Arrestin-independent Internalization of the m1, m3, and m4 Subtypes of Muscarinic Cholinergic Receptors*

Katharine B. LeeDagger §, Robin Pals-RylaarsdamDagger parallel , Jeffrey L. Benovic**, and M. Marlene HoseyDagger Dagger Dagger

From the Dagger  Department of Molecular Pharmacology and Biological Chemistry and the  Institute of Neuroscience, Northwestern University Medical School, Chicago, Illinois 60611 and the ** Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

To understand what processes contribute to the agonist-induced internalization of subtypes of muscarinic acetylcholine receptors, we analyzed the role of arrestins. Whereas the m2 mAChR has been shown to undergo augmented internalization when arrestins 2 and 3 are overexpressed (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), the agonist-induced internalization of m1, m3, and m4 mAChRs was unchanged when arrestins 2 or 3 were overexpressed in transiently transfected HEK-tsA201 cells. Furthermore, when a dominant-negative arrestin was used to interrupt endogenous arrestin function, there was no change in the internalization of the m1, m3, and m4 mAChR whereas the internalization of the beta 2 adrenergic receptor was completely blocked. Wild-type and GTPase-deficient dominant-negative dynamin were used to determine which endocytic machinery played a role in the endocytosis of the subtypes of mAChRs. Interestingly, when dynamin function was blocked by overexpression of the GTPase-deficient dynamin, agonist- induced internalization of the the m1, m3, and m4 mAChRs was suppressed. These results suggested that the internalization of the m1, m3, and m4 mAChRs occurs via an arrestin-independent but dynamin-dependent pathway. To ascertain whether domains that confer arrestin sensitivity and dynamin insensitivity could be functionally exchanged between subtypes of mAChRs, chimeric m2/m3 receptors were analyzed for their properties of agonist-induced internalization. The results demonstrated that the third intracellular loop of the m2 mAChR conferred arrestin sensitivity and dynamin insensitivity to the arrestin-insensitive, dynamin-sensitive m3 mAChR while the analogous domain of the m3 mAChR conferred arrestin resistance and dynamin sensitivity to the previously arrestin-sensitive, dynamin-insensitive m2 mAChR.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Sequestration or internalization is a process that participates in the agonist-dependent regulation of many G-protein-coupled receptors (GPRs)1 (1, 2). Within seconds to minutes of agonist exposure, many different types of GPRs are phosphorylated in an agonist-dependent manner, which leads to receptor/G-protein uncoupling and consequent desensitization of the receptors. Subsequently, certain GPRs are sequestered (internalized) away from their normal membrane environment. While certain receptors are reversibly sequestered and recycled back to the cell surface (3), others are targeted to lysosomes and degraded (4). Thus, sequestration may play different roles for different GPRs. For example, sequestration has been demonstrated to play a key role in the resensitization of the beta 2-adrenergic receptor following desensitization (5), whereas internalization has been suggested to prolong desensitization for the m4 muscarinic acetylcholine receptor (mAChR)(6).

The mAChRs are members of the GPR superfamily (7-11). There are five identified subtypes termed m1-m5. The m1, m3, and m5 subtypes couple predominantly to Gq and activate phospholipase C, while the m2 and m4 subtypes couple to Gi family members, inhibit adenylyl cyclase, and activate inwardly rectifying potassium channels (12-14). The m2 receptor has been used as a model of a Gi-linked receptor to understand the mechanisms underlying GPR regulation (15). Our laboratory has studied the regulation of the m2 mAChR subtype and has found that the receptor undergoes agonist-dependent phosphorylation, desensitization, and sequestration (16-18).

The processes underlying the internalization of GPRs have only recently begun to be understood. Arrestins are cytosolic proteins that have been shown to play a role in regulating receptor desensitization and, more recently, to also participate in internalization (1, 19, 20). Arrestins show specificity for binding to activated, phosphorylated receptors (21). There are four cloned arrestin family members. Arrestin 1 or visual arrestin is found predominantly in the retina and acts by preventing light-activated phosphorylated rhodopsin from interacting with transducin (22). Arrestin 2 (beta -arrestin) and arrestin 3 (beta -arrestin 2) regulate the interaction of non-visual GPRs with their G-proteins (1). X- or C-arrestin is a cone-specific arrestin isoform (23). Recently, studies have shown that arrestin 2 and arrestin 3 can facilitate internalization of the beta 2-adrenergic receptor (19, 24) and other GPRs (25, 26). Arrestin facilitates the internalization of these GPRs by binding to clathrin and acting as an adapter to bring the GPRs to clathrin-coated pits for subsequent endocytosis (19).

The process of clathrin-mediated endocytosis has been shown to require the protein dynamin, a GTPase-containing protein which oligomerizes and acts to pinch off the neck of the invaginated clathrin-coated pits (27, 28). Upon overexpression of a GTPase-deficient dominant-negative dynamin, endocytosis of the beta 2-adrenergic receptor has been shown to be inhibited (29). However, recent evidence has shown that some GPRs may also internalize in a dynamin-independent manner. The angiotensin II receptor (29) and the m2 mAChR (30) have been shown to undergo agonist-dependent internalization in HEK293 cells even when dynamin-dependent endocytosis is blocked. Interestingly, overexpression of arrestin 2 or 3 in HEK293 cells, COS cells, or JEG-3 cells can direct a fraction of m2 mAChRs to follow an arrestin- and dynamin-dependent pathway (30, 31). These results suggest that there are multiple mechanisms that can contribute to the regulation of the m2 mAChR and that different mechanisms may predominate in different cell types. The present study seeks to identify the modes of internalization utilized by other members of the mAChR family, namely the m1, m3, and m4 mAChR subtypes.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
Discussion
References

Materials-- Dulbecco's modified Eagle's medium (DMEM) and penicillin-streptomycin were purchased from Life Technologies, Inc. or Mediatech. Fetal bovine serum was purchased from Life Technologies, Inc. Hepes-buffered DMEM was obtained from Sigma. N-[3H]Methylscopolamine ([3H]NMS) was purchased from NEN Life Science Products.

Cell Culture and Transfection-- HEK-tsA201 cells (human embryonic kidney cells stably expressing simian virus 40 large T antigen) (32) were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/ml streptomycin, and 100 units/ml penicillin at 37 °C in a 5% CO2 environment. HEK-tsA201 cells were transfected using the calcium phosphate precipitation method followed by a 5-6-min shock with 30% ME2SO in DMEM as described previously (17).

The following plasmids were used in the amounts described below: 5 µg of bovine arrestin 2 (21) or arrestin 3 (33) in the pcDNA3 or pBC12B1 expression vectors, 5 µg of arrestin 2-(319-418) (34) in pcDNA3, 0.1 µg of human dynamin-1 (wild-type or dynK44A mutant) (28, 35) in pcDNA3; 10 µg of m1, m2, m3, or m4 mAChRs (7, 13) in pCR3 or pcDNA3. Hy9 and Hy10 chimeric receptors (36) were generous gifts from E. Peralta and were expressed from pcDNA3. The control conditions in each experiment were cells transfected with the receptor construct tested and empty vector (see figure legends).

Receptor Internalization Assay-- To measure the changes in the numbers of muscarinic receptors present on the cell surface as a function of time of agonist exposure, the hydrophilic radioligand, [3H]NMS, was used. One day post-transfection, cells from 100-mm plates were passed to 60-mm plates for use on the following day. Cells were incubated in the presence or absence of 1 mM carbachol (mAChR) or 10 µM isoproterenol (beta 2-adrenergic receptor) for 1 h at 37 °C. These cells were then washed with 5 × 3 ml of ice-cold PBS at 4 °C. The cells were removed from the plates with ice-cold Hepes-buffered DMEM and incubated with 2 nM [3H]NMS for 2 h at 4 °C. Nonspecific binding was defined by 10 µM atropine. For the beta 2-adrenergic receptor, internalization was measured as the reduction of binding sites for 10 nM [3H]CGP-12177 and nonspecific binding was defined as the binding observed in the presence of 10 µM alprenolol (30). Assays were performed for 4 h at 4 °C. Protein assays were performed to correct for any differences in cell number between plates.

Immunoblot Analysis-- Expression of arrestins and HA-tagged dynamin constructs were analyzed by Western blotting. In brief, 100 µg of protein from whole cell lysates was loaded onto SDS-acrylamide 8% gels and run at 60 mA constant current. The gels were then transferred overnight onto 0.2-µm nitrocellulose filters. The filters were then blocked in 5% milk in Tris-buffered saline (TBS) for 1 h. Primary antibodies were used at concentrations as described in the text and were diluted in milk/TBS and incubated with the filters for at least 1 h at room temperature and in some cases overnight at 4 °C. Blots were rinsed in TBS and then incubated with secondary antibody. Secondary antibody was used at a concentration of 1:2000 in milk/TBS for 1-2 h. Enhanced chemiluminescence was used to detect expressed proteins. The detection reagents were obtained from Pierce, and the film was purchased from NEN Life Science Products.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Effects of Arrestins 2 and 3 on Internalization of Subtypes of mAChR-- To elucidate the role arrestins may play in the sequestration of subtypes of mAChRs, we transiently transfected HEK293-tsA201 cells with either the m1, m3, or m4 mAChRs, in the presence or absence of arrestin 2 or arrestin 3. Western blotting was performed to confirm that the arrestins were expressed (Fig. 1A). As previously demonstrated, expression of arrestins was increased approximately 150 times above that of endogenous arrestin levels (30). Cells were treated with the agonist carbachol (1 mM) for 1 h, and the number of receptors remaining at the cell surface was measured using a saturating concentration (2 nM) of the hydrophilic ligand [3H]NMS. In the control cells (not overexpressing arrestin 2 or arrestin 3), each mAChR internalized as a consequence of agonist exposure (Fig. 1, B-D). Suprisingly, overexpression of either arrestin 2 or 3 had no effect on the internalization of the m1, m3, or m4 mAChR (Fig. 1, B-D). In the absence of overexpressed arrestins, the m1 mAChR internalized to 31 ± 2.8%, while in the presence of arrestins 2 or 3, the m1 mAChR internalized to 31 ± 8.8% or 31 ± 5.0%, respectively (Fig. 1B). The same pattern was observed for the m3 and m4 mAChR subtypes (Fig. 1, C and D). The m3 mAChR, when expressed alone, internalized 24 ± 3.7%, and in the presence of arrestin 2 or 3, internalization occurred to similar levels (30 ± 3.4% and 29 ± 3.5%, respectively) (Fig. 1C). Internalization of the m4 mAChR alone was 34 ± 5.0%. When in the presence of arrestins 2 and 3, the m4 mAChR internalized to levels of 37 ± 4.1% and 38 ± 6.4%, respectively (Fig. 1D). Overexpression of arrestin 2 or arrestin 3 has been shown to increase the ability of the m2 mAChR and the beta 2-adrenergic receptor to internalize under the same conditions used here (30). One possibility to explain the inability of overexpressed arrestins to increase the internalization of the m1, m3, and m4 mAChRs is that endogenous arrestins, although expressed at low levels (30), may be sufficient to mediate internalization of these mAChR subtypes. This idea led us to ask whether the endogenous arrestins within these cells were mediating the internalization of the subtypes.


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Fig. 1.   Effect of arrestin overexpression on m1, m3, and m4 mAChR internalization. A, representative Western blot of 100 µg of whole cell protein from transiently transfected cells that were either mock-transfected (lane 1), transfected with 5 µg of DNA for arrestin 2 (lane 2), or 5 µg of DNA for arrestin 3 (lane 3). Overexpressed arrestins were detected using a monoclonal antibody recognizing both arrestin 2 and 3 at a concentration of 1:2000 (30). The effect of overexpression of arrestin 2 or arrestin 3 on internalization of m1 (B), m3 (C), or m4 (D) was determined. HEK-tsA cells were transiently transfected with 10 µg of receptor plasmid and with 5 µg of empty vector (pBC12B1 or pcDNA3) or 5 µg of arrestin 2 or 3 in pBC12B1 or pcDNA3. Transfected cells were either untreated (not shown) or treated with 1 mM carbachol for 60 min, washed extensively in PBS, and incubated with 2 nM [3H]NMS in whole cell binding assays. Nonspecific binding was determined in the presence of 10 µM atropine. Data are represented as % receptor internalized as compared with the untreated control. Data are expressed as the mean ± S.E. for 3-5 experiments, each performed in duplicate.

We next performed experiments using a dominant-negative arrestin, arrestin 2-(319-418), which binds to clathrin constitutively and blocks the internalization of the beta 2-adrenergic receptor (34). Transiently transfected tsA cells expressing the beta 2-adrenergic receptor, m1, m3, or m4 mAChR in the presence or absence of arrestin 2-(319-418) were analyzed. Co-transfection of the arrestin 2-(319-418) construct was verified using immunoblot analysis with an antibody that recognizes the 15 amino acids at the C terminus of arrestin 2 (34) (Fig. 2, top). Expression of arrestin 2-(319-418) completely blocked the internalization of the beta 2-adrenergic receptor (Fig. 2). However, under identical conditions, the internalization of the m1, m3, and m4 mAChRs was not significantly reduced (Fig. 2). These data, together with the data presented in Fig. 1, suggested that the m1, m3, and m4 mAChRs may internalize in an arrestin-independent manner, and conceivably that a novel pathway may serve to regulate the internalization of the mAChR subtypes.


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Fig. 2.   Effect of dominant-negative arrestin on internalization. A dominant-negative arrestin, arrestin 2-(319-418) was used to disrupt arrestin-dependent endocytosis in HEK-tsA cells transfected with either 5 µg of empty vector pcDNA3 or arrestin 2-(319-418) in pcDNA3 along with 10 µg of beta 2-adrenergic receptor pcDNA3, m1 pCR3, m3 pcDNA3, or m4 pCR3. The top panel depicts a representative blot of 100 µg of whole cell protein that was probed for expression of arrestin 2-(319-418) using an antibody raised in rabbit against a C-terminal epitope of arrestin 2 as described previously (34). Transfected cells were either untreated (not shown) or treated with 1 mM carbachol (in the case of the mAChRs) or 10 µM isoproterenol (in the case of the beta 2-adrenergic receptor) for 60 min, washed extensively in PBS, and analyzed in ligand binding assays. Data are expressed as the mean ± S.E. for 5-7 experiments, each performed in duplicate. Data were analyzed using a paired Student's t test (*, significantly different from cells not expressing arrestin 2-(319-418), p < 0.01).

Does Dynamin-dependent Endocytosis Regulate the Ability of the mAChR Subtypes to Internalize?-- Because the mAChR subtypes described above appeared to internalize in an arrestin-independent manner, we postulated that the mAChR subtypes, like the m2 mAChR, may use an alternate endocytic machinery to the classical clathrin-dependent pathway to internalize. As a first step to test this possibility, we used strategies that interrupted dynamin-dependent endocytosis. Upon overexpression of a GTPase-deficient dynamin, dynaminK44A (dynK44A), clathrin-coated pits are unable to undergo fission, and proteins within the pit remain accessible to the external environment (37). Upon overexpression of the dominant-negative dynamin, we observed a complete inhibition in the ability of the m1, m3, and m4 mAChR subtypes to internalize (Fig. 3, A, B, and C). Overexpression of wild-type dynamin had no significant effect on the m3 and m4 mAChR subtypes, although the overexpression of wild-type dynamin caused an unexplained partial inhibition of internalization of the m1 mAChR (Fig. 3A). These results led us to conclude that the m1, m3, and m4 mAChRs utilize a dynamin-dependent, but arrestin-independent pathway to internalize.


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Fig. 3.   Effect of dynK44A on m1, m3, and m4 mAChR internalization. A dominant-negative mutant of dynamin I, dynK44A (27) was used to disrupt dynamin-dependent endocytosis in tsA201 cells co-transfected with the m1 (A), m3 (B), or m4 (C) mAChRs. Cells were transfected with 10 µg of receptor plasmid along with 0.1 µg of pcDNA3 or with wt-dynamin-HA or dynK44A-HA in pcDNA3. Transfected cells were either untreated (not shown) or treated with 1 mM carbachol for 60 min, washed extensively in PBS, and incubated with 2 nM [3H]NMS in whole cell binding assays. Overexpression of dyn or dynK44A was verified by Western blot using the HA antibody at a concentration of 1:1000. Data are expressed as the mean ± S.E. for 5-7 experiments, each performed in duplicate. Data were analyzed using a paired Student's t test (*, significantly different from cells not expressing dyn or dynK44A, p < 0.05).

Does the i3 Loop Regulate the Ability of the Different mAChR Subtypes to Utilize a Dynamin-sensitive and Arrestin-insensitive Pathway for Internalization?-- The experiments described above demonstrate that internalization of the m1, m3, and m4 mAChR subtypes could not be augmented by overexpression of arrestin 2 or 3, but could be blocked when dominant-negative dynamin is overexpressed. However, previous studies have demonstrated that arrestin 2 or 3 increased the extent of the internalization of the m2 mAChR and that a GTPase-deficient dynamin had no effect on m2 mAChR internalization (30).

To understand if the intracellular third loop of the m2 mAChR contains the determinants necessary for dynamin-independent endocytosis, chimeric m2/m3 receptors were used and analyzed for their ability to internalize in the presence and absence of overexpressed wild-type and dominant-negative dynamin (Fig. 4). The Hy9 chimera is an m2 mAChR containing an i3 loop entirely derived from the m3 mAChR, whereas the Hy10 chimera is an m3 mAChR with the i3 loop of the m2 mAChR (36). As described previously (30), the m2 mAChR was unaffected when wild-type or dominant-negative dynamin was overexpressed (compare 45 ± 2.5% versus 32 ± 7.8% or 33 ± 1.4% internalization of the m2 mAChR alone versus in the presence of wild-type or dominant-negative dynamin, respectively)(Fig. 4). When the Hy9 chimera was expressed alone, it internalized to 39 ± 5.0% (Fig. 4). Although overexpression of the wild-type dynamin had no effect on the ability of the Hy9 chimera to internalize, overexpression of the dominant-negative dynamin inhibited the internalization of the Hy9 chimeric receptor (36 ± 2.6% versus 15 ± 6.1% in the presence of wild-type versus dominant-negative dynamin, respectively). Interestingly, when the third loop of the m2 receptor was placed onto the body of the m3 mAChR (the Hy10 chimera), overexpression of dominant-negative dynamin had no effect on the ability of the Hy10 receptor to internalize (39 ± 7.4%, Hy10 alone versus 34 ± 7.4% and 41 ± 2.8% in the presence of overexpressed wild-type and dominant-negative dynamin, respectively). This was in marked contrast to the wild-type m3 mAChR (Fig. 3B). In all cases, overexpression of the dynamins was verified by Western blot (data not shown). Because it was possible to switch the dynamin sensitivity of the m3 mAChR to the previously dynamin-insensitive m2 mAChR, we postulated that the arrestin sensitivity could be switched as well.


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Fig. 4.   The role of the i3 loop on the dynamin dependence of m2 versus m3 mAChR internalization. Wild-type m2 mAChR or chimeric m2/m3 receptors were co-transfected with wild-type or dominant-negative dynamin and tested for their ability to internalize. 10 µg of wild-type m2 mAChR, chimeric m2/m3i3 loop (Hy9), or chimeric m3/m2i3 loop (Hy10) receptors (36) were either expressed alone (with 0.1 µg of pcDNA3) or 0.1 µg of wild-type or dynaminK44A. Transfected cells were either untreated (not shown) or treated with 1 mM carbachol for 60 min, washed extensively in PBS, and incubated with 2 nM [3H]NMS in whole cell binding assays. Overexpression of dynamins was verified by Western blot using an antibody recognizing the HA epitope at a concentration of 1:1000. Data are expressed as the mean ± S.E. for 5-7 experiments, each performed in duplicate. Data were analyzed using a paired Student's t test (*, significantly different from cells expressing wild-type dynamin, p < 0.05).

The third intracellular (i3) loop of the m2 mAChR has been shown to be important in mediating interaction with arrestins (30). Furthermore, when specific sites of phosphorylation were removed from this domain, the ability of the m2 mAChR to interact with arrestins was lost, suggesting that the i3 loop plays an important role in regulating arrestin binding in vivo and in vitro (30). In these experiments, we again used the Hy9 and Hy10 chimeras (36). These receptors were expressed in tsA201 cells alone or with arrestin 2 and arrestin 3 (Fig. 5). Cells expressing the m2 mAChR alone internalized 33 ± 4.4% after a 60-min exposure to 1 mM carbachol. Co-expression with arrestin 2 or arrestin 3 increased m2 mAChR internalization to 76 ± 4.3% and 69 ± 1%, respectively. Interestingly, when the i3 loop of the m2 mAChR was replaced with the i3 loop of the m3 mAChR (Hy9 receptor), the extent of internalization of the chimera was similar to the wild-type m2 mAChR; however, overexpression of either arrestin 2 or arrestin 3 did not enhance internalization (compare 39 ± 4.8% versus 46 ± 1% or 39 ± 9.7% with arrestin 2 or 3, respectively). Studies with the reciprocal chimera, Hy10, indicated that the m3 mAChR containing the i3 loop from the m2 mAChR was able to respond to overexpressed arrestin 2 or 3 (compare 33 ± 6.7% versus 59 ± 8.5% or 65 ± 6.3% with arrestin 2 or 3, respectively), in marked contrast to the behavior of the wild-type m3 mAChR (Fig. 1C). These results indicated that the region of the m2 mAChR that confers sensitivity to arrestin is found predominantly within the i3 loop and can convert a non-arrestin-sensitive receptor into an arrestin-sensitive receptor.


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Fig. 5.   The role of the i3 loop of the m2 mAChR on m3 mAChR internalization. Wild-type m2 mAChR or chimeric m2/m3 receptors were co-transfected with arrestins 2 or 3 and tested for their ability to internalize. 10 µg of wild-type m2 mAChR, chimeric m2/m3i3 loop (Hy9), or chimeric m3/m2i3 loop (Hy10) receptors were either expressed alone (with 5 µg of pBC12B1 or pcDNA3) or 5 µg of arrestin 2 or 3 in pBC12B1 or pcDNA3. Transfected cells were either untreated (not shown) or treated with 1 mM carbachol for 60 min, washed extensively in PBS, and incubated with 2 nM [3H]NMS in whole cell binding assays. Overexpression of arrestin 2 or 3 was verified by Western blot using an antibody recognizing both arrestin 2 and 3 at a concentration of 1:2000. Data are expressed as the mean ± S.E. for 5-7 experiments, each performed in duplicate. Data were analyzed using a paired Student's t test (*, significantly different from cells not expressing arrestin 2 or 3, p < 0.05).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The data presented in this study demonstrate that the agonist-induced internalization of the m1, m3, and m4 mAChR subtypes occurs via an arrestin-independent pathway. Overexpression of arrestins 2 and 3 had no effect on the agonist-induced internalization of the m1, m3, and m4 mAChRs. Furthermore, when endogenous arrestin proteins were inhibited from interacting with clathrin by overexpression of a dominant-negative arrestin construct (34), the m1, m3, and m4 mAChRs were still able to internalize even under conditions where the beta 2-adrenergic receptor was inhibited (Fig. 2). These results suggested that the m1, m3, and m4 mAChR receptors do not use endogenous arrestins to internalize and are insensitive to overexpression of arrestins 2 and 3. On the other hand, when dynamin-dependent endocytosis was blocked by overexpression of dynK44A, the m1, m3, and m4 mAChR subtypes were unable to internalize (Fig. 2, A, B, and C) suggesting that a dynamin-dependent pathway is used for subtype internalization. Thus, it appears that the m1, m3, and m4 mAChR subtypes utilize an arrestin-independent but dynamin-dependent pathway of internalization. We cannot rule out that an unidentified arrestin may be interacting with the receptors that is insensitive to the dominant-negative arrestin construct used here. Perhaps a more definitive approach would be to utilize antisense or ribozyme technologies to inhibit the expression of all endogenous arrestins. It is possible that the lack of inhibition by the dominant-negative arrestin on m1, m3, and m4 mAChR internalization could be due to a poor co-transfection efficiency; however, it should be noted that the internalization of the beta 2-adrenergic receptor was completely inhibited under identical conditions. The present results provide new insights into previously unappreciated differences in mAChR subtypes. While it appears that the m2 mAChR can utilize arrestin-independent and -dependent pathways for internalization (30), the m1, m3, and m4 mAChR subtypes examined in these studies did not utilize arrestins for their agonist-induced internalization. Interestingly, in the absence of overexpressed arrestins, the m2 mAChR also internalized via an arrestin-independent pathway, but this pathway is also dynamin-independent (30). Thus, multiple pathways appear to be used to achieve internalization of subtypes of mAChR in response to agonist stimulation. The present results suggest that arrestins do not appear to play a major role in mediating the internalization of the m1, m3, and m4 mAChR subtypes; however it is still possible that arrestins may play a role in uncoupling these receptors from G-proteins, i.e. in rapid desensitization. Future studies will examine the possible role of arrestins in mAChR subtype desensitization and the role internalization plays in mAChR subtype signaling and desensitization.

The present studies demonstrated that it is possible to switch the machinery utilized in receptor endocytosis. While the m2 mAChR internalized in a predominantly dynamin-independent manner, when the i3 loop from the dynamin-dependent m3 mAChR was placed onto the body of the m2 mAChR, the resulting chimera was unable to endocytose in the presence of dominant-negative dynamin (Fig. 4). The converse receptor, the Hy10 chimera, was insensitive to overexpressed dominant-negative dynamin. These results indicated that the i3 loop of the m2 mAChR contains an important domain (not found in the m3 mAChR) which mediates interaction with a component of dynamin-independent internalization machinery.

The results also suggested that the m1, m3, and m4 mAChRs utilize a different mechanism to internalize in tsA201 cells than the m2 mAChR. The portion of the m2 mAChR that interacts with arrestins 2 and 3 is likely to reside within the third intracellular loop of the m2 mAChR since chimeric m2 receptors lacking this domain were unable to internalize in an arrestin-dependent manner (Fig. 5). Furthermore, transfer of the i3 domain of the m2 mAChR to the m3 mAChR allowed the latter receptor to become arrestin-sensitive (Fig. 5). The i3 loop has been shown to play important roles in m2 mAChR regulation (17, 18). The m1 and m3 mAChRs have been shown to contain important domains regulating their internalization within their i3 loops (38, 39). Recently, our laboratory has identified certain key elements that contribute to the interaction of arrestins with the m2 mAChR (30). The m2 mAChR contains 2 clusters of serine/threonine residues located within the i3 loop (7, 40) that serve as substrates for agonist-mediated phosphorylation in vivo and in vitro (30). Removal of the serine/threonine (S/T) cluster at the N-terminal region (NAla-4) of the i3 loop had little effect on receptor phosphorylation, desensitization, and internalization (18). However, removal of the S/T cluster at the C-terminal region of the m2 i3 loop (CAla-4) produced a receptor that was unable to desensitize but still able to undergo phosphorylation and internalization (18). The CAla-4 mutant was also unable to interact with arrestin (30). This indicated that a specific domain in the m2 mAChR was necessary to allow for arrestin interaction. Interestingly, the m1, m3, and m4 mAChR subtypes tested here have similar clusters within their i3 loops. At present, little is known about the factors that are important in the regulation of the m1, m3, and m4 mAChRs. Future studies will concentrate on determining if these receptors are regulated by phosphorylation, and if so, what role phosphorylation plays in the regulation of these receptors.

In summary, the results presented here suggest that the m1, m3, and m4 mAChRs are internalized in an arrestin-independent manner via a dynamin-sensitive process. The data also suggest that the i3 loop of the m3 mAChR either lacks an arrestin binding domain or contains an inhibitory domain that precludes arrestin binding. The lack of effect of arrestins on the receptors studied here provide impetus to studies designed to elucidate the factors that do contribute to the regulation of these GPRs.

    FOOTNOTES

* This work was supported in part by Grants HL50121 (to M. M. H.) and GM44944 and GM47417 (to J. L. B.).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 National Service Research Award Training Grant T32 GM08061.

parallel Supported by a predoctoral fellowship from the Howard Hughes Medical Institute.

Dagger Dagger To whom correspondence should be addressed: Dept. of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 E. Chicago Ave. S-215, Chicago, IL 60611. Tel.: 312-503-3692; Fax: 312-503-5349; E-mail: mhosey{at}nwu.edu.

1 The abbreviations used are: GPR, G-protein-coupled receptor; mAChR, muscarinic acetylcholine receptor; DMEM, Dulbecco's modified Eagle's medium; NMS, N-[3H]methylscopolamine; PBS, phosphate-buffered saline; TBS, Tris-buffered saline.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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