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From the
We compared the G-protein requirements for coupling of human and
chicken m2 and m4 muscarinic acetylcholine receptors (mAChRs) to
inhibition of adenylyl cyclase, using a luciferase reporter gene under
the transcriptional control of a cAMP response element as a sensitive
monitor of intracellular cAMP levels. Previously, we used this system
to demonstrate that the chick m4 receptor preferentially coupled to
G
Muscarinic acetylcholine receptors (mAChR)
Heterotrimeric GTP-binding regulatory proteins (G-proteins)
couple mAChR to their intracellular effectors and consist of
Previously, we carried out studies using
a luciferase reporter gene under the transcriptional control of a cAMP
response element (CRE) as a sensitive monitor of intracellular cAMP
levels and cAMP-regulated gene expression. We have used this system to
examine both the functional responses of mAChRs (Migeon and Nathanson,
1994) and the function of various wild type and mutated G-protein
Reconstitution studies have provided information on in vitro mAChR-G-protein coupling capabilities. Chick cardiac
(predominantly cm2) mAChR receptors can activate G
In our
previous work, we demonstrated that the chicken m4 mAChR preferentially
coupled to G
We previously showed that G
In
summary, we have shown that the G-protein coupling specificity of the
m2 mAChR is different from that of the m4 receptor. Thus, we have shown
that their apparent functional redundancy is in fact more complicated
and that the two receptors can couple to AC through overlapping but
different sets of G-protein
We are grateful to Dr. Randy Reed (Johns Hopkins
University) for the gift of the rat G
Volume 270,
Number 27,
Issue of July 07, pp. 16070-16074, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
and G
Subunits (*)
ABSTRACT
INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-2 and G
over G-1
and G-3. We found that both the chick and human m2
mAChRs can couple to G-1, G-2,
G-3, and G, while the human m4 mAChR
preferentially couples to G-2 and G.
Both the G and G
forms of the
G
subunit were effective in reconstituting coupling of
the m2 and m4 mAChRs to inhibit adenylyl cyclase activity. The m2 and
m4 mAChRs thus couple to inhibition of adenylyl cyclase by overlapping
but different sets of G-protein subunits.
()are members of the superfamily of G-protein-coupled
receptors characterized by seven putative transmembrane domains. These
receptors regulate intracellular effectors such as ion channels and the
enzymes adenylyl cyclase (AC) and phospholipase C. Five subtypes of
mAChR have been identified; m1, m3, and m5 couple preferentially to
stimulation of phospholipase C, and m2 and m4 couple preferentially to
inhibition of AC. However, the specificity of mAChR functional coupling
is dependent both on levels of receptor expression and on the cell type
in which a given receptor subtype is expressed (Kubo et al.,
1986a, 1986b; Peralta et al., 1987a, 1987b; Bonner et
al., 1987, 1988; Shapiro et al., 1988; Ashkenazi et
al., 1989; Tietje et al., 1990; Tietje and Nathanson,
1991).
,
, and subunits. The
subunit is unique for each
G-protein and contains the site of GTP binding and hydrolysis as well
as sites for receptor and effector interaction. There are several
classes of G-proteins, defined by their subunits. The best
characterized are G
and G, named for their
abilities to stimulate and inhibit AC, respectively. Several forms of
and subunits have also been identified. Both the
and
subunits can regulate the activity of various effector
proteins (see Simon et al.(1991) and Spiegel et
al.(1992) for review).
subunits (Migeon et al., 1994). We found that the chick m4
(cm4) receptor had a surprising specificity for G-protein coupling: it
could use G-2 and G but not
G-1 or G-3 to mediate inhibition of
AC activity. In this study, we have compared the G-protein-coupling
requirements of human and chicken m2 and m4 subtype mAChRs. As m2 and
m4 muscarinic receptors both couple to inhibition of AC, we wanted to
determine whether m2 and m4 mAChRs required different G-protein
subunits in order to mediate inhibition of AC. Because only the m2
subtype of mAChR is expressed in the human heart while both m2 and m4
are expressed in the chicken heart, and because species-specific
differences in the coupling of receptors to G subtypes
have been observed (Jockers et al., 1994), we also compared
the chicken and human receptors. It has been shown that muscarinic and
somatostatin receptor coupling to a Ca current in
GH
cells is exquisitely sensitive to G-protein ,
, and subunit composition (Kleuss et al., 1991,
1992, 1993). The mAChR and the somatostatin receptors use
G
and G
,
respectively, to mediate inhibition of the Ca current. In this report, we also examined G
-mediated
inhibition of AC by mAChR to determine whether individual mAChR
subtypes require specific G subunits.
DNA and Expression Vectors
The chick m4 mAChR
genomic clone (Tietje et al., 1990), the chicken m2 mAChR
(Tietje and Nathanson, 1991), the human m2 mAChR (Bonner et
al., 1988), the human m4 (Bonner et al., 1988), the rat
G-1 (Jones and Reed, 1987), the rat
G-2 (Jones and Reed, 1987), the rat G
(Jones and Reed, 1987), the mouse G-1 (Strathmann et al., 1989), and the mouse G-2 (Strathmann et al., 1990). G-protein subunit cDNAs were expressed in
the expression vector pCD-PS (Bonner et al., 1988). The human
G-3 (Beals et al., 1987) cDNA was expressed
in the expression vector pNUT (Palmiter et al., 1987). The
-inhibin CRE-luciferase plasmid consisted of a CRE containing
74-base pair BstXI(-170) to NcoI(-96)
fragment from the -inhibin promoter (Pei et al., 1991)
blunt end ligated into the TK-105 luciferase plasmid. The
constitutively active RSV--galactosidase construct (Edlund et
al., 1985) has been described previously (Day et al.,
1989).
Cell Transfection and Culture
JEG-3 cells were
obtained from the American Type Culture Collection and were grown in
Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) supplemented with 10% fetal calf serum. Transient transfection of
JEG-3 cells and subsequent assays of luciferase and -galactosidase
activity were carried out as described elsewhere (Migeon and Nathanson,
1994). Cells seeded at 2.5 10
cells/15-mm well were
transiently transfected with between 200 and 300 ng total DNA by
calcium phosphate precipitation 72 h after plating. The transfection
mixes consisted of 15 ng/well of the -inhibin CRE-luciferase gene
construct, 40 ng/well of RSV--galactosidase, to normalize for
transfection efficiency, and the indicated amounts of receptor or
G-protein cDNAs. Carrier DNA was used to ensure that all transfections
within a given experiment have a constant amount of total DNA. The
medium was replaced 24 h after transfection and cells were treated with
the appropriate drug another 24 h later. Triplicate wells were treated
with various drugs for 5 h before harvesting of cells.
Assay of Luciferase Activity
After removal of
media, transfected cells were harvested by solubilization in 100 µl
of extraction buffer (100 mM KPO, 4 mM ATP, 1.5 mM MgSO, 1 mM dithiothreitol, 0.1% Triton X-100). 25 µl of cell extract were
added to 350 µl of luciferase assay buffer (100 mM KPO, 4 mM ATP, 1.5 mM
MgSO) in luminometer cuvettes. The luminometer injects 100
µl of D-luciferin (1 mM; Analytical Luminescence
Laboratories, Inc.) into each sample and determines luminescence over
30 s. Luciferase counts were divided by -galactosidase values (see
below) to determine normalized luciferase activity.
Assay of
25 µl of
cell extract were added to microtiter plate wells containing 200 µl
of -Galactosidase Activity-galactosidase assay buffer (60 mM NaPO,
10 mM KCl, 1 mM MgCl, 50 mM
-mercaptoethanol). 40 µl of the -galactosidase substrate o-nitrophenyl--galactopyranoside (2 mg/ml; Calbiochem)
were added to each well, and plates were colorimetrically assayed for
-galactosidase activity.
Determination of mAChR Expression Levels in Transient
Transfections
The level of mAChR expression in transiently
transfected cells was determined as described previously (Migeon and
Nathanson, 1994). In brief, the number of mAChR binding sites was
determined by the binding of the membrane-impermeable ligand
[H]NMS to intact cells, and the fraction of
transfected cells in the culture was estimated by in situ staining for -galactosidase activity with X-gal. The
transfection-specific level of [H]NMS binding was
divided by the percentage of transfected cells to yield the level of
extrapolated level of mAChR expression.
Immunoblot Analysis of G-protein
Expression
Immunoblot analysis of G-protein expression was
carried out as described previously (Migeon and Nathanson, 1994; Migeon et al., 1994).
Expression of mAChR and G-proteins in Transfected
Cells
The expression of mAChR and G-proteins in transiently
transfected JEG-3 cells has been described previously (Migeon and
Nathanson, 1994; Migeon et al., 1994). Transfection with the
G-protein expression vectors has been previously shown to result in
similar increases in the levels of each of the subunits (Migeon
and Nathanson, 1994; Migeon et al., 1994). The levels of cm2,
cm4, human m2 (hm2), and hm4 were quantitated by the binding of
[H]NMS to intact cells, and dividing the level of
specifically bound [H]NMS by the transfection
efficiency, as determined by cytochemical staining for
-galactosidase activity due to the cotransfected
-galactosidase expression vector. The extrapolated levels of
receptor expression were similar for all four receptors, in the range
of 5-12 pmol/mg of protein. As discussed previously (Migeon and
Nathanson, 1994), because the cytochemical staining probably
underestimates the actual fraction of transfected cells in the
cultures, these calculations probably overestimate the true levels of
receptor expression.
Chicken m2 and m4 mAChR Preferentially Couple to
Different G-proteins
We used a
luciferase reporter gene under the transcriptional control of a CRE to
measure intracellular cAMP levels and cAMP-regulated gene expression in
transiently transfected JEG-3 cells. This system allows detection of
physiologically relevant changes in intracellular cAMP levels without
requiring the presence of phosphodiesterase inhibitors or
supraphysiological concentrations of forskolin. In addition, it allows
measurement of responses following cotransfection of clones encoding
receptors and G-proteins in transiently transfected cells without
confounding effects due to the presence of untransfected cells in the
cultures. Previously we have used the JEG-3 cell CRE luciferase
expression assay to characterize cm4 mAChR-mediated inhibition of
forskolin-stimulated AC activity. We found that in JEG-3 cells, which
express G Subunits to Inhibit AC-1 and G-3 but not
G-2 or G (Montmayeur et al.,
1993), transiently transfected cm4 mAChR required coexpression of
either G-2 (Migeon and Nathanson, 1994) or
G (Migeon et al., 1994) in order to see
mAChR-mediated inhibition of CRE-driven luciferase expression. As both
the cm2 and cm4 can couple to inhibition of AC, we wanted to determine
which G-proteins couple the cm2 mAChR to this response. We found that,
while the cm4 mAChR preferentially couples to G-2 and
G (Fig. 1B), the cm2 receptor can
couple to G-1, G-2,
G-3, and G (Fig. 1A).
Thus, the cm2 receptor has a more promiscuous pattern of G-protein
coupling specificity than the cm4 receptor. The optimal EC values for carbachol for the cm2 and cm4 receptors are 1-2
10
M and 4-5
10
M, respectively. This is consistent with
previous work which demonstrated that, when stably expressed in either
CHO or Y1 cells, the cm4 receptor exhibited a higher sensitivity for
carbachol than the cm2 receptor (Tietje et al., 1990; Tietje
and Nathanson, 1991).
Figure 1:
Chicken m2 and m4 preferentially couple
to different sets of G-protein subunits for inhibition of
forskolin-stimulated AC activity. Control (open circles),
G-1 (filled circles), G-2 (open squares), G-3 (filled
squares), and G (filled triangles)
expression vectors (100 ng/well) were cotransfected with chicken m2 (A) (15 ng/well) or m4 (B) mAChR expression vector
(10 ng/well), -inhibin luciferase reporter gene (15 ng/well), and
RSV--galactosidase gene (40 ng/well). Transfected cells were
treated with 0.316 µM forskolin and varying concentrations
of carbachol. Data are shown as -fold increases in normalized
luciferase activity and values are means ± S.E., n = 3.
Human m2 and m4 mAChR Exhibit Different
G-protein Coupling Specificities for Inhibition of AC-The m2
mAChR is the only subtype expressed to a significant extent in
mammalian heart (Luetje et al., 1987; Peralta et al.,
1987a), while both the m2 and m4 mAChR subtypes are present in chicken
heart (Tietje and Nathanson, 1991). Species-specific differences in the
coupling of the human and bovine A1 adenosine receptors to G-proteins
have been recently reported (Jockers et al., 1994). We
compared the G-protein coupling patterns of the human and chicken m2
and m4 mAChR in order to determine whether species differences between
the receptor subtypes may account for the differing patterns of
receptor expression in mammalian and avian heart. We found no
differences in the G-protein coupling requirements between the human
and chicken receptors. G-1, G-2,
G-3, and G can all couple the hm2
mAChR to inhibition of AC (Fig. 2A), while the human m4
receptor requires G-2 or G (Fig. 2B). Thus, the differences in mAChR expression in
human and chicken heart do not result from different patterns of
G-protein coupling. The optimal EC values for carbachol
for the hm2 and hm4 receptors are 3-4
10
M and 3-4
10
M, respectively. The human and chick receptors are
therefore also similar in the increased functional sensitivity of the
m4 receptor compared to the m2 receptor.
Figure 2:
Human m2 and m4 preferentially couple to
different sets of G-protein subunits for inhibition of
forskolin-stimulated AC activity. Control (open circle),
G-1 (filled circles), G-2 (open squares), G-3 (filled
squares), and G (filled triangles)
expression vectors (100 ng/well) were cotransfected with human m2 (A) or m4 (B) mAChR expression vector (10 ng/well),
-inhibin luciferase reporter gene (15 ng/well), and
RSV--galactosidase gene (40 ng/well). Transfected cells were
treated with 0.316 µM forkolin and varying concentrations
of carbachol. Data are shown as -fold increases in normalized
luciferase activity and values are means ± S.E., n = 3.
We have previously
demonstrated that, in both transiently transfected and stably
transfected cells, the cm4 receptor can increase intracellular cAMP due
to ectopic coupling to the stimulatory G-protein G and
subsequent activation of adenylyl cyclase (Migeon and Nathanson, 1994;
Dittman et al., 1994). In the absence of transfected
G-proteins, cm4, hm2, and hm4 all mediate an increase in luciferase
expression ( Fig. 1and Fig. 2); as demonstrated previously,
expression of the appropriate G-protein subunits converts this
stimulation to inhibition. Interestingly, the cm2 receptor does exhibit
little if any stimulation of CRE-luciferase when expressed at a similar
level as the other receptors (Fig. 1A and 3A).
Both the G
There are two forms of G-1 and G-2
Forms of the G Subunit Can Couple m2 and m4 mAChR to
Inhibition of AC,
G-1 and G-2, which arise from
differentially splicing of a single gene. Inhibition of a
Ca current in GH3 cells by the m4 receptor has been
reported to be blocked by antisense oligonucleotides corresponding to
G
-1 but not G-2 (see below). The
experiments using G in Fig. 1and Fig. 2used rat G-1. In order to determine
whether mAChR inhibition of AC in JEG-3 cells requires a specific
G form, cells were transiently transfected with
combinations of human and chicken mAChR and either of the two
G subtypes (Fig. 3A-D). Because
rat G-2 is not available, we used cDNA clones
corresponding to murine G-1 and G-2.
Both the human and chick m2 and m4 receptors couple equally well to
both subtypes of G, thus demonstrating that both forms
of G can couple to either the m2 or the m4 receptor to
mediate inhibition of AC in JEG-3 cells.
Figure 3:
m2 and m4 mAChR can couple to both
G1 and G2 forms of G for
inhibition of forskolin-stimulated AC activity. Control DNA (open
circles), G-1 (filled circles), and
G-2 (filled squares) expression vectors (100
ng/well) were cotransfected with chicken m2 (A), chicken m4 (B), human m2 (C), or human m4 (D) mAChR
expression vector (A, 15 ng/well; B-D, 10
ng/well), the -inhibin luciferase reporter gene (15 ng/well), and
the RSV--galactosidase gene (40 ng/well). Transfected cells were
treated with 0.316 µM forkolin and varying concentrations
of carbachol. Data are shown as -fold increases in normalized
luciferase activity and values are means ± S.E., n = 3.
Diverse methods have been
used to examine muscarinic receptor G-protein coupling. These
techniques include reconstitution of purified proteins in lipid
vesicles and expression of exogenous receptors and G-proteins in well
characterized cell lines. Pertussis toxin (PTX) treatment,
subunit antisense oligonucleotides, and agonist-dependent subunit
labeling have also been used for determination of in situ G-protein coupling. PTX was one of the first tools used to study
the G-proteins involved in mAChR-mediated responses, particularly
inhibition of AC. While PTX treatment abolishes m2 and m4 mAChR
inhibition of AC (Shapiro et al., 1988; Peralta et
al., 1988; Jones et al., 1991), it does not distinguish
between the individual PTX-sensitive G-proteins. Transfected m2 mAChRs
were shown to couple to both G-2 and
G-3 in CHO cells (Dell'Acqua et al.,
1993) and to G-1, G-2, and
G-3 in transfected 293 cells (Offermanns et
al., 1994).
(Richardson et al., 1991), and activation of
reconstituted mammalian recombinant m2 receptors stimulates the binding
of the nonhydrolyzable GTP analog GTPS to G
-1,
G-3, G, and G
subunits (Tota et al. 1990; Parker et al., 1991).
Similarly, G-1, G-2, and
G-3 allow reconstitution of coupling of the m2 mAChR
to activation of the atrial inwardly rectifying K current. (Yatani et al., 1988). Thus, our results are
consistent with a variety of results demonstrating that the m2 receptor
can activate the entire PTX-sensitive G-protein family.
-2 and G (Migeon and
Nathanson, 1994; Migeon et al., 1994). Here we expand on those
studies by examining coupling of the chicken m2 mAChR to inhibition of
AC. We found that the m2 mAChR can couple to a larger set of G-protein
subunits, using G-1, G-2,
G-3, and G to inhibit AC. Thus while
m2 and m4 mAChR can couple to inhibition of AC, they can do so by
coupling to overlapping but different sets of G-protein subunits.
While the JEG-3 cells express endogenous G-1 and
G-3, they appear not to express enough of these
G-proteins to allow m2 mAChR-mediated inhibition of
forskolin-stimulated CRE-luciferase without cotransfection with
G-protein cDNA. In contrast to the difference in G-protein coupling
seen between the human and bovine A1-adenosine receptors (Jockers et al., 1994), both the m2 and m4 receptors exhibit identical
G-protein requirements between the human and chick homologues. While
the reason for the difference in subtype expression between mammalian
and avian hearts remains unclear, the presence of multiple subtypes in
chick heart raises the possibility that there may be differential
control of expression of receptor subtypes that would allow a higher
level of regulatory complexity in chick compared to mammalian heart.
-1 could couple the m4
receptor to inhibition of AC in JEG-3 cells (Migeon et al.,
1994). Before our report, the only direct evidence of mAChR functional
coupling through G was in GH cells where mAChRs
mediate inhibition of a Ca current. Injection of
GH
cells with antisense oligonucleotides against the
G-1 form of G abolished
mAChR-mediated inhibition of a Ca current (Kleuss et al., 1991), while injection of antisense oligonucleotides
against the G
-2 form did not. Specific forms of
and subunits are also required (Kleuss et al., 1992,
1993). We tested m2 and m4 coupling to G
-1 and
G-2. We found that chicken and human m2 and m4
receptors could couple equally well to the G-1 and
G-2 forms of G to inhibit
forskolin-stimulated CRE-luciferase activity. Thus, in contrast to the
mAChR-mediated Ca current in GH
cells,
there is no subtype specificity for mAChR inhibition of AC through
G in JEG-3 cells. These differences in coupling
specificities may be related to differences either in the
subunit composition between the two cell types or the regulatory
properties of calcium channels compared to adenylyl cyclase.
subunits. We have also shown that
mAChR-G-protein coupling is conserved across species and that different
patterns of G-protein coupling cannot explain differences in mAChR
expression in chicken and human cardiac tissue. Finally, we have shown
that m2 and m4 inhibition of AC in JEG-3 cells can utilize either
G-1 or G-2. The use of the
CRE-luciferase reporter gene system in JEG-3 cells has proved very
useful for examination of mAChR-G-protein coupling capabilities and has
given a greater understanding of possible receptor subtype function.
-galactopyranoside; RSV, Rous sarcoma
virus; CHO, Chinese hamster ovary; GTPS, guanosine
5`-3-O-(thio)triphosphate.
-1,
G-2, and G G-protein subunit
cDNAs, Drs. Roger Perlmutter and Chan Beals (University of Washington)
for the gift of the human G-3 cDNA, Dr. Kelly Mayo
(Northwestern University) for the gift of the -inhibin
CRE-luciferase reporter gene construct, Dr. Melvin Simon (University of
California, San Francisco) for the gift of the murine
G-1 and G-2. Dr. Tom Bonner (National
Institute of Mental Health) for the gift of the human m2 and m4 mAChR
clones and the expression vector pCD-PS. We also thank Michael Schlador
for his help in setting up some of these experiments and Dr. Stan
McKnight for his kind help and advice.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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