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(Received for publication, May 3,
1995; and in revised form, June 15, 1995) From the
We used solution hybridization, immunoprecipitation, and
immunoblot analyses to examine the developmental expression of chicken
m2 (cm2), cm3, and cm4 muscarinic acetylcholine receptor (mAChR) mRNA
and protein in embryonic and post-hatched chick heart and retina in
order to correlate developmental expression patterns with known
physiological events. cm2 is the predominant mAChR subtype expressed in
chick heart. cm3 and cm4 protein and mRNA expression is very low in
chick heart, and cm3 expression is highest early in development. The
decrease in cm3 expression correlates well with the developmental
decrease in mAChR-mediated activation of phospholipase C. cm4 is the
predominant mAChR subtype expressed in chick retina. The expression of
both cm4 protein and mRNA is highest early in development and decreases
as development progresses. cm2 and cm3 mAChR are expressed at
approximately equivalent levels and have similar patterns of
expression. The cm2 and cm3 protein levels increase throughout
development, while cm2 and cm3 mRNA levels peak at embryonic day 15 and
then decrease after hatching. Our data indicate that the three mAChR
subtypes are differentially regulated in chick heart and retina and
that the patterns of expression of mAChR may be important in the
development and physiology of these tissues.
Muscarinic acetylcholine receptors (mAChR) ( Muscarinic receptors undergo several changes in the developing chick
heart (reviewed in Nathanson(1989)). Atria from 3-4-day chick
embryos display a reduced responsiveness to the negative chronotropic
effects of muscarinic agonists even though the density of mAChR is
similar to that found in the atria of older embryos (Pappano and
Skowronek, 1974; Galper et al., 1977, Renaud et al.,
1980). The onset of the negative chronotropic response coincides with
structural and functional changes in G Several studies have shown
developmental changes in the density and molecular weight of the mAChR
during retinal development in chick embryos. Sugiyama et
al.(1977) reported a 30-fold increase in the concentration of
mAChR binding sites between E6 and E14, followed by a small decrease in
receptor binding sites after hatching. Autoradiographic studies of
radiolabeled mAChR showed two bands in the inner plexiform layer in E13
retina and three bands in the inner plexiform layer of adult chicken
retina (Sugiyama et al., 1977). SDS-polyacrylamide gel
electrophoresis analysis has shown two species of mAChR are present in
embryonic chick retina, 86 and 72 kDa (Large et al., 1985); it
was not known if these two molecular mass species were derived from
distinct genes or posttranslational modifications of a single gene
product. The larger form is predominant earlier in development, and
after synaptogenesis the smaller form is predominant. It has been
suggested that a secreted factor is involved in regulating the switch
in expression from the larger to the smaller species (Skorupa and
Klein, 1993). In this study we demonstrate that the expression of
cm2, cm3, and cm4 mAChR subtypes and their mRNAs in embryonic chick
heart and retina is differentially regulated throughout the development
of these tissues. The unique pattern of expression of each mAChR
subtype may play a role in the regulation of development of the
embryonic chick heart and retina.
Figure 1:
Immunoprecipitation demonstrating
specificity of mAChR antisera . Purified anti-cm2, anti-cm3, and
anti-cm4 antibodies were tested for specificity by the
immunoprecipitation assay described under ``Materials and
Methods.'' Receptors were solubilized from membranes of Y1 cells
stably expressing cm2 or cm4 and from membranes of COS-7 cells
transiently expressing cm3. Each antibody was used at a final
concentration of 6 µg/ml. Data are presented as percentage of cm2,
cm3, or cm4 mAChR immunoprecipitated from the total mAChR population in
Y1-cm2 cells, Y1-cm4 cells, or COS-7-cm3 cells. Values are mean
± S.D., n = 2-4. Solidbars, cm2; stripedbar, cm3; shadedbars, cm4.
Figure 2:
Immunoprecipitation assay of the
expression of cm2, cm3, and cm4 mAChR protein in embryonic and P7 chick
heart. Receptors solubilized from membranes of chick atria (A), ventricle (V), or whole heart (H) at
various stages of development were used in the immunoprecipitation
assay with purified anti-cm2 (a), anti-cm3 (b), and
anti-cm4 (c) antibodies. Antibodies were used at a final
concentration of 6 µg/ml. The data are presented as percentage of
cm2, cm3, or cm4 mAChR immunoprecipitated from the total mAChR
population in the chick heart tissues indicated. Values are mean
± S.E., n = 4-10. Statistically
significant differences between ventricle samples in panelb are denoted by asterisks (two-tailed t test: *, statistically different from 6V, p < 0.01;**,
statistically different from 9V, p <
0.05).
Figure 3:
Immunoprecipitation assay of the
expression of cm2, cm3, and cm4 mAChR protein in embryonic and P7 chick
retina. Receptors solubilized from membranes of chick retina (R) at several stages of development were used in the
immunoprecipitation assay with purified anti-cm2 (a), anti-cm3 (b), and anti-cm4 (c) antibodies. Antibodies were
used at a final concentration of 6 µg/ml. The data are presented as
percentage of cm2, cm3, or cm4 mAChR immunoprecipitated from the total
mAChR population in chick retina. Values are mean ± S.E., n = 5-11.
Figure 4:
Solution hybridization assay measuring the
expression of cm2, cm3, and cm4 mRNA in embryonic and P7 chick heart.
Total nucleic acids were extracted from chick atria (A),
ventricle (V), or whole heart (H) at various stages
of development and hybridized with subtype-specific riboprobes for cm2 (a), cm3 (b), and cm4 (c) in the solution
hybridization assay. The data are presented as the number of molecules
of cm2, cm3, or cm4 mRNA expressed per cell in chick heart. Values are
mean ± S.E., n = 3-12. Statistically
significant differences between atria and ventricle samples in panelb are denoted by asterisks (two-tailed t test: *, statistically different from 6A, p <
0.01;**, statistically different from 9A, p <
0.01).
Like cm2 mRNA expression, cm4 mRNA expression does not vary greatly
between atria and ventricle or over the course of embryonic development
except for a general decrease after E4 (Fig. 4c). cm4
mRNA was found to be expressed at 1-2.5 molecules/cell,
5-10 times lower than cm2 mRNA expression. The expression of cm3
mRNA was very low, with the mRNA levels being highest early in
development. As shown in Fig. 4b, cm3 mRNA is at its
highest level at E4 heart and E6 atria, with the difference in cm3 mRNA
levels between atria and ventricle at E6 and E9 being significant (p < 0.01). Our results suggest that the mAChR-stimulated
inositol phosphate production, which is greatest in E4 heart and
decreases as development progresses (Orellana and Brown, 1985; Barnett et al., 1990), is consistent with the pattern of expression of
the cm3 receptor shown here.
Figure 5:
Solution hybridization assay measuring the
expression of cm2, cm3, and cm4 mRNA in embryonic and P7 chick retina.
Total nucleic acids were extracted from chick retina (R) at
various stages of development and hybridized with subtype-specific
riboprobes for cm2 (a), cm3 (b), and cm4 (c)
in the solution hybridization assay. The data are presented as the
number of molecules of cm2, cm3, or cm4 mRNA expressed per cell in
chick retina. Values are mean ± S.E., n =
5-12.
Figure 6:
Immunoblot analysis of expression of mAChR
in transfected cell lines. Membranes were prepared from transfected
cell lines, and SDS gel electrophoresis was performed as described
under ``Materials and Methods.'' Antibodies were used at a
final concentration of 1.2 µg/ml. a, anti-cm2; b,
anti-cm3; c, anti-cm4. Membrane proteins were loaded in the
following order: Y1 cells (lane1), Y1 cells
expressing cm2 (lane2), Y1 cells expressing cm4 (lane3), COS-7 cells (lane4),
COS-7 cells expressing cm3 (lane5).
Figure 7:
Immunoblot analysis of expression of mAChR
in chick heart and retina. Membranes were prepared from chick tissues,
and SDS gel electrophoresis was performed as described under
``Materials and Methods.'' Antibodies were used at a final
concentration of 1.2 µg/ml. a, anti-cm2; b,
anti-cm3; c, anti-cm4. Membrane proteins were loaded in the
following order: E15 ventricle (lane1), E9 retina (lane2), E15 retina (lane3), P7
retina (lane4), E15 skeletal muscle (lane5).
We show here that the expression of cm2, cm3, and cm4 mAChR
subtypes is differentially regulated during embryonic development of
the chick heart and retina. While the mammalian heart only expresses
the m2 receptor (Peralta et al., 1987), chick heart expresses
at least three subtypes of mAChR. Both immunoprecipitation and solution
hybridization analyses indicate that cm2 is the predominant subtype of
mAChR expressed at the protein and mRNA levels. During embryonic
development the expression levels of cm2 protein and mRNA do not vary
greatly. However, P7 ventricle shows a large increase in cm2 mRNA
expression compared with P7 atria or embryonic atria and ventricle.
This large increase in cm2 mRNA is likely to be responsible for the
increase in mAChR binding sites in ventricle in newly hatched chicks
(Sullivan et al., 1985; Hosey et al., 1985). The
level of cm4 expression in embryonic and P7 chick heart is low compared
with that of cm2, in agreement with Northern blot analysis by Tietje
and Nathanson(1991), which showed that cm2 is more highly expressed
than cm4 in embryonic day 18 (E18) chick heart. The pattern of
expression of cm4 protein and mRNA is fairly constant in atria and
ventricle over the course of development. However, the expression of
cm4 may be important functionally during the development of cardiac
tissues expressing both cm2 and cm4 because these receptors have
different functional sensitivities: cm4 expressed in both CHO cells and
Y1 cells is more sensitive to carbachol than cm2 with respect to the
inhibition of adenylyl cyclase (Tietje et al., 1990; Tietje
and Nathanson, 1991). The third mAChR subtype expressed in chick
heart is cm3. The cm3 mRNA is expressed at higher levels early in
development. The expression of cm3 mRNA is greater in atria compared
with ventricle, in agreement with studies by Gadbut and Galper(1994),
which showed by RNase protection analysis that cm3 expression is higher
in E17 atria than in E17 ventricle. The cm3 receptor protein expression
is also highest early in development. These data indicate that the
mAChR-stimulated phosphoinositide hydrolysis in embryonic chick heart,
a response that is highest in E4 hearts and decreases in both atria and
ventricle as development progresses (Orellana and Brown, 1985; Barnett et al., 1990), is most likely mediated by the cm3 receptor.
Unlike the mammalian cardiac mAChR, both cm2 and cm4 exhibit high
affinity for pirenzepine and AF DX-116 (Tietje et al., 1990;
Tietje and Nathanson, 1991). The presence of cm3 in chick heart may
explain the mAChR-stimulated phosphoinositide hydrolysis in chick
heart, which is relatively insensitive to blockade by pirenzepine
(Brown et al., 1985), since cm3 expressed in Chinese hamster
ovary cells has low affinity for pirenzepine (Gadbut and Galper, 1994).
Muscarinic agonists can produce not only inhibitory but also
stimulatory effects on the heart. In chick heart, it has been shown
that the stimulatory action is mediated by a mAChR with low affinity
for pirenzepine (Portas, 1990) and that the developmental dependence of
the stimulatory effect correlates with the developmental changes in
mAChR-mediated stimulation of phospholipase C activity (Mubagwa et
al., 1992). It has been suggested that the stimulatory effect of
muscarinic agonists may be a mechanism to prevent excessive suppression
of cardiac activity (Pappano, 1991). Because of the presence of high
levels of acetylcholine in uninnervated heart early in development
(Coraboeuf et al., 1970), the high levels of cm3 early in
development may ensure that the heart maintains its contractile
activity prior to the onset of parasympathetic innervation. Large et al.(1985) identified two species of mAChR in embryonic
chick retina, with molecular masses of 86 and 72 kDa. The expression of
both 86 and 72 kDa increased between E9 and E17, and between E17 and
P10 the expression of 86 kDa decreased, while the expression of 72 kDa
continued to increase (Skorupa and Klein, 1993). This change in
expression was duplicated during culture of retinal cells for 6 days,
and conditioned medium from more mature retinal cell cultures caused
young retinal cells to switch their expression pattern after only 2
days in culture. It was suggested that a secreted factor from the older
retinal cells is involved in regulating the switch in expression from
the 86- to the 72-kDa protein (Skorupa and Klein, 1993). Our studies
demonstrate that there are at least three subtypes of mAChR expressed
in chick retina. The predominant receptor expressed in retina is cm4.
The pattern of expression of both cm4 protein and mRNA is similar to
that seen for the 86-kDa protein. The cm4 receptor expression level is
greatest early in development and steadily decreases through the P7
stage of development. The level and pattern of expression of the cm2
and cm3 receptors is very similar. Both cm2 and cm3 proteins are
expressed at very low levels in the earliest stage of development
studied and continually increase as development progresses. This
pattern of expression is identical to that seen for the 72-kDa protein. Immunoblot analysis of mAChR expression in chick retina using
subtype-specific antibodies demonstrates that the cm2 protein is
approximately 64-69 kDa, cm3 protein is approximately 92 kDa, and
cm4 protein is 86-95 kDa. While the differences in the molecular
masses found here compared with those reported by Large et al. (1985) are most likely due to differences in electrophoretic
conditions, the molecular weight of cm4 is greater than that of cm2,
and the immunoprecipitation data for these two proteins exactly
correlates with the affinity alkylation studies. We therefore conclude
that cm2 corresponds to the lower molecular weight receptor and cm4 to
the higher molecular weight receptor previously reported in chick
retina. Because of the lack of resolution inherent in counting gel
slices of a mixture of affinity-alkylated receptor subtypes, it is
likely that the radiolabeled bands representing cm3 and cm4 proteins
were not resolved, and therefore a third mAChR was not identified in
chick retina in the previous studies. The cm2 and cm3 mRNA
expression patterns in retina are also similar. The level of expression
is low early in development, peaks at E15, and then decreases through
the P7 stage. Interestingly, all three mAChR mRNAs present in chick
retina are most highly expressed at E15, which coincides with
synaptogenesis in the retina. The level of all three mRNA subtypes
decreases after synaptogenesis is complete. These results correlate
with the studies of Sugiyama et al. (1977), who reported an
increase in the concentration of mAChR binding sites in chick retina
between E6 and E14, followed by a small decrease in receptor binding
sites after hatching. In conclusion, we have demonstrated that the
mAChR subtypes expressed in chick heart and retina are differentially
regulated during development. The developmental expression patterns of
cm2, cm3, and cm4 correlate with physiological and biochemical changes
that take place during the development of these tissues. These results
suggest both a role for mAChR in the development of the heart and
retina and the possibility that there is a unique functional role for
each receptor in the developing chick heart and retina.
Volume 270,
Number 35,
Issue of September 01, pp. 20636-20642, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)are a
family of neurotransmitter receptors which belong to a superfamily of
proteins with seven putative transmembrane domains and elicit their
cellular signals through interactions with GTP binding regulatory
proteins (G proteins) (reviewed in Nathanson(1987)). Molecular cloning
studies have revealed five different subtypes of mammalian mAChR that
are products of distinct genes (Bonner et al., 1987, 1988;
Hulme et al., 1990). Subtypes m1, m3, and m5 have a relatively
high degree of similarity and preferentially couple to the stimulation
of phospholipase C, while m2 and m4 are similar and preferentially
couple to the inhibition of adenylyl cyclase. Three chicken mAChR
subtypes have recently been cloned and designated cm2 (Tietje and
Nathanson, 1991), cm3 (Gadbut and Galper, 1994), and cm4 (Tietje et
al., 1990). All three subtypes are expressed in chick heart and
brain as detected by Northern blot and RNase protection analyses. 
that increase
mAChR-G coupling (Halvorsen and Nathanson, 1984). The
ability of muscarinic agonists to stimulate phosphoinositide turnover
is greatest in embryonic day 4 (E4) hearts, and as development
progresses this response decreases (Orellana and Brown, 1985; Barnett et al., 1990). There is a developmental increase in the number
of mAChRs in chick atria, but not ventricles, between E10 and E12
(Kirby and Aronstam, 1983; Luetje et al., 1987a). This
increase in receptor number coincides with the functional innervation
of the atria by cholinergic neurons of the parasympathetic nervous
system. Blockade with atropine, a muscarinic antagonist (Kirby and
Aronstam, 1983) or disruption of innervation (Kirby et al.,
1985) can prevent the increase in mAChR number, suggesting that the
functional innervation stimulates the developmental increase in atrial
mAChR. There is a developmental increase in mAChR number in chick
ventricle at the time of hatching (Sullivan et al., 1985;
Hosey et al., 1985).
Cloning of the Third Cytoplasmic Loop of the cm3
mAChR
The third cytoplasmic loop of the cm3 mAChR gene was
amplified from chicken genomic DNA by the polymerase chain reaction
(PCR) with Taq polymerase (Promega) using degenerate
oligonucleotides complementary to sequences in the putative fifth and
sixth transmembrane domains and the third cytoplasmic loop of the
receptor. The following oligonucleotides were synthesized by the
University of Washington Molecular Pharmacology Facility. BamHI and EcoRI restriction sites are indicated by
brackets: 5` primer 1a:
5`-[GAATTCGGATCC]AC(A/C/T)AT(C/T)ATG(A/T)(G/C)CAT(C/T)CTGTA(C/T)TGG-3`;
5` primer 1b:
5`-[GAATTCGGATCC]AC(A/C/T)AT(C/T)ATGTC(C/T)AT(C/T)CTGTA(C/T)TGG-3`;
5` primer 2a: 5`-[GAATTCGGATCC]TT(C/T)TGGCTGAC(C/A/
T)ATGAA(G/A)(A/T)(G/C)CTGG-3`; 5`primer 2b:
5`-[GAATTCGGATCC]TT(C/T)TGGCTGAC(C/A/T)ATGAA(G/A)TC(C/T)TGG-3`;
3` primer 1:
5`-TGGAC(C/A/T)CC(C/A/T)TA(C/T)AA(C/T)AT(C/T)ATG[GGATCCGAATTC]-3`;
3` primer 2a: 5`-TGGCTGAC(C/A/T)ATGAA(A/G)(A/
T)(G/C)CTGGGA(A/G)[GGATCCGAATTC]-3`; 3` primer 2b:
5`-TGGCTGAC(C/A/T)ATGAA(A/G)TC(C/T)TGGGA(A/G)[GGATCCGAATTC]-3`.
Using 5` primer 1a, 5` primer 1b, and 3` primer 1 an 800-base pair
fragment was PCR-amplified, isolated, and ligated into the BamHI and EcoRI sites of pGEM3z (Promega), and
designated cm3-800. Using cm3-800 as a template, we
PCR-amplified a 597-base pair product using 5` primer 2a, 5` primer 2b,
and 3` primer 1, and a 234-base pair product using 5`primer 1a, 5`
primer 1b, 3` primer 2a, and 3` primer 2b. These PCR products were both
ligated into the BamHI and EcoRI sites of pGEM3z.
cm3-597 and cm3-234 were then sequenced by the dideoxy
chain termination method (Sanger et al., 1977) using Sequenase
(U.S. Biochemical Corp.). Sequence analysis indicated that these
fragments encoded the third cytoplasmic loop of an m3 subtype of mAChR.
Subsequent comparison with the recently published sequence of cm3
(Gadbut and Galper, 1994) confirmed that we had cloned the third
cytoplasmic loop of cm3.RNA Probe Construction
Subtype-selective
riboprobes were generated from the third cytoplasmic loop of each mAChR
gene. The probes for cm2 and cm4 were described previously (Habecker
and Nathanson, 1992). A 363-base pair fragment corresponding to
nucleotides 1135-1497 of the coding region of the cm3 gene was
PCR-amplified from cm3-597 and ligated into the BamHI
and EcoRI sites of pGEM3z. T7 and SP6 RNA polymerases
(Promega) were used to transcribe antisense and sense riboprobes,
respectively. Labeled riboprobes were separated from unincorporated
nucleotides on a Sephadex G-50 RNA spin column (Boehringer Mannheim).Isolation of Total Nucleic Acids
Total nucleic
acids were isolated according to the protocol of McKnight et al. (1988). Tissues were immediately frozen in liquid nitrogen upon
dissection and stored at -70 °C. Tissue (100-200 mg)
was homogenized in 50 µg/ml proteinase K in 1 SET (1% SDS,
5 mM EDTA, 10 mM Tris, pH 7.5) using a Polytron
homogenizer and digested for 1 h at 45 °C. Samples were then
phenol/chloroform/isoamyl alcohol-extracted, and nucleic acids were
precipitated in 70% EtOH with 150 mM NaCl. Total nucleic acid
pellets were resuspended in 0.1
SET and quantitated by UV
spectrophotometry. DNA concentration in the total nucleic acid samples
was measured by Hoechst stain (Sigma) (Labarca and Paigen, 1980) using
a fluorometer.
Solution Hybridization
mAChR mRNA present in
15-20 µg of each of the total nucleic acid samples was
quantitated by the solution hybridization assay as described by
Habecker and Nathanson (1992). Molecules of specific mAChR mRNA per
cell were calculated by comparing samples to a standard curve generated
by hybridization of 0.05-10 pg of the sense riboprobes with the
antisense riboprobes.Antibody Production
We designed glutathione S-transferase mAChR fusion proteins to generate
subtype-selective mAChR antibodies. PCR was used to amplify a
subtype-specific region of the third cytoplasmic loop of each mAChR.
Oligonucleotide primers were synthesized by the University of
Washington Molecular Pharmacology Facility. The sequence of cm2 that
was amplified corresponds to amino acids 253-345. For cm4 the
sequence amplified corresponds to amino acids 271-383. These two
fragments were each ligated into the BamHI site of the pGEX-2T
vector (Pharmacia Biotech Inc.). The sequences amplified for cm3 were
amino acids 379-499 and 379-434. These PCR fragments were
ligated into the BamHI and EcoRI sites of pGEX-2T.
Fusion proteins were purified from bacteria using a
glutathione-Sepharose (Pharmacia) column. Antisera were raised in New
Zealand White rabbits by R & R Rabbitry (Stanwood, WA). The
polyclonal cm2, cm3, and cm4 antisera were affinity-purified using the
mAChR fusion proteins coupled to CNBr-Sepharose (Pharmacia). Antibodies
were tested for specificity by an immunoprecipitation assay (described
below) using membranes from Y1 mouse adrenal carcinoma cells stably
expressing either cm2 or cm4 or using membranes from COS-7 green monkey
kidney cells transiently expressing cm3 (cm3 in pED was a gift from Dr.
Jonas B. Galper).Cell Culture and Transfection
The Y1 cell lines
stably expressing cm2 (Tietje and Nathanson, 1991) or cm4 (Tietje et al., 1990) were grown as described previously. COS-7 cells
were grown in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) containing 10% fetal bovine serum (Life
Technologies, Inc.), penicillin G (100 units/ml), and streptomycin
sulfate (0.1 mg/ml; Apothecon) in a 10% CO
environment at
37 °C. COS-7 cells plated on 150-mm culture dishes were transiently
transfected with 30 µg of cm3 in pED by the calcium phosphate
precipitation method (Sambrook et al., 1989).Immunoprecipitation Assay
Immunoprecipitation was
carried out as described by Luetje et al. (1987b). Tissues
were immediately frozen in liquid nitrogen upon dissection and stored
at -70 °C. Crude membrane fractions were prepared as
described by Luetje et al. (1987b). The mAChR in the membranes
were labeled with 2.4 nM
[
H]quinuclidinyl benzilate (47 Ci/mmol; Amersham
Corp.) at room temperature and solubilized as described (Luetje et
al., 1987b). Solubilized proteins were incubated overnight at 4
°C with goat anti-rabbit Immunobeads (Bio-Rad or Sigma) coated with
preimmune or subtype-specific mAChR antibodies. Following incubation,
the Immunobeads were pelleted and the supernatant was collected.
Radiolabeled mAChR remaining in the supernatant were precipitated with
2 volumes of 75% saturated ammonium sulfate, collected by filtration
over GF/C filters, and counted by liquid scintillation. The pelleted
Immunobeads were collected by filtration over GF/C filters and counted
by liquid scintillation to directly quantitate the percentage of mAChR
immunoprecipitated by the Immunobeads coated with the subtype-specific
antibodies. The percentage of mAChR precipitated from tissue extracts
was normalized by (a) subtraction of the percentage of counts
per second immunoprecipitated by Immunobeads coated with preimmune
serum and (b) correction for the fraction of receptor
immunoprecipitated by each antibody when tested against its respective
antigen in transfected cells.Membrane Preparation for SDS-Gel
Electrophoresis
Tissues were homogenized in cold
phosphate-buffered saline containing protease inhibitors, and membranes
were prepared and stored at -70 °C as described (Luetje et al., 1987a).Immunoblot Analysis of mAChR Expression
50 µg
of membrane protein were solubilized at room temperature for 15 min in
SDS-urea sample buffer (3.5% SDS, 5% 2-mercaptoethanol, 10% glycerol,
0.0005% bromphenol blue, 8 M urea, and 125 mM Tris-HCl, pH 6.8; prepared fresh using 5 concentrated
stock of SDS sample buffer without urea). Proteins were electrophoresed
on a 3.5% polyacrylamide stacking gel containing 4 M urea and
a 7% polyacrylamide running gel containing 4 M urea (Hunter
and Nathanson, 1986) and transferred electrophoretically to Immobilon-P
transfer membrane (Millipore Corp.). Immunoblot analysis was performed
as described previously (Luetje et al., 1987a). Transfer
membranes were blocked in 5% bovine serum albumin in TBST
(Tris-buffered saline with 0.1% Tween 20) overnight at 4 °C. After
washing in TBST, transfer membranes were incubated first with purified
anti-cm2, anti-cm3, or anti-cm4 antibody and then with horseradish
peroxidase-conjugated goat anti-rabbit IgG. Both the primary and
secondary antibodies were diluted in 1% bovine serum albumin in TBST
and incubated at room temperature for 60 min. ECL Western blotting
detection reagents (Amersham Corp.) were used for detection of mAChR
immunoreactivity. In some cases, transfer membranes were stripped in
100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris HCl,
pH 6.7, at 50 °C for 30 min. and reprobed using the same
antibodies; this stripping and reprobing was found to decrease
nonspecific labeling.
Specificity of mAChR Antibodies
The efficiency
and specificity of the subtype-specific mAChR antibodies was determined
by immunoprecipitation of radiolabeled mAChR solubilized from cell
lines expressing cm2, cm3, or cm4 (Fig. 1). The
affinity-purified cm2 antibody immunoprecipitates 80% of radiolabeled
cm2 receptors expressed in stably transfected Y1 cells.
Affinity-purified cm4 antibody immunoprecipitates 70% of cm4 receptors
from stably transfected Y1 cells, and affinity-purified cm3 antibody
immunoprecipitates 79% of cm3 receptors transiently expressed in COS-7
cells. These antibodies show minimal cross-reactivity with the other
receptor subtypes, confirming their specificity for their respective
antigens.
mAChR Protein Expression in Embryonic Chick
Heart
The developmental pattern of expression of mAChR proteins
in embryonic chick heart was determined by immunoprecipitation to
quantitate the percentage of cm2, cm3, and cm4 proteins in the total
mAChR population at each stage of development studied. The predominant
mAChR protein expressed throughout the development of the embryonic
chick heart is cm2 (Fig. 2a). The cm2 protein
represents 98-100% of all radiolabeled mAChR in both atria and
ventricles at each developmental stage examined. cm3 and cm4 mAChR
proteins represent a very small percentage of the total mAChR
population in embryonic chick heart, ranging from <1-5% of the
total mAChR protein (Fig. 2, b and c). While
there seems to be no obvious trend in the developmental expression
pattern of cm4 protein, there is a developmental decrease in cm3
protein expression. The level of cm3 in ventricle decreases during
development, with statistically significant differences in protein
expression between E6V and E9V, E15V, and post-hatched day 7 (P7)V and
between E9V and P7V. Although the atria exhibited a trend of decreased
expression of cm3 during development, the differences in expression
levels are not significant (p > 0.05). These studies are
consistent with the observations of Orellana and Brown(1985) and
Barnett et al.(1990) that the mAChR-stimulated
phosphoinositide turnover is greatest early in development and
decreases as development progresses. The cm3 protein is most likely
responsible for this phosphoinositide turnover seen in early
development.
mAChR Protein Expression in Embryonic Chick
Retina
We used the immunoprecipitation assay to determine the
pattern of expression of cm2, cm3, and cm4 mAChR subtypes during the
development of the chick retina. The expression of cm2 protein
increases as development progresses, as shown in Fig. 3a. At E9, the cm2 protein is undetectable, and by
P7 cm2 represents nearly 50% of total mAChR protein. In contrast, the
cm4 protein, which is more highly expressed than cm2 in the retina,
decreases as development proceeds. cm4 represents over 70% of mAChR at
E9 and decreases to 45% by P7 (Fig. 3c). The pattern of
expression of cm3 protein is similar to that seen for cm2: a continual
increase as development progresses. At E9 cm3 represents 15% of mAChR
protein in the retina and increases to approximately 45% by P7 (Fig. 3b). These data show that there are differential
expression patterns among the mAChR proteins expressed in embryonic and
post-hatched chick retina. It should be noted that in both the heart
and retina, the combined percentage of cm2, cm3, and cm4 mAChR
immunoprecipitated was greater than 100%. This is likely due to the
normalization in our calculations to correct for the inability of each
specific antibody to immunoprecipitate 100% of the respective antigen
in the transfected cell lines.
mAChR mRNA Expression in Embryonic Chick
Heart
Solution hybridization was used to measure the number of
molecules of cm2, cm3, and cm4 mRNA expressed per cell in embryonic
chick heart and retina. Consistent with the immunoprecipitation data,
the predominant mAChR mRNA in heart is that encoding cm2 (Fig. 4a). The level of cm2 mRNA expression ranged from
5 to 30 molecules/cell. Although the level of cm2 mRNA does not vary
greatly during the embryonic stages of development of the atria and
ventricle except for an overall decrease after E4, there is an increase
in the level of cm2 mRNA in P7 ventricle. In addition, the difference
in cm2 mRNA expression between P7 atria and ventricle is substantially
greater than any differences between embryonic atria and ventricle. The
increase in cm2 mRNA in P7 ventricle is consistent with previous
reports of an increase in mAChR binding sites in ventricle after
hatching (Sullivan et al., 1985; Hosey et al., 1985).
mAChR mRNA Expression in Embryonic Chick
Retina
The overall level of total mAChR mRNA expression in
retina is much lower than that seen in heart. The predominant mAChR
mRNA subtype in retina is cm4 (Fig. 5c), in agreement
with the immunoprecipitation data (Fig. 3). There were
0.5-3.5 molecules of cm4 mRNA per cell, with expression
decreasing in the later stages of development. Approximately
0.4-1.2 molecules of cm2 mRNA and 0.1-0.7 molecules of cm3
mRNA are expressed per cell in embryonic chick retina (Fig. 5, a and b). The peak level of expression of both of
these mRNAs occurs around E15. Interestingly, synaptogenesis in the
retina occurs at the time when all three mAChR mRNAs are at their peak
levels of expression, between E13 and E14 (Large et al.,
1985).
Immunoblot Analysis of mAChR Expression
The
subtype-specific mAChR antibodies were used to compare the molecular
weight of specific receptor subtypes with the species previously
identified in chick retina using affinity-alkylating ligands (Large et al., 1985). As a control the antibodies were tested on
immunoblots for specificity against the stably transfected Y1 cell
membrane proteins or the transiently transfected COS-7 cell membrane
proteins. The anti-cm2, anti-cm3, and anti-cm4 antibodies exhibit a
high level of specificity for their corresponding mAChR and show
minimal cross-reactivity (Fig. 6). Immunoreactivity to cm2, cm3,
and cm4 was all not detectable in skeletal muscle (Fig. 7). Fig. 7a shows that the cm2 protein is highly expressed
in E15 ventricle as expected from the previous data. cm2 is not
detectable in E9 retina but is found to be expressed in E15 retina and
at even higher levels in P7 retina. The approximate molecular mass of
cm2 ranges from 64 to 69 kDa. cm3 expression is too low to be detected
in E15 ventricle but is expressed in retina with the level increasing
as development continues. Its approximate molecular mass is 92 kDa (Fig. 7b). cm4 is also not detectable in E15 ventricle
but is present in the retina at a molecular mass ranging from 86 to 95
kDa, with the level of expression decreasing as development continues (Fig. 7c). The differences in molecular weights
compared with previous studies of mAChR in the retina (Large et
al., 1985) are most likely due to differences in SDS gel
electrophoresis protocols. Based on their sizes and patterns of
expression we conclude that cm2 corresponds to the lower molecular
weight species and cm4 corresponds to the higher molecular weight
species detected previously by affinity alkylation (Large et
al., 1985).
)
We thank Dr. Jonas B. Galper for the gift of cm3 in
pED.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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