From the 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
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ABSTRACT |
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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 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.
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INTRODUCTION |
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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 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 (-arrestin) and arrestin 3 (
-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
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 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.
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EXPERIMENTAL PROCEDURES |
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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 dynaminReceptor 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
(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
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.
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RESULTS |
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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
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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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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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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DISCUSSION |
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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
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
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.
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FOOTNOTES |
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* 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.
Supported by a predoctoral fellowship from the Howard Hughes
Medical Institute.
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.
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REFERENCES |
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