed by sequestration studies of m2 and m4 mAChRs in COS-7 and CHO cells. In COS-7 cells, overexpression of dynamin K44A inhibited m2 mAChR sequestration by 55%, whereas m4 mAChR sequestration was completely abolished. Similarly, sequestration of m4 mAChRs in CHO cells was blocked by 80-90% by overexpression of dynamin K44A, whereas m2 mAChR sequestration was not altered at all.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Sequestration of mAChR subtypes in HEK-293 tsA201, COS-7, and CHO cells and the effect of dynamin K44A
HEK-293 tsA201, COS-7, and CHO cells transiently transfected with m1, m2, m3, or m4 pCD-PS mAChR (12.5 or 25 µg of DNA/150-mm plate), together with pRK5 dynamin K44A, or control pRK5 (50 µg of pRK5 DNA/150-mm tissue culture plate) were incubated with 1 mM carbachol for 60 min at 37 °C. Thereafter, cells were washed, and cell surface receptor number was determined by specific [3H]NMS binding to intact cells. Transfection of COS-7 cells with 150 µg instead of 50 µg of pRK5 dynamin K44A essentially did not further decrease carbachol-induced m2 mAChR sequestration. Data are the means ± S.E. from three to nine independent experiments. Expression of m1, m2, m3, and m4 mAChRs in control HEK-293 cells was 315 ± 61, 39 ± 4, 274 ± 11, and 34 ± 5 fmol/mg protein, respectively. Expression of m2 and m4 mAChRs in control COS-7 cells was 211 ± 47 and 57 ± 7, and in control CHO cells 90 ± 37 and 39 ± 14 fmol/mg protein, respectively.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In view of the pivotal role of dynamin in regulating clathrin-coated vesicle endocytosis, we investigated whether dynamin is involved in mAChR sequestration, and in particular, whether the sequestration of the mAChR subtypes m1, m2, m3, and m4 is regulated differentially by this GTPase. Our study is the first systematic analysis of the regulation of receptor sequestration by dynamin within a particular family of G protein-coupled receptors. Whereas all four mAChR subtypes in HEK-293 cells sequestered rapidly and to a large extent, m2 mAChR sequestration was distinct from that of the other mAChR subtypes. Overexpression of the dominant-negative dynamin K44A mutant fully blocked m1, m3, and m4 mAChR sequestration, whereas m2 mAChR sequestration was not affected. The distinct sequestration of m2 mAChRs in HEK-293 cells was underscored by the finding that m2 mAChR sequestration was hardly reversible following agonist removal, whereas sequestered m1 mAChRs reappeared almost completely within 1-3 h. The dissimilarity in sequestration between m1 and m2 mAChRs was not related to their differential coupling properties. Also, the m4 mAChR subtype, which like the m2 mAChR subtype preferentially couples to pertussis toxin-sensitive G proteins, sequestered essentially in a dynamin-dependent manner in HEK-293 cells. The distinct sequestration of m2 mAChR was not confined to HEK-293 cells but also apparent in transiently transfected COS-7 and CHO cells. Overexpression of dynamin K44A in COS-7 cells blocked m4 mAChR sequestration completely, whereas m2 mAChR sequestration was inhibited by 55%. Similarly, while sequestration of m4 mAChRs in CHO cells could essentially be fully blocked by overexpressing dynamin K44A, m2 mAChR sequestration was completely dynamin-independent. During the processing of this manuscript, Hosey and co-workers (26) also showed that m2 mAChRs sequester in a dynamin-independent manner in HEK-293 cells. However, these investigators did not investigate the seminal question whether the other mAChR subtypes also utilize the dynamin-independent sequestration pathway.

Of particular interest was the observation that although sequestration of m2 mAChRs in HEK-293 cells was fully dynamin-independent, m2 mAChR sequestration in COS-7 cells was accomplished via both dynamin-dependent and dynamin-independent pathways. These findings may offer a molecular explanation for two contradictory studies on the regulation of m2 mAChR sequestration by GRK2 in COS-7 and HEK-293 cells (27, 28). In COS-7 cells, overexpression of a dominant-negative mutant of GRK2 was found to block phosphorylation and sequestration by about 50%, whereas in HEK-293 cells, overexpression inhibited m2 mAChR phosphorylation but not mAChR sequestration. On the basis of our study, it is therefore possible that these findings are related to the dissimilar sequestration pathways of m2 mAChRs in HEK-293 and COS-7 cells. Benovic and co-workers (29) and Caron and co-workers (16) have recently proposed the following early steps in the sequestration of beta 2-adrenergic receptors, which like m1 mAChRs sequester via dynamin-dependent clathrin-coated vesicles. Following agonist stimulation, receptors are phosphorylated by GRKs in an agonist-dependent manner and the subsequent binding of beta -arrestin to the phosphorylated receptor uncouples the receptor from its G protein. In turn, receptor-bound beta -arrestin may function as a high affinity clathrin adaptor, and on encountering a coated pit, beta -arrestin binds with high affinity to the assembled clathrin lattice, resulting in receptor internalization. Although this model is very attractive, it fails to explain why m2 mAChRs in HEK-293 cells do not sequester via dynamin-dependent clathrin-coated vesicles, yet are being phosphorylated by GRKs in HEK-293 cells (28) and are able to bind beta -arrestin efficiently (30). These studies lead us to conclude that GRK2-mediated phosphorylation of m2 mAChRs does not dictate whether mAChRs sequester via either dynamin-dependent or dynamin-independent pathways.

At present, the identity of the m2 mAChR sequestration pathway is unclear. A possible m2 mAChR sequestration pathway may be the endocytosis via caveolae. Caveolae are noncoated plasma membrane invaginations, which seem to have the ability to either internalize into the cell interior or to persist in the proximity of the plasma membrane (31). Previous data have suggested that cholecystokinin receptors in CHO cells as well as beta 2-adrenergic receptors in A431 cells may sequester via caveolae (5, 6). Very recently, Feron et al. (32) reported that m2 mAChRs in cardiac myocytes are targeted to caveolae following agonist binding. This observation led the authors to postulate that this receptor trafficking is required to cluster m2 mAChRs within the caveolae to initiate specific downstream signaling cascades, including activation of the resident caveolar isoform of nitric oxide synthase. Our study suggests that in the caveolae of cardiac myocytes, agonist-activated m2 mAChRs evidently also sequester and lose their ability to bind hydrophilic ligands. Further studies should be performed to test whether m2 mAChRs indeed sequester into caveolae in HEK-293 cells and cardiac myocytes.

Our study is the first demonstration of a receptor subtype-specific sequestration pathway within the family of mAChRs. In this context, it is interesting to note that, since receptor sequestration and recycling regulate receptor function (9-13), the m2 subtype-specific sequestration (and recycling) pathway may serve a regulatory mechanism of receptor responsiveness, distinct from that of the m1, m3, and m4 subtypes. At present, it is unknown whether activated m2 mAChRs are excluded from the dynamin-dependent clathrin-coated vesicle sequestration pathway, or are "specifically" recognized by a repertoire of as yet unidentified regulatory elements, which direct the activated m2 mAChRs into the dynamin-independent sequestration pathway. The observation that the m4 subtype, which has a striking homology in amino acid sequence and functional characteristics with the m2 subtype (1), as well as the less homologous m1 and m3 subtypes, sequester via the dynamin-dependent pathway, lends support to the latter possibility. The characterization of the molecular mechanisms that govern the distinct dynamin-independent m2 mAChR sequestration pathway in HEK-293 and other cells will provide new insights into the regulation of G protein-coupled receptors.

    ACKNOWLEDGEMENTS

We thank Riccarda Krudewig, Barbara Langer, and Nicole Markschies for expert technical assistance. We are indebted to Dr. S. Schmid for the gift of the human dynamin wild-type and dynamin K44A cDNA, Dr. A. Ullrich for pRK5, Dr. T. Bonner for pCD-PS and the human m3 mAChR cDNA, Dr. N. M. Nathanson for the mouse m1 mAChR DNA, Dr. D. J. Capon for the porcine m2 mAChR cDNA, and Dr. M. M. Hosey for providing HEK-293 tsA201 cells.

    FOOTNOTES

* This work was supported by a grant from the Deutsche Forschungsgemeinschaft and the IFORES program of the Universitätsklinikum Essen.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.

Dagger Recipient of a fellowship of the Heinrich Hertz-Stiftung.

§ To whom correspondence should be addressed. Tel.: 49-201-723-3462; Fax: 49-201-723-5968; E-mail: van_koppen{at}uni-essen.de.

1 The abbreviations used are: mAChR, muscarinic acetylcholine receptor; G protein, guanine nucleotide-binding protein; GRK, G protein-coupled receptor kinase; NMS, N-methylscopolamine; QNB, quinuclidinylbenzilate; CHO, Chinese hamster ovary.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Hulme, E. C., Birdsall, N. J. M., and Buckley, N. J. (1990) Annu. Rev. Pharmacol. Toxicol. 30, 633-673[CrossRef][Medline] [Order article via Infotrieve]
  2. Premont, R. T., Inglese, J., and Lefkowitz, R. J. (1995) FASEB J. 9, 175-182[Abstract/Free Full Text]
  3. Von Zastrow, M., Link, R., Daunt, D., Barsh, G., and Kobilka, B. K. (1993) J. Biol. Chem. 268, 763-766[Abstract/Free Full Text]
  4. Tolbert, L. M., and Lameh, J. (1996) J. Biol. Chem. 271, 17335-17342[Abstract/Free Full Text]
  5. Dupree, P., Parton, R. G., Raposo, G., Kurzchalia, T. V., and Simons, K. (1993) EMBO J. 12, 1597-1605[Abstract]
  6. Roettger, B. F., Rentsch, R. U., Pinon, D., Holicky, E., Hadac, E., Larkin, J. M., and Miller, L. J. (1995) J. Cell Biol. 128, 1029-1041[Abstract]
  7. Raposo, G., Dunia, I., Delavier-Klutchko, C., Kaveri, S., Strosberg, A. D., and Benedetti, E. L. (1989) Eur. J. Cell Biol. 50, 340-352[Medline] [Order article via Infotrieve]
  8. Roettger, B. F., Rentsch, R. U., Hadac, E. M., Hellen, E. H., Burghardt, T. P., and Miller, L. J. (1995) J. Cell Biol. 130, 579-590[Abstract]
  9. Yu, S. S., Lefkowitz, R. J., and Hausdorff, W. P. (1993) J. Biol. Chem. 268, 337-341[Abstract/Free Full Text]
  10. Pippig, S., Andexinger, S., and Lohse, M. J. (1995) Mol. Pharmacol. 47, 666-676[Abstract]
  11. Yang, J., Williams, J. A., Yule, D. I., and Logsdon, C. D. (1995) Mol. Pharmacol. 48, 477-485[Abstract]
  12. Bogatkewitsch, G. S., Lenz, W., Jakobs, K. H., and van Koppen, C. J. (1996) Mol. Pharmacol. 50, 424-429[Abstract]
  13. Holtmann, M. H., Roettger, B. F., Pinon, D. I., and Miller, L. J. (1996) J. Biol. Chem. 271, 23566-23571[Abstract/Free Full Text]
  14. Van der Bliek, A. M., Redelmeier, T. E., Damke, H., Tisdale, E. J., Meyerowitz, E. M., and Schmid, S. L. (1993) J. Cell Biol. 122, 553-563[Abstract]
  15. Takei, K., McPherson, P. S., Schmid, S. L., and de Camilli, P. (1995) Nature 374, 186-190[CrossRef][Medline] [Order article via Infotrieve]
  16. Zhang, J., Ferguson, S. S. G., Barak, L. S., Ménard, L., and Caron, M. G. (1996) J. Biol. Chem. 271, 18302-18305[Abstract/Free Full Text]
  17. Scherer, N. M., and Nathanson, N. M. (1990) Biochemistry 29, 8475-8483[Medline] [Order article via Infotrieve]
  18. Goldman, P. S., Schlador, M. L., Shapiro, R. A., and Nathanson, N. M. (1996) J. Biol. Chem. 271, 4215-4222[Abstract/Free Full Text]
  19. Daunt, D. A., Hurt, C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997) Mol. Pharmacol. 51, 711-720[Abstract/Free Full Text]
  20. Shapiro, R. A., Scherer, N. M., Habecker, B. A., Subers, E. M., and Nathanson, N. M. (1988) J. Biol. Chem. 263, 18397-18403[Abstract/Free Full Text]
  21. Peralta, E. G., Winslow, J. G., Peterson, G. L., Smith, D. H., Ashkenazi, A., Ramachandran, J., Schimerlik, M. I., and Capon, D. J. (1987) Science 236, 600-605[Medline] [Order article via Infotrieve]
  22. Bonner, T. I., Buckley, N. J., Young, A. C., and Brann, M. R. (1987) Science 237, 527-532[Medline] [Order article via Infotrieve]
  23. Van Koppen, C. J., Lenz, W., and Nathanson, N. M. (1993) Biochim. Biophys. Acta 1173, 342-344[Medline] [Order article via Infotrieve]
  24. Margolskee, R. F., McHendry-Rinde, B., and Horn, R. (1993) Biotechniques 15, 906-911[Medline] [Order article via Infotrieve]
  25. Van Koppen, C. J., Sell, A., Lenz, W., and Jakobs, K. H. (1994) Eur. J. Biochem. 222, 525-531[Abstract]
  26. Pals-Rylaarsdam, R., Gurevich, V. V., Lee, K. B., Ptasienski, J. A., Benovic, J. L., and Hosey, M. M. (1997) J. Biol. Chem. 272, 23682-23689[Abstract/Free Full Text]
  27. Tsuga, H., Kameyama, K., Haga, T., Kurose, H., and Nagao, T. (1994) J. Biol. Chem. 269, 32522-32527[Abstract/Free Full Text]
  28. Pals-Rylaarsdam, R., Xu, Y., Witt-Enderby, P., Benovic, J. L., and Hosey, M. M. (1995) J. Biol. Chem. 270, 29004-29011[Abstract/Free Full Text]
  29. Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450[CrossRef][Medline] [Order article via Infotrieve]
  30. Gurevich, V. V., Richardson, R. M., Kim, C. M., Hosey, M. M., and Benovic, J. L. (1993) J. Biol. Chem. 268, 16879-16882[Abstract/Free Full Text]
  31. Kurzchalia, T. V., and Parton, R. G. (1996) FEBS Lett. 389, 52-54[CrossRef][Medline] [Order article via Infotrieve]
  32. Feron, O., Smith, T. W., Michel, T., and Kelly, R. A. (1997) J. Biol. Chem. 272, 17744-17748[Abstract/Free Full Text]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.