From the Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195-7750
Received for publication, August 8, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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Muscarinic acetylcholine receptors (mAChRs) can
be differentially localized in polarized cells. To identify potential
sorting signals that mediate mAChR targeting, we examined the sorting of mAChRs in Madin-Darby canine kidney cells, a widely used model system. Expression of FLAG-tagged mAChRs in polarized Madin-Darby canine kidney cells demonstrated that the M2 subtype
is sorted apically, whereas M3 is targeted basolaterally.
Expression of M2/M3 receptor chimeras revealed
that a 21-residue sequence, Ser271-Ser291,
from the M3 third intracellular loop contains a basolateral sorting signal. Substitution of sequences containing the M3
sorting signal into the homologous regions of M2 was
sufficient to confer basolateral localization to this apical receptor.
Sequences containing the M3 sorting signal also conferred
basolateral targeting to M2 when added to either the third
intracellular loop or the C-terminal cytoplasmic tail. Furthermore,
addition of a sequence containing the M3 basolateral
sorting signal to the cytoplasmic tail of the interleukin-2 receptor
Targeting of newly synthesized proteins to their correct
subcellular locales is essential for cell function. Protein sorting is
particularly important in polarized cells such as neurons and epithelia, where cell-surface proteins must be specifically routed to
distinct plasma membrane subdomains. The mechanisms responsible for the
correct targeting of membrane proteins in polarized cells remain a
fundamental question in cell biology. Madin-Darby canine kidney
(MDCK)1 epithelial cells
provide a widely used and well characterized model system for studies
of protein targeting (1). Polarized MDCK cells establish apical and
basolateral plasma membrane domains with distinct protein and lipid
compositions. Many cell-surface proteins contain sorting signals that
direct them to the apical or basolateral domain. Apical sorting signals
can consist of a glycosylphosphatidylinositol anchor (2),
N-glycans (3, 4), or protein sequences in the extracellular,
transmembrane, and/or cytoplasmic domains (5-9). In contrast,
basolateral sorting signals are almost always found in the cytoplasmic
domain of transmembrane proteins and frequently contain a critical
tyrosine residue, a dihydrophobic motif, a cluster of acidic residues,
or a combination of these elements (10-13). Although much has been
learned about the sorting of single-pass transmembrane proteins, little
is known about signals that mediate the targeting of proteins with
multiple membrane-spanning domains.
Muscarinic acetylcholine receptors (mAChRs) are a family of
seven-transmembrane domain, G protein-coupled receptors composed of
five distinct subtypes (M1-M5). The
M1, M3, and M5 receptors preferentially couple to activation of phospholipase C via the Gq/11 family of G proteins, whereas the M2 and
M4 receptors preferentially couple to inhibition of
adenylyl cyclase via the Gi/o family (14). In addition to
their biochemical specificities, mAChR subtypes have unique cellular
and subcellular distributions (15). Muscarinic receptors are
asymmetrically distributed in polarized cells such as pancreatic and
lacrimal acinar cells (16, 17), lingual epithelial cells (18),
Xenopus oocytes (19, 20), and MDCK epithelial cells (21).
Furthermore, mAChR subtypes are differentially localized in a variety
of neuronal cells. For example, the M1 receptor is
expressed in the cell bodies and dendrites of hippocampal pyramidal
neurons and granule cells in the dentate gyrus, where it mediates
postsynaptic responses to acetylcholine (22). In contrast,
M2 is found mainly in the axon terminals of cholinergic and
non-cholinergic septohippocampal projection neurons and hippocampal interneurons, where it modulates neurotransmitter release (23, 24). The
M3 receptor is found both on cell bodies and dendrites of
hippocampal granule and pyramidal neurons and on axon terminals in the
hippocampal molecular layer and striatum (25, 26). Despite the
differential localization of mAChR subtypes in a variety of polarized
cells, little is known about the mechanisms by which their precise
subcellular distributions are achieved.
To begin to elucidate the signals and mechanisms that govern mAChR
targeting, we have utilized the MDCK cell system to identify sorting
determinants for mAChR subtypes. Although the follicle-stimulating hormone receptor possesses a basolateral sorting signal in its C-terminal cytoplasmic tail (27), and basolateral targeting information
for the Construction of Epitope-tagged and Chimeric mAChRs--
A
modified FLAG epitope (DYKDDDDA) was added to the extracellular N
termini of the M1-M5 mAChR coding sequences
immediately after the initiator methionines using PCR to generate
pFM1, pFM2, pFM3, pFM4,
and pFM5. The mouse M1 (29), porcine
M2 (clone Mc7) (30), human M3 (31), human
M4 (32), and human M5 (31) mAChR cDNAs in
the mammalian expression vector pCDPS (31) were used as templates. For
M1, M2, and M4, the forward primer
encoded the FLAG epitope, and the forward and reverse primers contained unique restriction sites to facilitate subcloning into pCDPS. PCR
fragments were as follows: M1,
KpnI-NheI, nt 1-676 of M1 coding sequence; M2, KpnI-MscI, nt 1-689 of
M2 coding sequence; and M4, SacI-NheI, nt 1-1229 of M4 coding
sequence. The M3 and M5 receptors were
FLAG-tagged using a sequential PCR approach as described (33), with the
FLAG epitope encoded by internal primers. The M3 PCR
product (NcoI-SnaBI) contained 306 nt of pCDPS
vector sequence and nt 1-462 of M3 coding sequence. The
M5 PCR product (NcoI-EcoRI) contained
367 base pairs of pCDPS vector sequence and nt 1-1021 of
M5 coding sequence. PCR products were subcloned into the
parental plasmids to generate epitope-tagged mAChRs. The ability of the FLAG-tagged receptors to bind the muscarinic antagonist
[3H]quinuclidinyl benzilate (47 Ci/mmol; Amersham
Pharmacia Biotech) was verified by transient expression in COS-7 or
JEG-3 cells. The presence of the FLAG epitope was then verified by
immunoprecipitation from transfected cell membranes using the anti-FLAG
M2 monoclonal antibody (Sigma). Studies of FLAG-M2
mAChR-mediated inhibition of adenylyl cyclase, receptor
desensitization, and sequestration have been reported previously
(34).
M2/M3 chimeric mAChRs were constructed using
sequential PCR as described (33) to replace parts of the M2
coding sequence with the homologous regions of M3 coding
sequence as aligned in Ref. 31. pFM2, pFM3, or
M2/M3 chimeric constructs were used as PCR
templates for subsequent chimeras. All PCR-amplified constructs were
engineered with BglII and EcoRI sites at their
5'- and 3'-ends, respectively, and cloned into the BglII and
EcoRI sites of pCDPS. The sequences comprising the
M2/M3 chimeras are as follows, with the numbers
in parentheses representing the amino acid residues of M3
that were substituted into M2:
M2/M3-(240-590), coding nt 1-579 of
M2 and nt 694-1779 of M3;
M2/M3-(486-590), coding nt 1-1143 of
M2 and nt 1456-1779 of M3;
M2/M3-(240-485), coding nt 1-579 of
M2, nt 694-1455 of M3, and nt 1144-1404 of
M2; M2/M3-(384-485), coding nt
1-1014 of M2, nt 1150-1455 of M3, and nt
1144-1404 of M2; M2/M3-(240-383),
coding nt 1-579 of M2, nt 694-1149 of M3, and
nt 1015-1404 of M2;
M2/M3-(240-309), coding nt 1-579 of
M2, nt 694-927 of M3, and nt 793-1404 of
M2; M2/M3-(253-296), coding nt
1-621 of M2, nt 757-888 of M3, and nt
754-1404 of M2; M2/M3-(297-309), coding nt 1-753 of M2, nt 889-927 of M3, and
nt 793-1404 of M2; M2/M3-(253-269), coding nt 1-621 of
M2, nt 757-807 of M3, and nt 673-1404 of
M2; and M2/M3-(266-296), coding nt
1-660 of M2, nt 796-888 of M3, and nt
754-1404 of M2.
Fusion proteins in which M3 sequences were added to either
the i3 loop or the C terminus of M2 were generated by
sequential PCR using pFM2 and either
M2/M3-(266-296) or pFM3 as
templates, respectively. The
M2+M3-(i3:266-296) PCR product was cloned into the BglII and EcoRI sites of pCDPS, whereas the
M2+M3 C-terminal fusion constructs were
subcloned into the MscI and EcoRI sites of the
parental pFM2 plasmid. The sequences comprising the
M2+M3 fusion proteins are as follows:
M2+M3-(i3:266-296), coding nt 1-660 of
M2, nt 796-888 of M3, and nt 661-1404 of
M2; M2+M3-(C-term:266-296), coding
nt 1-1398 of M2 and nt 796-888 of M3;
M2+M3-(C-term:271-296), coding nt 1-1398 of
M2 and nt 811-888 of M3;
M2+M3-(C-term:266-291), coding nt 1-1398 of
M2 and nt 796-873 of M3;
M2+M3-(C-term:271-291), coding nt 1-1398 of
M2 and nt 811-873 of M3; and
M2+M3-(C-term:570-590), coding nt 1-1398 of
M2 and nt 1708-1779 of M3.
The interleukin-2 receptor/M3 fusion protein was generated
by sequential PCR using the human interleukin-2 receptor Cell Culture--
MDCK (strain II) cells were obtained from Dr.
Keith Mostov (University of California, San Francisco, CA). JEG-3 human
choriocarcinoma and COS-7 cells were obtained from American Type
Culture Collection (Manassas, VA). All cell lines were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 100 units/ml penicillin G, and 0.1 mg/ml streptomycin
sulfate at 37 °C in a humidified 10% CO2 environment.
Transfection and Immunocytochemical Analysis of Chimeric mAChR
Constructs--
To analyze the targeting of mAChR constructs, MDCK
cells seeded at near-confluency (3.5 × 105
cells/well) on 2-well glass chamber slides (4.2 cm2/well;
Nalge Nunc International, Naperville, IL) were transfected the
following day using the calcium phosphate precipitation method (36)
with 4 µg of receptor cDNA/well. Cells were fixed at confluence (36-48 h post-transfection) with paraformaldehyde solution (4% (w/v)
paraformaldehyde and 4% (w/v) sucrose in phosphate-buffered saline
(PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.5 mM
KH2PO4), pH 7.4) for 30 min at room temperature
and processed for immunocytochemistry. Fixed cells were rinsed twice
with PBST (PBS containing 0.1% (v/v) Tween 20), permeabilized with
0.25% (v/v) Triton X-100 (in PBS) for 5 min at room temperature, and blocked with 10% (w/v) bovine serum albumin in PBST containing 0.25%
Triton X-100 for 2 h at room temperature. After blocking, cells
were incubated with anti-FLAG M2 (1.2 µg/ml), anti-IL-2R
Quantitation of the apical/basolateral distributions of mAChR
constructs was performed using the public domain NIH Image program (developed at the National Institutes of Health). The mean pixel intensity/unit area (pixel values 0-255) of staining in the apical and
basolateral domains was determined by manually outlining the areas of
interest in the raw (unprocessed) x-z images. Data were processed using Microsoft Excel.
Functional Assays--
Muscarinic receptor-mediated changes in
forskolin-stimulated cAMP levels in transiently transfected JEG-3 cells
were analyzed as described previously (37). Transfection mixtures
contained (per well) 30 ng of receptor cDNA, 25 ng of
N-[3H]Methylscopolamine Binding
Assays--
Cell-surface expression of mAChR constructs in transfected
JEG-3 cells was determined by the binding of
N-[3H]methylscopolamine, a
membrane-impermeable muscarinic antagonist, to intact cells as
previously described (41) with the following modifications.
Transfection mixtures contained (per 100-mm culture dish) 1.2 µg of
receptor cDNA, 1.0 µg of Differential Localization of mAChR Subtypes in MDCK
Cells--
Previous studies demonstrated that mAChRs are
asymmetrically distributed in a variety of polarized cells (16-22).
Despite many observations of mAChR localization, little is known
concerning the cellular mechanisms and molecular signals that underlie
the sorting of mAChRs to specific subcellular domains. To identify sorting signals for mAChR subtypes, we examined their targeting in MDCK
epithelial cells, a widely used and well characterized model system for
protein sorting. For these studies, recombinant mAChRs were FLAG-tagged
at their N termini to enable immunochemical detection. The FLAG-tagged
M1, M2, and M3 receptors were
expressed at levels similar to their non-tagged counterparts when
transfected into COS-7 or JEG-3 cells, whereas the expression of
FLAG-M4 and FLAG-M5 was significantly lower
than that of the non-tagged
receptors.2
For receptor targeting studies, the steady-state distributions of
recombinant mAChRs were analyzed in confluent MDCK cells by
immunocytochemistry and confocal microscopy. The M2 and
M3 receptors displayed reciprocal polarized distributions.
Although M2 was highly enriched on the apical membrane,
M3 was localized to the basolateral domain (Fig.
1, B and C).
Although some basal M3 staining was evident, most
M3 immunoreactivity was restricted to the lateral
subdomain. In contrast, the M1, M4, and
M5 receptors exhibited non-polarized distributions, with
labeling apparent throughout the cells (Fig. 1, A,
D, and E). MDCK cell polarity was verified by
examining the distribution of the endogenous E-cadherin-associated protein The N-terminal Portion of the M3 Third Intracellular
Loop Contains a Basolateral Sorting Signal--
The differential
targeting of the M2 and M3 mAChRs allowed us to
test the feasibility of using receptor chimeras to identify regions of
the receptors important for either apical sorting of M2 or
basolateral targeting of M3. Since basolateral sorting
signals can be dominant over apical signals when present in the same
molecule (4, 10, 44), we analyzed M2/M3
receptor chimeras in a gain-of-function approach to identify regions of
M3 sequence that would confer basolateral targeting to the
otherwise apical M2 receptor. Schematic diagrams of the
initial set of chimeric constructs are presented in Fig.
2. Fig. 3
shows the steady-state localizations of these hybrid receptors. The
first construct, M2/M3-(240-590), contains M3 Phe240-Leu590, encompassing the
C-terminal half of the fifth transmembrane domain (TM5), the i3 loop,
the sixth and seventh transmembrane domains (TM6 and TM7,
respectively), and the C-terminal tail in the context of the
M2 receptor. The M2/M3-(240-590)
chimera displayed a primarily basolateral localization in MDCK cells
similar to wild-type M3 (Fig. 3, B and
C). This result suggests that a region of sequence in the
C-terminal half of M3 is sufficient for basolateral targeting.
We next sought to identify the sequence containing the basolateral
sorting signal by substitution of smaller regions of M3 into the homologous positions of M2. M3
Glu486-Leu590 did not confer basolateral
targeting to M2, with the chimera having an apical
distribution very similar to wild-type M2 (Fig. 3,
A and D). This indicates that the M3
basolateral sorting signal does not lie in TM6 or TM7 or in the
C-terminal tail. M2/M3-(240-485), which
contains the C-terminal half of TM5 and the i3 loop of M3 in the context of M2, did exhibit a primarily basolateral
distribution that was very similar to wild-type M3 (Fig. 3,
B and E), suggesting that the basolateral sorting
signal lies within the M3 i3 loop. Further dissection of
the M3 sequence confirmed this possibility. Substitution of
M3 Phe240-Leu383, containing the
N-terminal half of the i3 loop, into M2 conferred a mainly
basolateral localization to the receptor molecule, although minor
apical staining was also apparent (Fig. 3G). In contrast, substitution of M3 Pro384-Lys485,
comprising the C-terminal half of the i3 loop, did not confer basolateral localization, and this chimera displayed an apical distribution similar to wild-type M2 (Fig. 3, A
and F). This suggests that the M3 basolateral
sorting signal lies in the N-terminal half of the i3 loop. Furthermore,
when M3 Phe240-Gly309 was
substituted into M2, the receptor exhibited a basolateral distribution virtually indistinguishable from wild-type M3
(Fig. 3, B and H). These data indicate that a
70-amino acid region from TM5 and the i3 loop of M3
contains a basolateral sorting signal that is sufficient to redirect
the M2 receptor to the basolateral domain of MDCK cells.
Having identified a region of M3 sequence containing a
putative basolateral sorting signal, we next wanted to test the role of
the transmembrane residues in basolateral targeting. As shown in Fig.
4 (C and D),
M2/M3-(253-296), which contains TM5 from M2, displayed a basolateral localization similar to
wild-type M3, suggesting that transmembrane residues are
not necessary for basolateral targeting. This observation allowed us to
focus on residues located in the N-terminal portion of the
M3 i3 loop to identify a minimal sequence that provides
basolateral targeting information. Since M3
Arg253-Gln296 confers basolateral targeting,
we also tested Gln297-Gly309 for basolateral
sorting activity. As shown in Fig. 4 (B and E), M2/M3-(297-309) exhibited an apical
distribution similar to wild-type M2, suggesting that the
basolateral targeting activity lies within M3
Arg253-Gln296. M3
Arg253-Gln269 did not confer basolateral
targeting to M2 (Fig. 4F), with the chimera
having an apical distribution. However,
M2/M3-(266-296) displayed a primarily
basolateral localization very similar to wild-type M3 (Fig.
4, C and G). These results strongly suggest that
M3 Ala266-Gln296 contains a
basolateral sorting signal that is sufficient to redirect the apical
M2 receptor.
The M3 Sorting Signal Confers Basolateral Targeting to
Both M2 and IL-2R
One important property of basolateral sorting signals is that they are
autonomous, i.e. they can confer basolateral targeting to an
unrelated, heterologous protein. To test whether the M3 basolateral sorting signal acts in an autonomous fashion, we added M3 Ala266-Gln296 to the C terminus
of IL-2R
The above results demonstrate that a 31-amino acid sequence,
Ala266-Gln296, from the N-terminal portion of
the M3 i3 loop contains a basolateral sorting signal in
MDCK cells. In an attempt to further define the basolateral targeting
determinant, we created M2+M3 fusion proteins
in which shorter segments of M3 sequence were fused to the
C-terminal coding residue of M2 to investigate whether
basolateral targeting is lost upon removal of critical amino acids.
Analysis of the steady-state distributions of these fusion proteins
revealed that addition of just 21 residues of the M3 i3
loop to the C terminus of M2 could still redirect it to the
basolateral domain (Fig. 6). Three fusion
proteins, M2+M3-(C-term:271-296),
M2+M3-(C-term:266-291), and
M2+M3-(C-term:271-291), showed a primarily
basolateral localization (Fig. 6, D-F) similar to the
wild-type M3 receptor (Fig. 6C). To exclude the
possibility that addition of any 21-amino acid sequence to the C
terminus of M2 would disrupt its apical targeting and
result in a basolateral distribution, M3
Lys570-Leu590 was fused to the C-terminal
coding residue of M2. This sequence comprises the
C-terminal 21 amino acids of M3, which did not alter the
apical sorting of M2 when included in a substitution
construct (Fig. 3D).
M2+M3-(C-term:570-590) exhibited an apical
distribution very similar to wild-type M2 (Fig. 6,
B and G), indicating that addition of a random
21-amino acid sequence does not disrupt the apical targeting of
M2. These data show that a 21-amino acid peptide, Ser271-Ser291 from the i3 loop of
M3, provides a basolateral sorting signal capable of
rerouting the otherwise apical M2 receptor.
Quantitation of mAChR Apical/Basolateral Distributions--
To
confirm the basolateral targeting results described above, quantitation
of the immunofluorescence signals for selected mAChR constructs in the
apical and basolateral domains of MDCK cells was performed using NIH
Image. As shown in Fig. 7, ~80% of
M3 receptor immunoreactivity was found in the basolateral
domain, similar to the value obtained for the basolateral marker
Functional Analysis of M2/M3
Chimeras--
The sequence containing the M3 basolateral
sorting signal overlaps a region of the i3 loop
(Arg252-Thr272 of rat M3) shown to
be important for functional coupling to the Gq family of G
proteins (45, 46). Therefore, we tested whether addition or
substitution of the M3 basolateral sorting signal into
M2 would either confer M3-like coupling to
Gq or interfere with M2 coupling to the
Gi family of G proteins. Coupling of mAChRs to the
Gq family of G proteins was assessed by examining their ability to activate phospholipase C in response to the muscarinic agonist carbamylcholine (carbachol). In COS-7 cells transfected with
the M3 mAChR, treatment with a maximal concentration of
carbachol (1 mM) led to an ~2-fold stimulation of
phospholipase C activity relative to untreated controls, whereas
carbachol treatment of M2-transfected cells resulted in
only a 1.2-fold increase in phospholipase C activity (2.36 ± 0.21- and 1.24 ± 0.06-fold stimulation of phospholipase C
activity normalized for receptor expression for M3 and
M2, respectively; mean ± S.E., n = 4). Three M2/M3 chimeric receptors were tested: M2/M3-(266-296) (substitution construct),
M2+M3-(i3:266-296), and
M2+M3-(C-term:266-296) (addition constructs to
either the i3 loop or the C-terminal tail of M2,
respectively). None of these chimeras stimulated phospholipase C
activity to a significant extent following treatment with 1 mM carbachol (1.05 ± 0.02-, 1.14 ± 0.07-, and
0.99 ± 0.04-fold stimulation for
M2/M3-(266-296), M2+M3-(i3:266-296), and
M2+M3-(C-term:266-296), respectively; mean ± S.E., n = 4). These data indicate that the
M3 basolateral sorting determinant is not sufficient to
confer Gq coupling to the M2 mAChR.
M2 receptor coupling to the Gi family of G
proteins was assessed in JEG-3 human choriocarcinoma cells by
determination of the carbachol-mediated regulation of expression of a
luciferase reporter gene under the transcriptional control of a
promoter containing a cAMP response element (CRE-luciferase). This
system has been used extensively to measure M2- and
M4-mediated inhibition of forskolin-stimulated adenylyl
cyclase activity and cAMP production (33, 34, 37, 41, 47). Consistent
with previous results (33, 34, 41), the M2 receptor showed
a concentration-dependent inhibition of
forskolin-stimulated CRE-luciferase activity (Fig. 8). In contrast, the M3 mAChR
showed stimulation of CRE-luciferase activity (Fig. 8), presumably due
to the inability of M3 to couple to Gi and to
ectopic coupling of M3 to Gs. Similar results
have been observed previously for the M1 receptor in this
system (33, 41). All three chimeric receptors tested inhibited
forskolin-stimulated CRE-luciferase activity in a
concentration-dependent manner (Fig. 8). Whereas both
substitution and addition of M3 sequence to the M2 i3 loop resulted in inhibition of CRE-luciferase
activity to a similar extent as wild-type M2 (51 ± 4, 57 ± 4, and 58 ± 4% inhibition by
10 The goal of this study was to characterize the molecular
mechanisms and signals involved in the polarized targeting of mAChR subtypes. Examination of the steady-state distributions of mAChRs in
MDCK cells revealed that the M2 and M3
receptors are targeted to opposite domains (Fig. 1). This is the first
demonstration of differential sorting of highly homologous members of a
single G protein-coupled receptor family in MDCK cells. We utilized a gain-of-function approach to identify a basolateral sorting signal in
the M3 receptor by analysis of
M2/M3 chimeric receptor constructs. The use of
chimeras between closely related proteins with opposite phenotypes is
advantageous over studies using deletion or truncation mutagenesis
because it greatly reduces the possibility that a loss of receptor
targeting is due to a generalized effect on protein structure. This
consideration is especially important for polytopic membrane proteins
such as G protein-coupled receptors.
The M3 basolateral sorting signal is contained within a
21-amino acid sequence, Ser271-Ser291, from
the N-terminal portion of the M3 i3 loop. Addition of this signal to the M2 receptor in either the i3 loop or the
C-terminal tail caused M2 to be redirected from the apical
domain to the basolateral domain of MDCK cells, whereas addition of an
irrelevant 21-residue sequence did not alter the apical distribution of
M2. Furthermore, substitution of a 70-amino acid region of
M3 containing the basolateral determinant also conferred
basolateral targeting to the otherwise non-polarized M1
mAChR.3 Together, the data
show that M3 Ser271-Ser291
contains a basolateral sorting signal that acts in a
position-independent manner and is dominant over targeting signals in
other mAChR subtypes. Interestingly, this sequence conferred partial
basolateral targeting when transferred to the apical IL-2R The M3 basolateral sorting signal identified in this study
is sufficient to confer basolateral targeting to other mAChR subtypes. However, an M3 deletion mutant lacking
Ala266-Gln296 displayed a basolateral
distribution virtually identical to that of the wild-type
M3 receptor.2 Thus, the
Ser271-Ser291 domain is not the only region of
M3 that can mediate its basolateral targeting. Our results
suggest that the sequence identified here is the strongest in terms of
conferring basolateral targeting activity to heterologous proteins, but
other elements of the M3 receptor may mediate its
basolateral sorting in the absence of this signal. These other
M3 basolateral sorting elements may not be strong enough to
counteract the apical signals in M2 and so would not be
detected in our experimental approach, but may mediate basolateral
targeting of M3 in the absence of any additional signals. Consistent with this idea, it has been reported that the polarized sorting of other proteins can be mediated by multiple, independent targeting motifs (8, 10, 49).
One common feature of basolateral sorting signals is that they
sometimes overlap with sequences involved in endocytosis from the
plasma membrane (10-12, 44, 50). However, the M3
basolateral sorting signal, which resides in the N-terminal portion of
the i3 loop, does not coincide with known internalization motifs for M3 or the highly homologous M1 receptor, which
are located in the middle and the immediate membrane-proximal portions
of the loop (51, 52). Targeting studies of other proteins have also revealed that basolateral sorting signals can be spatially distinct from endocytosis signals (4, 13, 27, 53, 54).
The M3 basolateral sorting signal has a 3-amino acid
overlap (Ser271-Thr273) with a
membrane-proximal region of the i3 loop implicated in M3
receptor coupling to the phospholipase C pathway via the Gq family of G proteins (45, 46). For this reason, we tested whether the
basolateral targeting motif could also confer coupling to phospholipase
C. M2/M3 chimeras containing the basolateral sorting signal did not stimulate phosphatidylinositol turnover, demonstrating that it is not sufficient for coupling to Gq
proteins. This is not surprising, as there is minimal overlap between
the two motifs, and G protein coupling of mAChRs is thought to be mediated by a multisite domain (46). The basolateral targeting motifs
for other G protein-coupled receptors are also distinct from their
functional G protein-coupling domains (27, 53, 55).
Basolateral targeting signals have now been identified in many
transmembrane proteins. Although no consensus sequence exists, structural determinants such as tyrosine-based (10, 27, 49, 50, 56) or
dihydrophobic (11-13, 49) motifs are often found in basolateral
sorting signals. However, some basolateral targeting sequences act
independently of these motifs (7, 54, 57, 58) or do not contain them at
all (4, 59). The M3 basolateral sorting signal
(Ser271-Ser291) does not contain a critical
tyrosine or a dihydrophobic motif. Thus, the sequence identified here
may represent a novel basolateral targeting determinant. Either the
continuous amino acid sequence itself or a three-dimensional epitope
formed by noncontiguous elements within the sequence may form the
actual signal for basolateral targeting.
Heterologous protein expression studies have suggested that epithelial
cells and neurons use common cellular mechanisms to generate a
polarized distribution of membrane proteins. Based on the sorting of
viral glycoproteins, it was initially proposed that the apical domain
of epithelial cells corresponds to neuronal axons, whereas the
basolateral domain corresponds to cell bodies and dendrites (60).
Although this parallel does not hold true for all proteins (61, 62),
recent studies have confirmed that some basolateral proteins in MDCK
cells are restricted to the somatodendritic domain of cultured
hippocampal neurons and that the same signals used for basolateral
targeting are also likely to mediate somatodendritic targeting (63).
However, proteins that are apical in MDCK cells are not restricted to
the axon, but instead are distributed uniformly throughout the axon and dendrites of cultured hippocampal neurons (63). These and other studies
have suggested the existence of "axon-including" signals, rather
than signals that mediate targeting to the axon exclusively (64). The
differential targeting of the M2 and M3 mAChRs
in MDCK cells (Fig. 1) and neurons in vivo (24, 25) suggests that similar signals may operate to achieve polarized sorting of mAChRs
in epithelial cells and neurons. While the basolateral sorting signals
in M3 may mediate its localization to the somatodendritic domain, the apical targeting information in M2 may allow
its inclusion in axons. It will be of interest to determine whether the
signals that mediate mAChR targeting in MDCK cells are also important for the differential localization of mAChR subtypes in neurons.
In conclusion, we have identified a novel 21-amino acid sequence from
the N-terminal portion of the M3 mAChR i3 loop that mediates basolateral targeting in MDCK cells. This determinant, although not uniquely necessary for the basolateral targeting of
M3, is dominant over apical sorting signals in the
M2 mAChR and can be transferred to a heterologous protein,
IL-2R-chain caused significant basolateral targeting of this heterologous
apical protein. The results indicate that the M3
basolateral sorting signal is dominant over apical signals in
M2 and acts in a position-independent manner. The
M3 sorting signal represents a novel basolateral targeting
motif for G protein-coupled receptors.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
áRESULTS
DISCUSSION
REFERENCES
2A-adrenergic receptor appears to be in a domain
composed of multiple transmembrane sequences (28), sorting information
for G protein-coupled receptors in polarized cells remains largely
unknown. In this report, we used chimeric receptor constructs in a
gain-of-function approach to identify a basolateral sorting signal for
the M3 mAChR in MDCK cells. The M3 basolateral sorting signal lies in a 21-amino acid sequence from the N-terminal portion of the third intracellular (i3) loop, is dominant over apical
signals in the M2 receptor, and can act in a
position-independent manner. This M3 sequence represents a
novel basolateral sorting motif for G protein-coupled receptors.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
áRESULTS
DISCUSSION
REFERENCES
-chain (IL-2R
; Tac antigen) cDNA (pIL2R3; kindly provided by Dr. Warren J. Leonard, National Institutes of Health, Bethesda, MD) (35) and
pFM3 as templates. This fusion protein consists of coding nt 796-888 (Ala266-Gln296) of M3
fused to the C terminus of IL-2R
. Both the fusion protein and
wild-type IL-2R
were cloned into the BglII and
EcoRI sites of pCDPS. PCR-amplified DNA sequences were
verified using an Applied Biosystems Model 373A automated sequencing system.
(1:100;
Upstate Biotechnology, Inc., Lake Placid, NY), or anti-
-catenin (1:100; Transduction Laboratories, Lexington, KY) monoclonal antibody in PBST containing 3% bovine serum albumin and 0.25% Triton X-100 overnight at 4 °C in a humid chamber. Following four washes with PBST, cells were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody (1:250; Cappel Research Products, Durham, NC) in PBST containing 3% bovine serum albumin and 0.25% Triton X-100 for 2-3 h at room temperature. After four more washes with PBST, slides were coverslipped with Vectashield (Vector Labs, Inc., Burlingame, CA). Staining was visualized using a Nikon Optiphot 2 microscope equipped with a 60× Nikon oil immersion objective. Fluorescent images were collected in both the
x-y and x-z planes using a Bio-Rad MRC600 laser scanning confocal microscope. For each
x-y image, a z-series of ~20 optical
sections was taken at 0.7-µm intervals from the apical to the
basolateral regions of the cells. Images were projected and analyzed
using Adobe Photoshop.
168-CRE-luciferase plasmid (38), 40 ng of Rous sarcoma
virus-
-galactosidase plasmid (39), 100 ng of G
i2 (40)
in pCDPS, and 55 ng of pCDPS carrier to achieve a total of 250 ng of
DNA/well. The medium was changed 20-24 h after transfection; cells
were treated with 0.4 µM forskolin and various
concentrations of carbamylcholine (carbachol) an additional 20-24 h
later as described (41) and lysed; and assays of luciferase and
-galactosidase activities were performed (37). Muscarinic receptor-mediated stimulation of phosphatidylinositol hydrolysis was
determined in COS-7 cells as previously described (41) using 5 µg of
receptor DNA/100-mm dish for transfection.
168 CRE-luciferase plasmid, 1.6 µg
of Rous sarcoma virus-
-galactosidase plasmid, 4.0 µg of G
i2, and 2.2 µg of pCDPS carrier to achieve a total of
10.0 µg of DNA/dish. Cells from each dish were subcultured onto one
6-well plate 20-24 h after transfection and allowed to attach for an additional 24 h. N-[3H]Methylscopolamine
binding assays were performed as described (41), except that protein
content was determined by the method of Lowry et al.
(42).
áRESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
áRESULTS
DISCUSSION
REFERENCES
-catenin, a basolateral marker (4, 43).
-Catenin was
exclusively localized to the lateral subdomain (Fig. 1F), indicating that the cells are correctly polarized under our
experimental conditions. These results demonstrate that the
M2 and M3 mAChRs are targeted to opposite
domains of MDCK cells at steady state and suggest that they possess
sorting signals that direct them to distinct subcellular locations.
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Fig. 1.
Differential localization of mAChR subtypes
in MDCK cells. MDCK cells were transfected with cDNA encoding
the FLAG-tagged M1 (A), M2
(B), M3 (C), M4
(D), or M5 (E) mAChR as described
under "Experimental Procedures." Cells were fixed, stained with the
anti-FLAG antibody, and visualized by confocal microscopy.
Untransfected cells were stained with an antibody against the
endogenous basolateral protein -catenin (F). For each set
of images, the upper panel shows images collected in the
x-y plane (projected z-series), and
the lower panel shows a vertical (x-z)
section taken at the level of the white line in the
corresponding x-y image. Bar = 15 µm.
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Fig. 2.
Schematic representation of
M2/M3 receptor chimeras. The
M2 (white) and M3 (black)
mAChRs were used as the parent constructs from which
M2/M3 chimeric receptors were derived as
described under "Experimental Procedures." For each chimeric
construct, M2 sequence is represented in white
and M3 sequence in black. Numbers in
parentheses represent the amino acids of M3 that
were substituted into the homologous region of the M2
receptor.
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Fig. 3.
Localization of M2/M3
chimeric receptors in MDCK cells. MDCK cells transfected with
constructs encoding FLAG-tagged M2, M3, or
M2/M3 receptor chimeras were fixed and stained
with anti-FLAG antibody as described under "Experimental
Procedures." x-y (upper panels;
projected z-series) and x-z
(lower panels; vertical section taken at the level of the
white line) images for each construct are shown.
A, M2; B, M3;
C, M2/M3-(240-590); D,
M2/M3-(486-590); E,
M2/M3-(240-485); F,
M2/M3-(384-485); G,
M2/M3-(240-383); H,
M2/M3-(240-309). Bar = 15 µm.
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Fig. 4.
M3
Ala266-Gln296 contains a basolateral sorting
signal. A, presented is a schematic diagram of
the N-terminal juxtamembrane region of the M3 i3 loop
showing Arg253-Gly309 in single-letter code.
Lines and numbers represent sequences and
specific residues, respectively, of M3 that were
substituted into the homologous regions of M2 to create the
chimeras shown in D-G. AP, apical;
BL, basolateral. B-G, MDCK cells transfected
with constructs encoding FLAG-tagged M2, M3, or
M2/M3 chimeras were stained with anti-FLAG
antibody as described under "Experimental Procedures."
x-y (upper panels; projected
z- series) and x-z (lower
panels; vertical section taken at the level of the white
line) images for each construct are shown. B,
M2; C, M3; D,
M2/M3-(253-296); E,
M2/M3-(297-309); F,
M2/M3-(253-269); G,
M2/M3-(266-296). Bar = 15 µm.
and Is Position-independent--
The
results above show that M3
Ala266-Gln296 contains a signal that, when
substituted into the M2 receptor, can redirect this apical receptor to the basolateral domain of MDCK cells. However, basolateral targeting of the substitution constructs could be due either to addition of a basolateral signal from M3 or to removal of
an apical signal from M2. To distinguish between these
possibilities, M3 Ala266-Gln296
was added either to the homologous position in the i3 loop of M2 (M2+M3-(i3:266-296)) or at the
C terminus, following the last amino acid of the M2 coding
sequence (M2+M3-(C-term:266-296)). When
analyzed by confocal microscopy, both of these receptors showed a
predominantly basolateral distribution similar to wild-type M3 (Fig. 5, B-D).
Thus, the redirection of M2 is due to addition of the
M3 basolateral sorting signal rather than elimination of apical targeting information. Furthermore, the data show that the
M3 basolateral sorting signal acts in a
position-independent manner and is dominant over any apical targeting
information present in the M2 receptor.
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Fig. 5.
The M3 basolateral sorting signal
is position-independent and can be transferred to a heterologous
protein. MDCK cells transfected with constructs encoding
FLAG-tagged M2 (A), M3
(B), M2+M3-(i3:266-296)
(C), or M2+M3-(C-term:266-296)
(D) or with IL-2R (E) or the
IL-2R
/M3 fusion protein (F) were stained with
anti-FLAG antibody (A-D) or with anti-IL-2R
antibody
(E-F) as described under "Experimental Procedures."
x-y (upper panels; projected
z- series) and x-z (lower
panels; vertical section taken at the level of the white
line) images for each construct are shown. Bar = 15 µm.
(Tac antigen), a single-pass transmembrane protein with a
short cytoplasmic tail (35). Consistent with previous results (13),
wild-type IL-2R
had a predominantly apical distribution when
expressed in MDCK cells (Fig. 5E), similar to wild-type
M2 (Fig. 5A). By contrast, a substantial
fraction of the IL-2R
/M3 fusion protein was found in the
basolateral domain, with strong immunoreactivity in the lateral
subdomain, although a significant portion of the fusion protein was
still detected apically (Fig. 5F). Thus, the M3
basolateral sorting signal can at least partially redirect IL-2R
to
the basolateral domain, suggesting that it can confer basolateral
targeting to a heterologous protein.
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Fig. 6.
Localization of M2+M3
C-terminal fusion proteins in MDCK cells. A, presented
is a schematic diagram showing M3 i3 loop
Ala266-Gln296 in single-letter code.
Lines and numbers represent sequences and
specific residues, respectively, of M3 that were fused to
the C-terminal cytoplasmic tail of M2 to create the fusion
proteins shown in D-F. BL, basolateral.
B-G, MDCK cells transfected with constructs encoding
FLAG-tagged M2, M3, or
M2+M3 C-terminal fusion proteins were stained
with anti-FLAG as described under "Experimental Procedures."
x-y (upper panels; projected
Z-series) and x-z (lower
panels; vertical section taken at the level of the white
line) images for each construct are shown. B,
M2; C, M3; D,
M2+M3-(C-term:271-296); E,
M2+M3-(C-term:266-291); F,
M2+M3-(C-term:271-291); G,
M2+M3-(C-term:570-590). Bar = 15 µm.
-catenin (85%), whereas only 30% of M2
immunoreactivity was basolateral, reflecting the predominantly apical
distribution of M2. All M2/M3 chimeras that contain either a substitution or addition of the M3 basolateral sorting signal,
Ser271-Ser291, displayed a basolateral
enrichment of at least 70%, confirming the ability of the sorting
signal to redirect M2 to the basolateral domain. In
contrast, M2/M3 chimeras that lack these amino
acids showed ~30% basolateral immunoreactivity, similar to wild-type M2. Additionally, staining for wild-type IL-2R
was only
~30% basolateral, whereas that for the IL-2R
/M3
fusion protein was 57% basolateral. This result confirms that the
M3 basolateral targeting determinant provides a basolateral
sorting signal to IL-2R
, although in this case, the basolateral
targeting activity is not sufficient to fully override the endogenous
apical signals. These data support the immunocytochemical results
discussed above and confirm the ability of the M3
basolateral sorting signal to cause significant targeting of both
M2 and IL-2R
to the basolateral domain of MDCK
cells.
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Fig. 7.
Quantitation of the steady-state
distributions of mAChR constructs in MDCK cells. Quantitation of
the immunofluorescence signals for selected mAChR constructs and
-catenin in the apical and basolateral domains of MDCK cells was
performed using NIH Image as described under "Experimental
Procedures." Data represent the mean fluorescence intensity per unit
area in the basolateral domain and are expressed as the percentage of
total fluorescence intensity in the apical and basolateral domains.
Data are plotted as the mean of two or the mean ± S.E. of three
to seven images for each construct.
5 M carbachol for
M2, M2/M3-(266-296), and
M2+M3-(i3:266-296), respectively; mean ± S.E., n = 4), addition of M3 sequence to the C terminus of M2
(M2+M3-(C-term:266-296)) resulted in 35 ± 6% inhibition by 10
5 M
carbachol (mean ± S.E., n = 4). This difference
could be due either to reduced functional coupling or to lower
cell-surface expression of the fusion protein compared with wild-type
M2. To distinguish between these possibilities, we
determined the level of each receptor at the cell surface using
N-[3H]methylscopolamine, a
membrane-impermeable muscarinic antagonist. Under the same transfection
conditions used in the functional assay, the
M2/M3 chimeras were expressed at similar levels
(358 ± 17, 381 ± 2, and 356 ± 52 fmol/mg protein for
M2/M3-(266-296), M2+M3-(i3:266-296), and
M2+M3-(C-term:266-296), respectively; mean ± S.E., n = 3), which were slightly higher
than those for wild-type M2 and M3 (215 ± 41 and 261 ± 30 fmol/mg protein, respectively; mean ± S.E.,
n = 3). Therefore, the slightly reduced ability of the
M2+M3-(C-term:266-296) receptor to inhibit
forskolin-stimulated CRE-luciferase activity is likely due to a
slightly reduced efficiency of coupling to G
i2. Taken
together, the data suggest that the M2/M3
chimeric receptors fold correctly, reach the cell surface, and
stimulate a functional response similar to the wild-type M2 mAChR. Thus, addition or substitution of the M3 basolateral
sorting signal does not appear to substantially alter M2
receptor conformation or function.
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Fig. 8.
Inhibition of forskolin-stimulated adenylyl
cyclase activity by M2/M3 chimeric receptors in
JEG-3 cells. JEG-3 cells were transfected with constructs encoding
the M2 ( ), M3 (
),
M2/M3-(266-296) (
),
M2+M3-(i3:266-296) (
), or
M2+M3-(C-term:266-296) (
) receptor and
treated with the indicated concentrations of carbachol as described
under "Experimental Procedures." Data are expressed as the
percentage of forskolin-stimulated luciferase activity measured in the
absence of carbachol and represent the mean ± S.E. of four
experiments performed in triplicate.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
áRESULTS
DISCUSSION
REFERENCES
; although
a substantial fraction of the chimeric molecules were redirected to the
basolateral domain, a detectable fraction remained apical. The
incomplete basolateral targeting of IL-2R
by the M3
basolateral sorting signal suggests that the targeting activity of this
basolateral determinant is not sufficient to completely override the
activity of the apical signals in IL-2R
, although it can completely
counteract the apical signals in M2. Thus, the apical
targeting information in IL-2R
may be stronger than that in
M2, perhaps due either to higher signal strength or to a
greater number of apical targeting determinants. This notion is
consistent with recent suggestions that basolateral sorting signals may
not always be dominant over apical signals and that the overall
targeting phenotype of a protein may be determined by the relative
strength (9) or valence (48) of multiple sorting signals.
Alternatively, we cannot rule out the possibility that incomplete
basolateral targeting of the IL-2R
/M3 fusion protein is
due to saturation of the basolateral targeting pathway and spillover of
excess protein into the apical pathway, perhaps resulting from higher
expression of IL-2R
/M3 as compared with the
M2/M3 chimeras.
, in an autonomous fashion. The findings reported here add
significantly to our knowledge of the signals that underlie the
polarized targeting of G protein-coupled receptors. Future work will be
aimed at further elucidation of the molecular signals and cellular
machinery involved in mAChR sorting in both epithelial cells and neurons.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Keith Mostov for the gift of MDCK cells, Dr. Warren J. Leonard for the gift of pIL2R3 cDNA, Paulette Brunner (Keck Center for Advanced Studies in Neural Signaling, University of Washington) for assistance with confocal microscopy, and Dr. Michael Schlador and Renee Chmelar for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant NS26920 (to N. M. N.) and by a postdoctoral fellowship in pharmacology/morphology from the Pharmaceutical Research and Manufacturers of America Foundation (to L. S. N.).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.
To whom correspondence should be addressed: Dept. of Pharmacology,
University of Washington, P. O. Box 357750, Seattle, WA 98195-7750. Tel.: 206-543-9457; Fax: 206-616-4230; E-mail:
nathanso@u.washington.edu.
Published, JBC Papers in Press, January 2, 2001, DOI 10.1074/jbc.M007190200
2 L. S. Nadler and N. M. Nathanson, unpublished observations.
3 H. A. Iverson, L. S. Nadler, and N. M. Nathanson, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
MDCK, Madin-Darby
canine kidney;
mAChR, muscarinic acetylcholine receptor;
i3 loop, third
intracellular loop;
PCR, polymerase chain reaction;
nt, nucleotides;
IL-2R, interleukin-2 receptor
-chain;
PBS, phosphate-buffered
saline;
CRE, cAMP response element;
TM, transmembrane domain.
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