0022-3565/08/3253-947–953$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics
JPET 325:947–953, 2008
Vol. 325, No. 3
135095/3340704
Printed in U.S.A.
Muscarinic M4 Receptor Recycling Requires a Motif
in the Third Intracellular Loop
Yuichi Hashimoto, Kanoko Morisawa, Hiroyuki Saito, Eri Jojima, Norihiro Yoshida,
and Tatsuya Haga
Institute for Biomolecular Science, Faculty of Science, Gakushuin University, Tokyo, Japan
Received December 5, 2007; accepted March 11, 2008
Prolonged exposure to agonists of G protein-coupled receptors (GPCRs) usually results in attenuation of cellular responses (desensitization). One molecular mechanism of desensitization involves the phosphorylation of the receptors by
G protein-coupled receptor kinases (GRKs) and increased
binding of the inhibitory protein ␤-arrestin to the phosphorylated receptors, thereby inhibiting their coupling with G
proteins (Claing et al., 2002). Another mechanism is the
internalization of receptors by which they become inaccessible to hydrophilic, membrane-impermeable ligands, including agonists. The internalization of receptors occurs via
clathrin-coated vesicles, caveolae, or noncoated vesicles
(Feron et al., 1997; Pals-Rylaarsdam et al., 1997; Vögler et
al., 1999). After internalization, some GPCRs can be recycled
back to the plasma membrane as functional receptors to
allow resensitization of desensitized receptors. Receptor inThis work was supported by the Ministry of Education, Culture, Sports,
Science, and Technology of Japan (Grants-in-Aid for Scientific Research on
Priority Area 15083207 to T. H.).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.107.135095.
recycled after agonist removal, and the mutant M4del-V373A393 was also internalized to half the extent of the wild type but
was not recycled back to the cell surface after agonist removal.
When the sequence corresponding to Val373-Ala393 was grafted
onto the i3 portion of a recycling-negative mutant of muscarinic
M2 receptor with deletion of almost the whole of the i3 sequence, approximately 40% of the chimeric receptor on the cell
surface was internalized, and more than 65% of the internalized
receptors were recycled back to the cell surface. These results
indicate that the regions including Leu272-Arg338 and Val373Ala393 are involved in internalization of the M4 receptor, and the
region including Val373-Ala393 is indispensable for its recycling,
whereas the other regions of i3 are dispensable for internalization and recycling.
ternalization may also be an intermediate step preceding
degradation in the lysosomes (down-regulation).
The muscarinic acetylcholine receptor (mAChR) family
consists of five subtypes and can be subdivided into two
functional groups: M1, M3, and M5 subtypes, which couple to
the Gq family of G proteins; and the M2 and M4 subtypes,
which couple to the Gi family of G proteins (Caulfield and
Birdsall, 1998). It has been reported that M4 receptors undergo internalization via clathrin-coated pits after activation
by agonist and a large proportion of internalized M4 receptors can be recycled back to the plasma membrane after
agonist removal (Bogatkewitsch et al., 1996), whereas M2
receptors, which are functionally and structurally homologous to M4 receptors, undergo internalization via a nonclathrin pathway and a small proportion of internalized M2 receptors is recycled (Vögler et al., 1998; Delaney et al., 2002).
The molecular mechanism of mAChR desensitization remains unclear, although several reports have indicated the
involvement of various proteins, such as GRK, arrestin,
clathrin, dynamin, and Rab GTPases in the internalization
and recycling of muscarinic receptors (Pals-Rylaarsdam et
ABBREVIATIONS: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; mAChR, muscarinic acetylcholine receptor; i3,
third intracellular loop; CHO, Chinese hamster ovary; NMS, N-methylscopolamine; PBS, phosphate-buffered saline; ORF, open reading frame;
PCR, polymerase chain reaction; HEK, human embryonic kidney; CCh, carbamylcholine.
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ABSTRACT
The present study was performed to identify sequence(s) in the
third intracellular loop (i3) of the muscarinic acetylcholine receptor M4 subtype (M4 receptor) involved in its internalization
and recycling. In transiently transfected human embryonic kidney 293-tsA201 cells, 40 to 50% of cell-surface M4 receptors
are internalized in an agonist-dependent manner, and approximately 65% of internalized receptors are recycled back to the
cell surface after removal of the agonist. We examined the
internalization and recycling of M4 receptor mutants with partial
deletion in i3 and found that various mutants (M4del-K235-K240,
M4del-T241-K271, and M4del-W339-N372) showed internalization
and cell-surface recycling in a similar manner to the M4 receptor. We also found that the mutant M4del-L272-R338 was internalized to only half the extent of the M4 receptor and was
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Hashimoto et al.
Materials and Methods
Materials. N-[3H]Methylscopolamine ([3H]NMS, 80 Ci/mmol) was
purchased from GE Healthcare (Chalfont St. Giles, UK), the mammalian expression vector pcDNA3 was from Invitrogen (Carlsbad, CA),
Dulbecco’s phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl,
8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.5) was from Sigma-Aldrich
(St. Louis, MO), and restriction enzymes were from Toyobo Corp.
(Osaka, Japan). A cDNA of human muscarinic acetylcholine receptor
M4 subtype (M4 receptor) was provided by Dr. T. I. Bonner (National
Institutes of Health, Bethesda, MD), a cDNA of human muscarinic
acetylcholine receptor M2 subtype (M2 receptor) was provided by Dr. W.
Sadée (University of California, San Francisco, CA).
Construction of Mammalian Expression Vectors. The open
reading frames (ORFs) of human muscarinic acetylcholine receptor
M4 and M2 subtypes were subcloned into pcDNA3 (Invitrogen) using
BamHI/NotI and EcoRI/NotI sites, respectively. The M4 receptor
consists of coding nucleotides 1 to 1440. An M4 receptor mutant (Fig.
1) with deletion of most of the i3 (M4del-i3 ⫽ M4del-K235-A393) was
generated by overlap extension polymerase chain reaction (PCR).
The deleted region corresponds to coding nucleotides 703 to 1179.
One pair of primers [P1, 5⬘-GTCAC TTTGC GCTCC CGCGG GCCCT
CGGGC CGGTG CTTG-3⬘, (1196 –1180) ⫹ (702– 681); P2, 5⬘-TACGG
TGGGA GGTCT ATATA AGCAG AGCTC-3⬘, ⫺123 to ⫺94] was used
to amplify the DNA containing both coding nucleotides [(1–702) ⫹
(1180 –1196)] and the 5⬘-upstream region sequences (⫺123 to ⫺1) of
Fig. 1. Schematic representation of the i3 regions of various mutants of
M4 and M2 receptors and chimeric mutants between M4 and M2 receptors. Black, M4 receptor sequence; gray, M2 receptor; white, deleted
regions of sequences.
the M4 receptor ORF. The second pair of primers [P3, 5⬘-ATGGC
TGGCA ACTAG AAGGC ACAGT CGAGG-3⬘, 1542–1513; P4, 5⬘GAGGG CCCGC GGGAG CGCAA AGTGA CACGA ACG-3⬘, (694 –
702) ⫹ (1180 –1203)] was used to amplify the DNA containing both
coding nucleotides [(694 –702) ⫹ (1180 –1440)] and 3⬘-downstream
region sequences (1441–1542) of the M4 receptor ORF. Primer P4
was designed to have 26 bases exactly complementary to primer P1.
The two sets of primers were used in two separate PCRs to amplify
overlapping DNA fragments. The overlapping fragments were mixed
and used as the template for the third round of PCR, in which a
full-length DNA encoding the M4 receptor mutant, M4del-K235-A393,
was generated using two primers (P2 and P3). The resulting DNA
fragment was excised with BamHI and NotI and ligated into the
BamHI/NotI sites of pcDNA3. Expression constructs of a series of M4
receptor mutants with a partial deletion in i3 were generated by the
same method as described for construction of the M4del-K235-A393
expression vector, although overlapping sequence lengths of the first
and second PCR products varied from 21 to 37 bases. To construct a
chimeric mutant of the M2 receptor with the i3 of the M4 receptor,
designated M2(M4i3)M2, the ORF of the M2 receptor cDNA was
subcloned into the HindIII/EcoRI site of pUC18 to produce a vector
designated pUC-m2. To replace the majority of the i3 of the M2
receptor (Arg211-Thr388, coding nucleotides 631-1164) with the corresponding region of the M4 receptor (Leu220-Thr401, coding nucleotides 658 –203), silent mutations were introduced to generate an
NruI site and an MluI site at 5⬘- and 3⬘-terminal region of M2-i3 in
pUC-m2, respectively. The cDNA encoding M4 receptor (Leu220Thr401) was amplified by PCR using primers with NruI and MluI
sites at their termini and inserted into pUC-m2 to produce a cDNA
encoding M2(M4i3)M2, and the resulting construct was designated
pUC18-M2(M4i3)M2. The cDNA encoding full-length M2(M4i3)M2
was excised and subcloned into pcDNA3. A chimeric mutant of the
M2 receptor with the carboxyl-terminal half of M4-i3, in which a
region of M2-i3 (Ser283-Thr388) was replaced by the corresponding
region of M4-i3 (Ser297-Thr401), was generated as follows. The cDNA
encoding the amino-terminal half of M2-i3 (Arg211-Ser283) was amplified by PCR using two primers with NruI and SacI sites at their
5⬘ termini, respectively, and ligated into the NruI/SacI sites of
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al., 1997; Lee et al., 1998; Vögler et al., 1999; Schlador et al.,
2000; Delaney et al., 2002). Little is known about which
domain(s) of receptors participate in endocytic trafficking,
especially in endocytic recycling, although the third intracellular loop (i3) was suggested to be involved in agonist-dependent internalization of M1 and M2 receptors (Maeda et al.,
1990; Goldman et al., 1996; Tsuga et al., 1998). i3 is thought
to be indispensable for agonist-induced internalization of M2
receptors, because with deletion of i3, internalization of the
M2 receptor mutant was abolished in CHO cells (Tsuga et al.,
1998). In the case of M2 receptors, i3 presents sites for
phosphorylation by GRK2 in an agonist-dependent manner,
and its phosphorylation facilitates M2 receptor internalization (Pals-Rylaarsdam and Hosey, 1997). On the other hand,
little is known about the role of M4 receptor i3 in internalization and recycling, with the exception of the report by Van
Koppen et al. (1994) in which deletion of a large portion of i3
had little effect on its internalization.
In the present study, we screened for motif(s) in i3 of M4
receptors involved in agonist-dependent internalization and
recycling and found a sequence that is indispensable for its
recycling. This motif is completely distinct from sequences
reported to be indispensable for recycling of other GPCRs
(Gage et al., 2001; Kishi et al., 2001; Tanowitz and von
Zastrow, 2003; Vargas and von Zastrow, 2004; Paasche et al.,
2005). The location of this motif, i3 of the M4 receptor, is also
distinct from those of previously identified motifs, all of
which reside in the carboxyl-terminal tails of the receptors.
To the best of our knowledge, results in the present study
provide the first evidence for the presence of a sequence in
mAChR, which is functionally similar to “the recycling motif”
but structurally distinct from the one reported for other
GPCRs. The M4 receptor is known to be a target for antiparkinsonian drugs. Detailed knowledge on desensitization/resensitization of the M4 receptor may be useful for drug treatment of the disease, understanding of tolerance to drugs, and
development of new drugs.
Motif Required for M4 Muscarinic Receptor Recycling
Results
Muscarinic M4 or M2 receptors were transiently transfected into HEK293-tsA201 cells and subjected to agonistdependent internalization by exposure to 100 ␮M carbamylcholine. Forty to 50% of cell-surface M4 receptors underwent
internalization with t1/2 of 16 ⫾ 2.0 min (mean ⫾ S.E.) and
65% of internalized M4 receptors recycled back to the cell
surface after agonist removal (Fig. 2A). Under essentially the
same conditions, approximately 90% of cell-surface M2 receptors underwent internalization, and approximately 33% of
Fig. 2. Agonist-induced internalization of M4 and M2 receptors and their
recycling induced by removal of agonist. HEK293-tsA201 cells were transiently transfected with one of the genes encoding wild-type M4 receptor
(M4 WT; A), wild-type M2 receptor (M2 WT; B), i3-deleted M4 receptor
(M4del-i3; C), or i3-deleted M2 receptor (M2del-i3; D) and were treated
with CCh (100 ␮M) for the times indicated. After an induction period of
60 min, CCh was washed out, and the cells were further incubated in
fresh medium with (closed symbols) or without (open symbols) CCh (100
␮M) for the times indicated. The amounts of M2 or M4 receptors on the
cell surface were measured using the membrane-impermeable ligand,
[3H]NMS, as described under Materials and Methods. The results in each
panel are shown as percentages of the levels observed in untreated cells.
The data shown in each panel are means ⫾ S.D. of two to four independent experiments, each performed in triplicate or quadruplicate. Absolute
values of specific and nonspecific binding of [3H]NMS per well in 24-well
plate for untreated cells are as follows: A, specific, 4513 ⫾ 1278 dpm
(mean ⫾ S.E.) and nonspecific, 191 ⫾ 20 dpm; B, 3611 ⫾ 281 and 269 ⫾
21 dpm; C, 3492 ⫾ 897 and 209 ⫾ 25 dpm; and D, 3902 ⫾ 1142 dpm and
187 ⫾ 5 dpm. Statistical analyses were performed using one-way ANOVA
with a Newman-Keuls test. ⴱⴱⴱ, significantly different from the value at
60 min, p ⬍ 0.001.
internalized M2 receptors recycled back to the cell surface
after agonist removal (Fig. 2B).
The role of i3 in internalization and recycling was examined for both M2 and M4 receptors using mutants with deletion of i3 of M4 and M2 receptors. We have prepared an M4
receptor mutant with deletion of most of i3 (M4del-K235-A393
or M4del-i3), in which only 18 and 8 amino acid residues at
the amino- and carboxyl-terminal junctions of i3 are retained, respectively (Fig. 1). Affinities of CCh for wild-type
M4 receptor (M4 WT) and M4del-i3 were estimated from the
displacement by CCh of [3H]NMS binding and were shown to
be similar to each other (Fig. 3). The IC50 values of CCh for
M4 WT and M4del-i3 were estimated to be 29 ⫾ 4.1 (mean ⫾
S.E.) and 21 ⫾ 0.72 ␮M (mean ⫾ S.E.), respectively, and
there was no significant difference in these two values.
M4del-i3 showed significant internalization with a t1/2 of
21.8 ⫾ 1.8 min (mean ⫾ S.E.), although the proportion of
internalized receptors (20 –30%) decreased to approximately
half of that of the M4 WT (Fig. 2, A and C). In contrast, an M2
receptor mutant with deletion of most of i3 (M2del-S234-R381
or M2del-i3), in which 26 and 7 amino acid residues at the
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pUC18-M2(M4i3)M2. The resulting plasmid was excised and ligated
into the pcDNA3 vector and designated pcDNA3-M2(M2i3(208 –
283)-M4i3(298 – 401))M2. An M2 receptor mutant with deletion of
most of i3 (deletion of residues from Ser234 to Arg381) (M2del-S234R381 ⫽ M2del-i3) was generated by the same method as described for
construction of M4del-K235-A393. The deleted region of M2del-i3 was
identical to that of the i3-deleted M2 mutant described previously
(Tsuga et al., 1998). Chimeric mutants of the M2 receptor [M2(M4(S298A393))M2 and M2(M4(V373-A393))M2], with M4i3 (Ser298-Ala393) or M4i3
(Val373-Ala393) in place of M2i3 were prepared from pcDNA3M2(M2i3(208 –283)-M4i3(298 – 401))M2 with three steps of PCR as described for preparation of M4del-K235-A393.
Nucleotide sequences of all constructs were confirmed by DNA sequencing using a 3100-Avant Genetic Analyzer (Applied Biosystems,
Foster City, CA). All plasmids were amplified and purified by using
QIAfilter Plasmid Midi Kits (QIAGEN GmbH, Hilden, Germany).
Cell Culture. Human embryonic kidney (HEK) 293-tsA201 (Margolskee et al., 1993) cells were cultured in Dulbecco’s modified Eagle’s medium/F-12 (Invitrogen) supplemented with 10% fetal bovine
serum (Invitrogen), 100 ␮g/ml penicillin, and 100 U/ml streptomycin
(Invitrogen) at 37°C in a 5% CO2 environment.
Transfection of Mammalian Expression Vectors and Radioligand Binding Assay. HEK293-tsA201 cells (approximately 50 –
60% confluence) were transiently transfected with muscarinic receptor expression vectors on 10-cm-diameter dishes using Lipofectamine
2000 (Invitrogen) essentially according to the manufacturer’s instructions. The amounts of DNA used ranged from 0.25 to 6 ␮g/dish.
Twenty-four hours after transfection, the cells were plated (2 ⫻ 105
cells/well) onto 24-well culture dishes coated with poly-D-lysine (BD
Biosciences, Franklin Lakes, NJ). Forty-eight hours after transfection, the cells were stimulated with carbamylcholine (CCh; final, 100
␮M; Sigma-Aldrich) for various periods up to 180 min and then used
for examination of receptors on the cell surface. For the recycling
experiments, the cells were incubated with 100 ␮M CCh for 1 h and
then washed with PBS supplemented with 0.8 mM CaCl2 and 0.33
mM MgCl2 [PBS(⫹)] followed by incubation in CCh-free medium for
various periods. After incubation, the cells were washed with PBS(⫹)
and incubated on ice for 2 h with 1 nM [3H]NMS in HEPES-buffered
Dulbecco’s modified Eagle’s medium/F-12 (Invitrogen) in the presence or absence of 1 ␮M atropine sulfate (Wako Pure Chemical
Industries, Osaka, Japan) to measure nonspecific and total binding,
respectively. After incubation with [3H]NMS, cells were washed with
icecold PBS(⫹) three times and lysed in 0.25 ml of 1% (v/v) Triton
X-100 (Nacalai Tesque, Kyoto, Japan) and mixed with 3.5 ml of
liquid-scintillation cocktail [70% (v/v) toluene (Nacalai Tesque), 30%
(v/v) Triton X-100, 0.4% (w/v) 2,5-diphenyloxazole (Sigma-Aldrich),
and 0.01% (w/v) 1,4-bis-2-(methyl-5-phenyloxazolyl)benzene (Wako
Pure Chemical Industries)] (Tsuga et al., 1998), and the radioactivity
of the suspension was measured using a liquid scintillation counter.
Triplicate or quadruplicate measurements were performed for each
point. Internalization and recycling of receptors were estimated as
the decrease and increase, respectively, of binding sites for the membrane-impermeable muscarinic ligand, [3H]NMS (Galper et al.,
1982).
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Hashimoto et al.
amino- and carboxyl-terminal junctions of i3 are retained,
respectively (Fig. 1), underwent internalization with a longer
t1/2 of 47.2 ⫾ 4.0 min (mean ⫾ S.E.) (Fig. 2D). This value for
t1/2 was approximately 5-fold higher than that for internalization of the wild-type M2 receptor (M2 WT), which was
8.5 ⫾ 1.2 min (Fig. 2B). Little recycling of internalized receptors after agonist removal was observed for either M2del-i3
or M4del-i3, in contrast to M2 and M4 receptors (Fig. 2, C and
D). Figure 4 shows effects of carbamylcholine concentrations
on the proportion of internalized wild-type M4 receptors (Fig.
4A) and M4 receptor mutants with deletion of most of i3 (Fig.
4B). There was no significant difference in the potency of
carbamylcholine to induce internalization between M4 WT
[half-maximal value of 5.36 ⫾ 0.73 ␮M (mean ⫾ S.E.)] and
M4del-i3 [half-maximal value of 4.32 ⫾ 0.97 ␮M (mean ⫾
S.E.)].
To determine whether the region in i3 is involved in agonist-dependent internalization and recycling of M4 receptors,
we constructed various M4 receptor mutants with partial
deletion in i3 (Fig. 1) and examined their internalization and
recycling. All of these M4 mutants (Fig. 5A, M4del-K235-K240;
Fig. 5B, M4del-T241-K271; Fig. 5C, M4del-L272-R338; Fig. 5D,
M4del-W339-N372; Fig. 5E, M4del-V373-A393; Fig. 5F, M4delS298-A393) underwent internalization in an agonist-dependent manner. The proportion of internalized receptors did
not change significantly for M4del-K235-K240, M4del-T241K271, or M4del-W339-N372 but was reduced to approximately
half for M4del-L272-R338, M4del-V373-A393, and M4del-S298A393 compared with wild-type M4 receptors. These results
indicate that the regions including Lys235-Lys240, Thr241Lys271, and Trp339-Asn372 are dispensable for internalization
of M4 receptors, but the regions including Leu272-Arg338 and
Val373-Ala393 are involved in their internalization.
On the other hand, recycling was observed for M4 receptor
Fig. 4. Effect of carbamylcholine concentrations on internalization of
wild-type M4 receptor and M4 receptor mutant with deletion of most of i3.
HEK293-tsA201 cells were transiently transfected with one of the genes
encoding wild-type M4 receptor (M4 WT; A) or i3-deleted M4 receptor
(M4del-i3; B) and were treated with indicated concentrations of CCh for
60 min and then subjected to radioligand binding assay as described
under Materials and Methods. The results in each panel are shown as
percentages of the levels observed in untreated cells. The data shown in
each panel are means ⫾ S.D. of three independent experiments, each
performed in triplicate. Absolute values of specific and nonspecific binding of [3H]NMS per well in 24-well plate for untreated cells are as follows:
A, specific, 2592 ⫾ 245 dpm (mean ⫾ S.E.) and nonspecific, 190 ⫾ 3 dpm;
and B, 6907 ⫾ 996 and 195 ⫾ 2 dpm.
mutants, M4del-K235-K240, M4del-T241-K271, M4del-L272R338, and M4del-W339-N372 (Fig. 5, A–D), but not for M4delV373-A393 or M4del-S298-A393 (Fig. 5, E and F). The level of
internalization was 25 to 60% for the former mutants and 20
to 25% for the latter. It is possible that a certain proportion
of internalized receptors needs to be reached before it enters
a recycling pathway. This possibility, however, is not likely to
be the case because the significant recycling of M4del-L272R338 receptors with 23% internalization was observed,
whereas no significant recycling of M4del-i3 receptors with
30% internalization was observed (Fig. 2C). In the former
case, we took advantage that the level of internalization is
decreased by the increase in expression level (data not
shown). These results indicate that the recycling capability
depends on particular sequence in i3, but not in the level of
internalization, and that a sequence from Val373 to Ala393 is
required for the recycling of M4 receptors.
Next, we examined whether the partial sequences of M4-i3,
Val373-Ala393 or Ser298-Ala393, are sufficient to enable recycling of the recycling-negative receptor. The whole or partial
i3 sequences of M4 receptors were incorporated into M2deli3, which shows a low level of internalization at a slow rate
(Fig. 2D). All chimeric mutants, M2(M4i3)M2, M2(M4(V373-
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Fig. 3. Displacement by carbamylcholine of [3H]NMS binding to whole
cells expressing wild-type M4 receptor and M4 receptor mutant with
deletion of most of i3. HEK293-tsA201 cells were transiently transfected
with one of the genes encoding wild-type M4 receptor (M4 WT; F) or
i3-deleted M4 receptor (M4del-i3; f) and were incubated with [3H]NMS
on ice 2 h in the absence or presence of indicated concentrations of CCh
with or without atropine sulfate. After incubation, the amounts of
[3H]NMS bound to cells were measured as described under Materials and
Methods. The results are shown as percentages of the binding in the
absence of CCh as 100%. The data shown are means ⫾ S.D. of three
independent experiments, each performed in duplicate. Absolute values
of specific and nonspecific binding of [3H]NMS per well in 24-well plate in
the absence of CCh are as follows: M4 WT, specific, 835 ⫾ 31 dpm
(mean ⫾ S.E.) and nonspecific, 220 ⫾ 22 dpm; an M4del-i3, 2731 ⫾ 84
dpm and 242 ⫾ 34 dpm.
Motif Required for M4 Muscarinic Receptor Recycling
A393))M2, and M2(M4(S298-A393))M2, were found to internalize to a similar extent to the M4 receptors, and the internalized receptors were recycled after removal of agonist (Fig. 6,
A–C). The proportions of recycled receptors were 100, 65.8 ⫾
5, and 70.7 ⫾ 3.5% for M2(M4i3)M2, M2(M4(V373-A393))M2,
and M2(M4(S298-A393))M2, respectively. The values for these
chimeric mutants are similar to or greater than those for the
M4 receptor. These results indicate that a sequence, Val373Ala393, confers the ability to undergo recycling on M2del-i3.
Discussion
Muscarinic acetylcholine receptors as well as ␣2A-2C adrenergic, dopaminergic D2– 4, and serotonergic 5HT1A receptors
possess a long i3 with more than 150 residues, but the func-
Fig. 6. Enhancement of internalization and recycling of i3-deleted M2
receptors by incorporating all or partial i3 sequences of the M4 receptor
into the i3 portion of the M2 receptor mutant with deletion of most of i3.
All or partial i3 sequences (Ser298-Ala393 and Val373-Ala393) of the M4
receptor were incorporated into the i3 portion of the M2 receptor mutant
with deletion of i3. Resulting M2-M4 chimeric receptors were designated
M2(M4i3)M2 (A), M2(M4(S298-A393))M2 (B), and M2(M4(V373-A393))M2
(C), respectively. The genes encoding these receptors were transfected
into HEK293-tsA201 cells, and the internalization and recycling experiments were performed as described in the legend to Fig. 2. The results in
each panel are shown as percentages of the levels observed in untreated
cells. The data shown in each panel are means ⫾ S.D. of three independent experiments, each performed in triplicate or quadruplicate. Absolute
values of specific and nonspecific binding of [3H]NMS per well in 24-well
plate for untreated cells are as follows: A, specific, 4976 ⫾ 716 dpm
(mean ⫾ S.E.) and nonspecific, 292 ⫾ 30 dpm; B, 2163 ⫾ 308 and 205 ⫾
11 dpm; and C, 1207 ⫾ 150 and 217 ⫾ 4 dpm. Statistical analyses were
performed using one-way ANOVA with a Newman-Keuls test. ⴱⴱ and ⴱⴱⴱ,
significantly different from the value at 60 min; ⴱⴱ, p ⬍ 0.01; and ⴱⴱⴱ, p ⬍
0.001.
tional role of these long i3 in receptors has not been fully
elucidated. Only membrane-proximal regions in the i3 of M1
and M2 muscarinic receptors are known to be required for
the interaction with G proteins (Maeda et al., 1990; Kameyama et al., 1994; Wess et al., 1997), and most of the central
part of i3 of M1, M2, and M3 muscarinic receptors can be
deleted without losing ligand-binding ability or G proteinactivating activity (Shapiro and Nathanson, 1989; Moro et
al., 1993; Kameyama et al., 1994). On the other hand, the
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Fig. 5. Effects of partial i3 deletion on agonist-induced internalization of
M4 receptors and recycling induced by agonist removal. HEK293-tsA201
cells were transiently transfected with one of the genes encoding M4
receptor mutants with partial deletion of i3. Experiments were performed
as described in the legend to Fig. 2. The results in each panel are shown
as percentages of the levels observed in untreated cells. The data shown
in each panel are means ⫾ S.D. of two to five independent experiments,
each performed in triplicate or quadruplicate. Absolute values of specific
and nonspecific binding of [3H]NMS per well in 24-well plate for untreated cells are as follows: A, specific, 2115 ⫾ 996 dpm (mean ⫾ S.E.) and
nonspecific, 202 ⫾ 122 dpm; B, 8653 ⫾ 5483 and 291 ⫾ 49 dpm; C, 1616 ⫾
192 and 204 ⫾ 95 dpm; D, 2751 ⫾ 262 and 274 ⫾ 93 dpm; E, 2465 ⫾ 208
and 252 ⫾ 55 dpm; and F, 3200 ⫾ 1447 and 238 ⫾ 9 dpm. Statistical
analyses were performed using one-way ANOVA with a Newman-Keuls
test. ⴱ and ⴱⴱ, significantly different from the value at 60 min; ⴱ, p ⬍ 0.05;
and ⴱⴱ, p ⬍ 0.01.
951
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Hashimoto et al.
of two internalization pathways, the latter mutant with a
deletion in Leu272-Arg338 is thought to be internalized
through the i3-independent pathway and to be recycled back
to the cell surface through the i3-dependent pathway because
the mutant with deletion of most of i3 does not undergo
recycling. It is likely that the sequence Val373-Ala393 is responsible for recycling of receptors internalized through either the i3-dependent or -independent pathway. This was
supported by the observation that insertion of the M4 sequence (Val373-Ala393) into an i3-deleted M2 receptor mutant
enabled the resulting chimeric receptor to be internalized
and recycled back to the cell surface in an agonist-dependent
manner. The simplest interpretation of these results is that
the sequence Val373-Ala393 is necessary and sufficient for
internalization and recycling of the M2-M4 chimeric receptor, although the possibility remains that other regions, such
as the first and second intracellular loops and carboxyl-terminal tail, or amino- or carboxyl-terminal regions of i3, are
also involved.
M4 receptor mutants with deletions in various regions of i3
were expressed at approximately the same level as the wildtype M4, bound to muscarinic agonists or antagonists, and
were internalized in an agonist-dependent manner. These
results are not consistent with the suggestion that the central part of i3 forms a rigid structure that is responsible for
internalization but are consistent with the idea that it adopts
a flexible structure with short segments that are responsible
for internalization and recycling. Recently, the i3 of M2 receptor has been shown to have a flexible structure with no
secondary structure (Ichiyama et al., 2006). It is reasonable
to assume that the i3 of M4 also has a flexible structure.
It is interesting that the identified recycling sequence
Val373-Ala393 within the i3 of the M4 receptor has no homology to previously identified signal sequences present at the
extreme carboxyl terminus or in a distal portion of the carboxyl-terminal tail of several GPCRs (Gage et al., 2001; Kishi
et al., 2001; Tanowitz and von Zastrow, 2003; Vargas and von
Zastrow, 2004; Paasche et al., 2005). The regulatory mechanism of recycling of GPCRs with a large i3 may be different
from that of GPCRs, the recycling of which is regulated by
the carboxyl-terminal tail of the receptor.
It will be reasonable to assume that the sequence, Val373Ala393, interacts with some protein(s), thereby modulating
receptor recycling. One of possible candidates for interaction
with the sequence is ␤-arrestin 1 (Vögler et al., 1999). We
examined the effects of wild-type and dominant-negative
(V53D) ␤-arrestin 1 on agonist-dependent internalization
and recycling. Overexpression of either V53D ␤-arrestin 1 or
wild-type ␤-arrestin 1 did not appreciably affect recycling or
internalization of wild-type M4 receptor, although more than
severalfold of expression of ␤-arrestin 1 proteins were confirmed by Western blot analysis (data not shown). Dominantnegative (V54D) ␤-arrestin 2 (Ferguson et al., 1996) also did
not affect internalization of wild-type M4 receptor. Another
candidate for interaction with the sequence might be elongation factor 1A, which was reported to interact with i3 of M4
receptors (McClatchy et al., 2002) and inhibit recycling of M4
receptors (McClatchy et al., 2006). It remains to be elucidated
whether elongation factor 1A may interact with the region,
Val373-Ala393, and compete with the putative protein(s) that
interact with the region and facilitate the recycling.
In the present study, we identified a novel recycling signal
Downloaded from jpet.aspetjournals.org at ASPET Journals on July 31, 2017
central part of i3 appears to be responsible for regulation of
receptors. The i3 of M2 has been shown to be the site for
phosphorylation (Nakata et al., 1994) and for interaction
with G␤␥ (Wu et al., 1998); i3 of M3 has been shown to be the
site for interaction with proteins, such as ␤-arrestin, G␤␥,
casein kinase 1␣, and SET (Wu et al., 1997, 2000; Budd et al.,
2000; Simon et al., 2006); i3 of M1 has been shown to be the
site for interaction with regulators of G protein signaling
(Bernstein et al., 2004); and i3 of M4 has been shown to be
the site for interaction with elongation factor (McClatchy et
al., 2002).
It has been suggested that the i3 is involved in agonistdependent internalization of M1, M2, M3, and M4 muscarinic
receptors (Lameh et al., 1992; Lee and Fraser, 1993; Moro et
al., 1993; Van Koppen et al., 1994; Goldman et al., 1996;
Tsuga et al., 1998). Consistent with previous work (Tsuga et
al., 1998), i3 deletion of M2 receptors was shown to impair its
rapid internalization (Fig. 2D). Van Koppen et al. (1994)
reported that deletion of a large portion of i3 (residues from
Glu264 to Arg394) decreased the rate and proportion of M4
receptor internalization. Our findings are essentially consistent with their results (Fig. 2C), although the proportions of
internalized M4 or i3-deleted M4 receptors were different
between the two studies. This may be due to differences in
the cells used. The results of both the present study and those
reported by Van Koppen et al. (1994) indicate that M4 receptors can internalize without most of i3 in contrast to M2
receptors, which are hardly internalized without most of i3.
Without most of i3, M4 receptors are internalized with a
similar potency of CCh (Fig. 4), but the proportion of internalization was reduced to approximately half of that observed for M4 receptors with i3 (Fig. 2), and no recycling was
observed (Fig. 2). M4del-i3 as well as M4 WT has the ability
to interact with G proteins, Go (data not shown), and then the
apparent decrease in internalization of M4del-i3 is not due to
the loss in G protein-activating ability followed by the decreased availability of G␤␥ subunit and the reduction of
GRK2 activity. Thus, using a series of M4 receptor mutants
with partial deletions of i3, we searched for portions of the i3
that are involved in its agonist-dependent internalization
and recycling. We found that the proportion of internalized
receptors did not change appreciably for mutants with deletion in Lys235-Lys240, Thr241-Lys271, or Trp339-Asn372 and
decreased to approximately half for mutants with deletion in
either Leu272-Arg338 or Val373-Ala393 as well as for a mutant
with deletion of most of i3 (Lys235-Ala393). These results are
consistent with the idea that several types of mechanism are
active in M4 receptor internalization: an i3-independent
mechanism and an i3-dependent mechanism. Based on this
assumption, the i3-independent internalization does not require most part of i3, and the i3-dependent internalization
requires the two regions in i3 [(Leu272-Arg338) and (Val373Arg393)] but not the other regions in i3 [(Lys235-Lys271) and
(Trp339-Asn372)].
The recycling of internalized receptors after agonist removal was abolished for an M4 receptor mutant with a deletion in Val373-Ala393, indicating that the sequence (Val373Ala393) is necessary for recycling of internalized receptors.
On the other hand, appreciable recycling was observed for an
M4 receptor mutant with a deletion in Leu272-Arg338, indicating that the sequence (Leu272-Arg338) is not required for
recycling of internalized receptors. Based on the assumption
Motif Required for M4 Muscarinic Receptor Recycling
sequence present in the i3 of the M4 receptor. To our knowledge, this is the first report of a recycling sequence present in
the i3, although it has already been reported that a tyrosine
residue in the i3 is involved in the recycling of mouse neurotensin type 2 receptor, which has a short i3 of 56 amino acid
residues (Martin et al., 2002). Our study provides a clue to
the molecular mechanism of mAChR desensitization/resensitization. It also strengthens the emerging concept that
GPCR recycling is a regulated process using a short segment
in intracellular sites of receptors. Further studies are required to identify a cytoplasmic protein that interacts with
this signal sequence and facilitates M4 receptor recycling.
Acknowledgments
We thank S. Hirasaka for preparing some constructs.
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Address correspondence to: Dr. Yuichi Hashimoto, Institute for Biomolecular Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan. E-mail: [email protected]
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