Am J Physiol Cell Physiol
280: C884–C896, 2001.
G protein coupling to M1 and M3 muscarinic receptors
in sublingual glands
W. LUO, L. R. LATCHNEY, AND D. J. CULP
Center for Oral Biology and the Department of Pharmacology and Physiology,
University of Rochester Medical Center, Rochester, New York 14642
Received 2 May 2000; accepted in final form 30 October 2000
salivary glands; muscarinic cholinergic receptors; mucous
cells; m1-toxin; G␣q/11; exocrine secretion
of the oral cavity are numerous and
diverse and are composed of either one or both of two
principal acinar exocrine cell types, serous and mucous
cells. In rodents and humans, mucous exocrine cells
predominate within acinar structures and receive robust parasympathetic innervation with a paucity of
sympathetic innervation. Correspondingly, unlike serous glands, both fluid secretion (20) and exocrine
secretion of mucin glycoproteins by mucous glands (5)
are primarily under muscarinic cholinergic control.
Previously, we identified equivalent amounts of M1
and M3 muscarinic receptor subtypes in membranes
from either whole rat sublingual glands or isolated
acini (27). Furthermore, both muscarinic receptor subtypes appear to be associated directly with mucous
acinar cells and are required for maximal exocrine
secretion (5). The coexpression and regulatory function
SALIVARY GLANDS
Address for reprint requests and other correspondence: D. J. Culp,
Center for Oral Biology, 601 Elmwood Ave., Box 611, Rochester, NY
14642-8611 (E-mail: [email protected]).
C884
of muscarinic receptors within mucous acini may or
may not extend to signaling events at the postreceptor
level. For example, M1 and M3 receptors may each
couple potentially to different guanine nucleotide binding proteins (G proteins), even when expressed in the
same cell (8).
At least 19 separate G protein ␣-subunits have been
identified in mammalian tissues and are segregated
into four families on the basis of primary sequence
homology: G␣s, G␣i, G␣q, and G␣12 (11). Cholera toxin
(CTX) catalyzes the ADP ribosylation of the G␣s family
and G␣t(1 and 2), resulting in the loss of intrinsic GTPase
activity (14). Pertussis toxin (PTX) ADP ribosylates a
cysteine residue near the carboxyl terminus of G␣i,
G␣o, G␣t, and G␣gus, preventing subsequent G protein
and receptor interactions (14). In studies of cell lines
expressing M1 or M3 receptors, both receptor subtypes
are linked predominantly to G␣q/11 to activate phospholipase C (PLC) (8, 14). In addition, both receptor
subtypes may activate PTX-sensitive G proteins (21)
and stimulate PLC through G␤␥ dimers (16). In rat
parotid glands, M3 receptors couple to both G␣q and
G␣i1 (6). Thus, in sublingual glands, coupling of either
M1 or M3 receptors to other G proteins in sublingual
acini cannot be ruled out, especially given the predominance of muscarinic receptors in regulating mucous
acinar cell functions.
To provide insights into the functional role of receptor coexpression in sublingual glands, we therefore
investigated the coupling of M1 and M3 receptors in
sublingual membranes to G protein ␣-subunits. The
coupling and toxin sensitivity (PTX and CTX) of receptors to G proteins was assessed by high-affinity
GTPase activity and the GTP-induced shift in carbachol high-affinity binding sites. Photoaffinity labeling
by [32P]GTP-azidoaniline and antisera specific for
␣-subunits was used to detect candidate subunits coupled to muscarinic receptors and to identify ␣-subunits
linked to carbachol-induced phosphoinositide hydrolysis. The contribution of M1 receptors to carbacholinduced responses was determined by using the
pseudoirreversible and highly specific M1 receptor antagonist m1-toxin, a 64-amino acid peptide isolated
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0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society
http://www.ajpcell.org
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Luo, W., L. R. Latchney, and D. J. Culp. G protein
coupling to M1 and M3 muscarinic receptors in sublingual
glands. Am J Physiol Cell Physiol 280: C884–C896,
2001.—Rat sublingual gland M1 and M3 muscarinic receptors each directly activate exocrine secretion. To investigate
the functional role of coreceptor expression, we determined
receptor-G protein coupling. Although membrane proteins of
40 and 41 kDa are ADP-ribosylated by pertussis toxin (PTX),
and 44 kDa proteins by cholera toxin (CTX), both carbacholstimulated high-affinity GTPase activity and the GTP-induced shift in agonist binding are insensitive to CTX or PTX.
Carbachol enhances photoaffinity labeling ([␣-32P]GTPazidoaniline) of only 42-kDa proteins that are subsequently
tractable to immunoprecipitation by antibodies specific for
G␣q or G␣11 but not G␣12 or G␣13. Carbachol-stimulated
photoaffinity labeling as well as phosphatidylinositol 4,5bisphosphate (PIP2) hydrolysis is reduced 55% and 60%,
respectively, by M1 receptor blockade with m1-toxin. G␣q/11specific antibody blocks carbachol-stimulated PIP2 hydrolysis. We also provide estimates of the molar ratios of receptors
to G␣q and G␣11. Although simultaneous activation of M1
and M3 receptors is required for a maximal response, both
receptor subtypes are coupled to G␣q and G␣11 to stimulate
exocrine secretion via redundant mechanisms.
RECEPTOR-G PROTEIN COUPLING IN SUBLINGUAL GLANDS
from the venom of the Eastern green mamba, Dendroaspis angusticeps (23).
MATERIALS AND METHODS
nizer. The homogenates were filtered through 250-␮m nylon
mesh (Small Parts) and spun at 25,000 g for 25 min. The
resulting pellets were further homogenized (6 ⫻ 30 s) with a
BioHomogenizer (Biospec Products) at a high setting, pelleted as before, and washed with and resuspended in TrisEDTA buffer. Protein content was determined by the method
of Bradford, as described previously (5), with the use of
bovine serum albumin (BSA) as standard. Aliquots (2 mg/ml)
were stored at ⫺80°C until use.
ADP ribosylation. PTX and CTX were preactivated for 30
min at 30°C with 20 and 50 mM DTT, respectively, dissolved
in 20 mM Tris-EDTA buffer (pH 7.5). Activated toxin and
[32P]NAD (5 ␮Ci/assay) were mixed with ADP-ribosylation
buffer consisting of 1 mM EDTA, 2.5 mM MgCl2, 100 mM
NaCl (for PTX only), 1 mM ATP, 10 ␮M GTP, 2.5 mM DTT,
10 mM thymidine, 10 ␮M NAD, 10 ␮g/ml leupeptin, 5 ␮g/ml
aprotinin, 0.2 mM PMSF, and 20 mM Tris 䡠 HCl, pH 7.5, and
the reaction was initiated by adding the membrane suspension (3 mg/ml resuspended in ADP-ribosylation buffer). The
reaction was terminated after 60 min with ice-cold TrisEDTA buffer, followed by spinning (12,000 g for 10 min, 4°C).
Membrane pellets were subjected to SDS-PAGE and autoradiography. In competition, photoaffinity labeling and GTPase
assays for bacterial toxin pretreatment of membranes were
conducted with and without [32P]NAD at final concentrations
of 25 ␮g/ml PTX or 10 ␮g/ml CTX, and the ratios of toxins to
membrane proteins were kept constant at 10 ␮g PTX/mg
protein and 4 ␮g CTX/mg protein.
GTPase assay. Sublingual membranes were washed twice
by centrifugation (12,000 g for 10 min, 4°C) with 30 volumes
of ice-cold assay buffer (50 mM triethanolamine-HCl, pH 7.4,
0.2 mM EGTA, 10 ␮g/ml leupeptin, 5 ␮g/ml aprotinin, and
0.2 mM PMSF). To assay GTPase activity, we used the
method described by Ghodsi-Hovsepian et al. (10). Membranes (5 ␮g) were incubated at 25°C for 5 min with or
without muscarinic agonist or antagonist in assay buffer
supplemented with 100 mM NaCl, 5 mM MgCl2, 0.2% BSA, 5
mM phosphocreatine, 1 mM App(NH)p, 50 U/ml creatine
phosphokinase, 0.25 mM ATP, and 1 mM DTT. The reaction
was started by addition of 2 ␮l of 12.5 ␮M GTP containing 0.5
␮Ci [␥-32P]GTP (final volume 50 ␮l) and stopped with 1 ml of
5% (wt/vol) Norit A suspended in ice-cold 50 mM NaH2PO4
(pH 4.5). Tubes were centrifuged (12,000 g for 10 min, 4°C),
and a 60-␮l aliquot of each supernatant was counted in 10 ml
of scintillation fluid. Results are presented as high-affinity
GTPase activity, which is defined as the difference between
total and nonspecific hydrolysis of [␥-32P]GTP. Nonspecific
GTPase activity was determined in the presence of 50 ␮M
unlabeled GTP. In control experiments, total GTPase activity
in the presence of 1 mM carbachol was linear for at least 10
min (not shown). As a result, all subsequent assays were
conducted for 10 min. Spontaneous release of 32Pi (in the
absence of membranes) accounted for ⬍1.5% of total added
radioactivity.
Synthesis and purification of [␣-32P]GTP-azidoaniline.
[␣-32P]GTP-azidoaniline was synthesized and purified as described by Fields et al. (9). Briefly, [␣-32P]GTP was evaporated under a mild nitrogen stream and dissolved with 3.6%
EDAC-HCl in 0.1 M MES (pH 5.6). Nonradiolabeled GTP was
added, and the mixture was allowed to set for 10 min. We
then added 4% 4-azidoaniline-HCl suspended in peroxidefree 1,4-dioxane and incubated the solution for 16 h at room
temperature in the dark with constant rotation. The reaction
was terminated by extraction of unreacted azidoaniline three
times with 200 ␮l of water-saturated ethyl acetate. The water
phase was spotted onto a flexible polyethylenimine-cellulose
plate (5 ⫻ 20 cm), and thin-layer chromatography (TLC) was
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Materials. Specific pathogen and sialodacryoadenitis virus-free male Wistar rats (2 mo old, 150–175 g) were obtained from Charles River Laboratories (Kingston facility,
Stone Ridge, New York). PTX and CTX were from List Biological Laboratories. Carbachol, atropine, ATP, GDP, NAD,
phosphocreatine, creatine phosphokinase, 5⬘-adenylyl-␤,␥imidodiphosphate (AppNHp), thymidine, benzamidine,
EDTA, EGTA, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl (EDAC-HCl), triethylammonium bicarbonate, 2-(Nmorpholino) ethanesulfonic acid (MES), HEPES, 14C-methylated protein markers, and phosphatidylinositol 4,5bisphosphate (PIP2) were from Sigma Chemical. Dithiothreitol
(DTT) was obtained from United States Biochemical, and
GTP, guanosine 5⬘-O-(3-thiotriphosphate) (GTP␥S), aprotinin, leupeptin, and phenylmethylsulfonyl fluoride (PMSF)
were from Boehringer Mannheim Biochem. Protein A-agarose was obtained from Calbiochem. Immobilon-PVDF (polyvinylidene difluoride) was obtained from Millipore. Cappel
Rabbit IgG was obtained from ICN Pharmaceuticals, polyethylenimine-cellulose plates were from J. T. Baker, and
4-azidoaniline-HCl was from Fluka. Phosphatidylserine (PS)
and phosphatidylethanolamine (PE) were from Avanti Polar
Lipids; [125I]-labeled rProtein A (81.1 ␮Ci/␮g), [3H]PIP2 (6.0
Ci/mmol), [␣-32P]GTP (3,000 Ci/mmol), [␥-32P]GTP (6,000
Ci/mmol), and [32P]NAD (800 Ci/mmol) were from Dupont
NEN; and [3H]-N-methyl-scopolamine (NMS) was from Amersham International. Recombinant proteins, rat G␣q, and
human G␣11 were obtained from Chemicon International.
Rabbit antibodies (IgG fractions, 200 ␮g IgG/ml) D17, E17,
S20, and A20, as well as their blocking proteins, were obtained from Santa Cruz Biotechnology. E17 was raised to
amino acids 13–29 of the amino-terminal domain unique to
G␣q. D17 was raised to amino acids 13–29 of the aminoterminal domain unique to G␣11. A20 and S20 were raised to
amino acids 2–21 of the amino-terminal domain of G␣13 and
G␣12, respectively. Rabbit antisera W082, B825, and Z811
were generous gifts from Dr. Paul Sternweis (Department of
Pharmacology, University of Texas Southwestern Medical
Center, Dallas, TX). W082 was raised to a synthetic peptide
representing the internal amino acid sequence 115–133,
unique to G␣q (22). Z811 was raised to a synthetic peptide of
the common carboxy-terminal sequence 345–359 for G␣q and
G␣11 (22). B825 was raised to a peptide spanning the internal
sequence 114–133 of G␣11 (13). Antibody CT92, kindly provided by Dr. Dianqing Wu (Department of Pharmacology,
University of Rochester Medical Center, Rochester, NY), was
raised against a synthetic peptide to the internal sequence
116–132 of G␣14 (1). Antisera AS233 and AS343 (25) were
raised to synthetic peptides sharing distinct carboxy-terminal sequences unique to G␣12 (370–379) and G␣13 (367–377),
respectively, and were kind gifts from Dr. Karsten Spicher
(Institut für Pharmakologie, Freie Universität Berlin,
Thielallee, Berlin, Germany).
Membrane preparation. Rats were killed by exsanguination after CO2 anesthesia. Sublingual glands were removed
quickly and kept in ice-cold L-15 medium until all glands
were obtained. The glands were dissected at 4°C to remove
remaining fat, connective tissue, and the hilum region and
were then rinsed briefly in 20 mM Tris 䡠 HCl, pH 7.5, 1 mM
EDTA, 10 ␮g/ml leupeptin, 5 ␮g/ml aprotinin, and 0.2 mM
PMSF (Tris-EDTA buffer), minced, and homogenized in 10
volumes of Tris-EDTA buffer with a glass Dounce homoge-
C885
C886
RECEPTOR-G PROTEIN COUPLING IN SUBLINGUAL GLANDS
performed at room temperature in the dark by using 0.8 M
triethylammonium bicarbonate buffer (pH 7.5) as the mobile
phase. TLC-purified [␣-32P]GTP-azidoaniline was detected
by autoradiography, eluted with 0.5 M NaCl, lyophilized, and
reconstituted. Radioactivity was determined, and aliquots
were stored at ⫺70°C. Similar procedures were followed for
synthesis and purification of nonradiolabeled GTP-azidoaniline, with the exception that the TLC-separated GTP analog
was detected with ultraviolet (UV) light (360 nm).
Photoaffinity labeling.
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Photoaffinity labeling was performed as described by
Fields et al. (9) with minor modifications. Sublingual membranes were resuspended to 1 mg/ml in photolabeling buffer
(20 mM Tris 䡠 HCl, pH 7.5, 1 mM EDTA, 10 mM MgCl2, 100
mM NaCl, and 1 mM benzamidine) and incubated for 5 min
at 30°C with 100 ␮M of GDP plus an additional 5 min in the
presence or absence of agonist or antagonist, as indicated. In
some cases, membranes (1 mg/ml photolabeling buffer) were
preincubated for 30 min at room temperature with 100-fold
molar excess of m1-toxin (relative to membrane high-affinity
pirenzepine sites). We then added 2 ␮l of 750 ␮M GTP
containing 0.2–0.6 ␮Ci of [␣-32P]GTP-azidoaniline to initiate
each reaction (final volume 50 ␮l). After 2 min at 30°C,
reactions were terminated by placing tubes on ice and adding
500 ␮l of ice-cold stopping solution (5 mM DTT in photolabeling buffer). Unbound [␣-32P]GTP-azidoaniline was removed by centrifugation (12,000 g for 30 min, 4°C), and
supernatants were discarded. Pellets were resuspended in 50
␮l of stopping solution and exposed on ice to UV light (300
nm, 60 W; Fotodyne Transilluminator) for 2 min (3 cm from
light source) and prepared for SDS-PAGE.
SDS-PAGE and autoradiography. Electrophoresis was as
described previously (4) by using 4% stacking and 10% running gels in a Hoeffer minigel system. In some cases, a lower
concentration of bisacrylamide (0.12%) was used to obtain
better resolution of the isoforms of ␣-subunits. Gels were
stained with Coomassie blue and/or silver as described (4),
dried at 60°C with a gel dryer, and exposed to Kodak XAR-5,
Biomax MS, or MR film at ⫺70°C. Autoradiograms were
scanned with either a laser densitometer (LKB 2202 UltroScan XL) or a Digital Imaging System (IS-1000; Alpha
Innotech).
Western blotting. Membrane proteins (50 ␮g) were subjected to SDS-PAGE, and proteins were transferred to Immobilon-PVDF membranes at 200 mA overnight with a Hoeffer mini transfer apparatus (TE 22). Under these conditions,
no proteins of ⬍70 kDa remained in the gel, as assessed by
extensive silver staining. Membrane strips were rinsed twice
and incubated in blotting solution (0.2% Triton X-100 and 5%
nonfat dry milk in PBS) at room temperature for 1 h before
overnight incubation with individual antisera diluted in blotting solution. After three rinses with blotting solution, strips
were incubated 2–3 h with 500,000 cpm/ml [125I]-Protein A,
rinsed, and subjected to autoradiography with Kodak Biomax
MS or MR film.
Immunoprecipitation of photolabeled G proteins. Sublingual membranes were photolabeled as described in Photoaffinity labeling, and two aliquots were subjected to SDS-PAGE
and autoradiography as controls in each experiment to verify
carbachol-enhanced photolabeling of 42-kDa proteins. The
resultant membrane pellets (90 ␮g per condition) were solubilized by first being incubated in 15 ␮l of 2% SDS for 10 min
at room temperature. We then added 48 ␮l of ice-cold solubilization buffer (10 mM Tris 䡠 HCl, pH 7.4, 1% wt/vol Nonidet
P-40, 1% wt/vol deoxycholate, 0.2% SDS, 150 mM NaCl, 1
mM DTT, 1 mM EDTA, 0.2 mM PMSF, 1 ␮M GDP, and 10
␮g/ml aprotinin), and the mixture was repipetted. Membrane
solubilization appeared quantitative, because no specific G
proteins were detectable in the subsequent pellets when
assessed in preliminary experiments by Western analysis for
each antibody used. After centrifugation (14,000 g for 10
min), the supernatant was precleared by adding 3.3 ␮l of 5
mg/ml purified rabbit IgG in PBS and incubating for 4 h at
4°C, followed by addition of 20 ␮l of Protein A-agarose (50%
suspension, preequilibrated and washed with solubilization
buffer ⫹ 0.5% SDS), and the mixture was incubated for 1 h at
4°C with continuous rocking. After centrifugation (14,000 g
for 10 min), we added 20 ␮g of antibody IgG (with or without
blocking peptide) and 1 ml of cold solubilization buffer to the
supernatant and incubated overnight at 4°C with continuous
rocking. Protein A-agarose (10 ␮l of a 50% suspension) was
added, and incubation continued for 4–6 h. The mixture was
centrifuged (14,000 g for 10 min), and the pellet was washed
with 1 ml of solubilization buffer plus 0.5% SDS and then
subjected to SDS-PAGE and subsequent autoradiography
with Kodak Biomax MS film. In all cases, blocking antigen
peptides were used at a 10:1 molar ratio with respect to
antibody IgG in the solution. In controls consisting of nonimmune rabbit IgG in place of antibody, we were unable to
detect radiolabeled proteins in the final immunoprecipitate,
and no IgG was detected in the supernatant when subjected
to SDS-PAGE and extensive silver staining.
PIP2 hydrolysis assay. Measurement of PLC activity was
as described by Gutowski et al. (12) but with minor modifications. Membranes were diluted to 0.5 mg/ml with buffer A
(1 mM EDTA, 3 mM EGTA, 100 mM NaCl, 5 mM MgCl2, 3
mM DTT, 10 ␮M GDP, and 50 mM HEPES, pH 7.2), and 10
␮l of membrane suspension (5 ␮g protein) were added to 40
␮l of buffer B (3 mM EGTA, 80 mM KCl, 1 mM DTT, 1 ␮M
GDP, and 50 mM HEPES, pH 7.2) containing phospholipid
vesicles. The final vesicle suspension was prepared by sonication and contained 1.5 mM PE, 1.5 mM PS, 0.15 mM PIP2,
and 7,000–10,000 dpm [3H]PIP2. In some cases, buffer B also
contained GTP␥S, carbachol, and/or atropine. Ten microliters of 9 mM CaCl2 were then added, and the reaction was
started by transferring tubes to a 30°C water bath. Reactions
were terminated after 12 min by placing the tubes in ice
water, followed by rapid addition of ice-cold 1% BSA (100 ␮l)
and 10% trichloroacetic acid (200 ␮l). After 5 min, the precipitates were pelleted (12,000 g for 5 min), and 300 ␮l of the
supernatants were removed for determination of radioactivity. In parallel assays, membranes were preincubated with
m1-toxin or antibody Z811 against G␣q/11. In the case of
antibody Z811, membrane aliquots (0.5 mg/ml buffer A) were
preincubated for 20 min at room temperature, followed by 30
min on ice in the absence (control) or presence of either Z811
(1 ␮M IgG), Z811 premixed with 10 ␮M of the antigen
peptide, or 1 ␮M rabbit IgG. Portions of each membrane
aliquot were then assayed in the absence or presence of
GTP␥S (1 ␮M) with and without carbachol (1 mM). In the
case of m1-toxin, two equal membrane aliquots (0.5 mg/ml
buffer A) were preincubated for 30 min at room temperature
with or without m1-toxin (100-fold molar excess of m1-toxin
relative to membrane high-affinity pirenzepine sites) and
then assayed in the presence of GTP␥S (1 ␮M), carbachol (1
mM) plus GTP␥S (1 ␮M), or atropine (100 ␮M) plus carbachol
(1 mM) plus GTP␥S (1 ␮M).
Radioligand binding. Membranes (20–50 ␮g) were resuspended in 0.5–1 ml of binding buffer (20 mM Tris 䡠 HCl, pH
7.5, 1 mM EDTA, 3 mM MgCl2, and 0.1 mM PMSF) and
incubated with [3H]NMS at 4°C for 3 h to achieve steadystate binding. Incubations were stopped by addition of 4 ml of
RECEPTOR-G PROTEIN COUPLING IN SUBLINGUAL GLANDS
C887
Statistics. Unless indicated, variability is expressed as
means ⫾ SE. Statistical comparisons of results was by the
Student’s t-test with P ⬍ 0.05 being significant.
RESULTS
ice-cold binding buffer, followed by vacuum filtration through
Whatman GF/C filters. Atropine (10 ␮M) was included in the
incubation for determination of nonspecific binding. In competition assays, nonradiolabeled agonist (with or without
guanine nucleotide) was added simultaneously with 0.5 nM
[3H]NMS. Results were analyzed by using the computer
programs EBDA and LIGAND, as described previously (27).
The dissociation constant (Kd) for [3H]NMS in membrane
preparations ranged from 0.7 to 1.1 nM, and the proportion of
high-affinity pirenzepine sites ranged from 42 to 48% of total
binding sites, similar to results reported previously (27).
Preparation of m1-toxin. Toxin was isolated from lyophilized venom of the Eastern green mamba, D. angusticeps
(Sigma Chemical), according to the protocol described by
Potter et al. (23), except that material from the initial Sephadex G-50 column was further fractionated by gel filtration
chromatography on a column (1.5 ⫻ 170 cm) of Sephadex
G-25 Superfine eluted with 0.1 M ammonium acetate, pH 6.8,
at 4°C. This additional step serves to enrich the preparation
for m1-toxin and removes a component possessing anti-rat
M3 receptor activity before reverse-phase HPLC. Preparations of m1-toxin were pure as determined by SDS-PAGE and
subsequent reverse-phase HPLC. One unit of m1-toxin was
similar to that defined by Potter et al. (23): the minimum
amount required to block 95% of the specific binding of 0.1
nM [3H]NMS to 0.2 pmol M1 receptors [rat M1-Chinese
hamster ovary (CHO) cell membranes] in 10 ml of 50 mM
NaH2PO4 plus 1 mM EDTA, pH 7.4, at 25°C for 45 min.
Under these standard assay conditions, 1 unit of m1-toxin is
equivalent to ⬃30 ng toxin protein and corresponds to a
molar ratio of m1-toxin to receptor of ⬃20:1 (23). Preparations of m1-toxin were specific for the inhibition of M1 receptors as assessed under the standard radioligand binding
assay conditions described above, which include 1 unit of
m1-toxin and membranes from CHO cell lines expressing
either human M1-M5 receptor subtypes (kindly provided by
Dr. Mark R. Brann) or rat M1 or M3 receptors. In addition, 10
units of m1-toxin had no inhibitory effects on [3H]NMS binding to rat M3 receptors (200:1 molar ratio of m1-toxin to
receptors).
Fig. 2. ADP ribosylation of sublingual membrane proteins by cholera
toxin (CTX) in the presence of [32P]NAD, followed by SDS-PAGE and
autoradiography. A: SDS-PAGE gel stained with Coomassie blue
before autoradiography to demonstrate protein loading in each lane.
Arrows indicate positions of molecular mass markers (kDa). B:
radiolabeling of ⬃44 kDa proteins (arrowhead) by increasing concentrations of CTX.
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Fig. 1. Inhibition by carbachol of [3H]N-methylscopolamine (NMS;
0.5 nM) binding to sublingual membranes (50 ␮g protein) in the
absence (control) or presence of 100 ␮M guanosine 5⬘-O-(3-thiotriphosphate) (GTP␥S). Nonspecific binding was determined in the
presence of 10 ␮M atropine. Values are means from single representative experiments performed in triplicate.
To initially evaluate the effective coupling between
muscarinic receptors and G proteins in sublingual
membrane preparations, we studied the shift induced
by GTP␥S in the affinity of the agonist carbachol for
[3H]NMS binding sites. As shown in a representative
experiment in Fig. 1, GTP␥S induced a rightward shift
in the carbachol competition curve. Binding data from
five separate experiments were fit to models of one,
two, and three binding sites, and the best fit was
chosen when P ⬍ 0.05 by the F test. In the absence of
GTP␥S, the best fits were to a two-site model with
inhibition constant (Ki) values (means ⫾ SE) of 5.4 ⫾
1.1 and 99.8 ⫾ 17.0 ␮M, representing 54 ⫾ 3 and 46 ⫾
3% of the total binding sites, respectively. In the presence of 100 ␮M GTP␥S, the best fits were to a one-site
model with a mean Ki of 129 ⫾ 14 ␮M.
A series of experiments were then conducted to determine whether muscarinic receptors in sublingual
membranes are coupled to G proteins sensitive to PTX
or CTX. We first determined the ADP ribosylation of
membrane proteins as a function of toxin concentration. CTX induced the concentration-dependent ADP
ribosylation primarily of 44-kDa proteins within sublingual membranes as resolved by SDS-PAGE and
subsequent autoradiography. Maximal radiolabeling
was achieved at 10 ␮g/ml CTX (Fig. 2B). Similar experiments were conducted with PTX, and, as shown in
C888
RECEPTOR-G PROTEIN COUPLING IN SUBLINGUAL GLANDS
Therefore, to test coupling of sublingual muscarinic
receptors to PTX-dependent G proteins, we incubated
sublingual membranes before ADP ribosylation with or
without agonist (1 mM carbachol) for 15 min at room
temperature in ADP-ribosylation buffer that contained
10 ␮M GTP. Agonist was maintained in the buffer
during subsequent ADP ribosylation by PTX in the
presence of [32P] NAD. Under these conditions, a proportion of those G proteins that couple to muscarinic
receptors would be expected to be activated. Accordingly, if muscarinic receptors are indeed coupled to
PTX-sensitive G proteins, then the pool of receptorcoupled ␣-subunits in the heterotrimeric state, and
hence available to PTX-catalyzed ADP ribosylation,
should be less than in the absence of carbachol. In
contrast, carbachol had no apparent effect on PTXdependent radiolabeling of either 40- or 41-kDa proteins (Fig. 3C, compare lanes 2 and 3). As a control,
PTX-catalyzed ADP ribosylation was completely inhibited when membranes were incubated in the presence
of 100 ␮M GTP␥S (Fig. 3C, lane 4). In additional
competition binding experiments, we found that PTXcatalyzed ADP ribosylation of sublingual membrane
proteins also had no detectable effect on the shift in
carbachol binding affinity induced by GTP␥S (see
Fig. 4).
As another means to distinguish coupling of sublingual muscarinic receptors to G proteins sensitive to
PTX or CTX, we determined whether either toxin attenuated agonist-enhanced high-affinity GTPase activity. In an initial experiment, GTPase activity was in-
Fig. 3. ADP ribosylation of sublingual membrane proteins by PTX in the presence of [32P]NAD, followed by
SDS-PAGE and autoradiography. A: SDS-PAGE gel stained with Coomassie blue before autoradiography. Arrows
indicate positions of molecular mass markers (kDa). B: radiolabeling of ⬃40 kDa proteins (arrowhead) by
increasing concentrations of pertussis toxin (PTX). C: before ADP ribosylation, sublingual membranes were
incubated in the presence or absence of carbachol (1 mM) or GTP␥S (100 ␮M) for 15 min at room temperature in
ADP-ribosylation buffer. Agonist or GTP␥S was maintained in the buffer during subsequent incubation (60 min)
with PTX (25 ␮g/ml) and [32P]NAD. The gel was prepared with a lower concentration of bisacrylamide (0.12%) and
was run until the 29-kDa molecular mass marker was near the bottom of the gel to help separate proteins of ⬃40
and 41 kDa.
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Fig. 3B, proteins of ⬃40 kDa were radiolabeled in a
concentration-dependent manner with maximal radiolabeling at 25 ␮g/ml PTX. If proteins were allowed to
migrate further into the gel, PTX-dependent proteins
began to resolve into two bands of ⬃40 and 41 kDa,
presumably representing at least two distinct ␣-subunits (Fig. 3C, lane 2). To verify that PTX at 25 ␮g/ml
and CTX at 10 ␮g/ml were saturating for ribosylation
of sublingual membrane proteins, we treated membrane aliquots with and without toxin in the absence of
[32P]NAD and then reexposed them to toxin in the
presence of [32P]NAD. As a control, membrane aliquots
were treated with or without toxin in the presence of
[32P]NAD. Samples were subjected to SDS-PAGE and
autoradiography to first verify toxin-enhanced radiolabeling of membrane proteins of appropriate mass and
to determine residual radiolabeling of proteins in membranes pretreated with toxin. In two separate experiments, autoradiographs (1-day exposure) of control
samples demonstrated radiolabeling similar to results
shown in Figs. 2 and 3, whereas radiolabeled proteins
were barely detectable by autoradiography after a 10day exposure of samples from membranes pretreated
with each toxin (not shown). On the basis of these
combined results, we used PTX and CTX at 25 and 10
␮g/ml, respectively, in further experiments to distinguish whether toxin-sensitive G proteins play a role in
carbachol-stimulated signaling events in sublingual
membranes.
PTX selectively ADP ribosylates ␣-subunits of target
G protein holotrimers rather than free ␣-subunits (29).
RECEPTOR-G PROTEIN COUPLING IN SUBLINGUAL GLANDS
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average maximal intensity of radiolabeling induced by
carbachol was nearly 60% above basal levels as assessed by densitometric scanning of autoradiographs (
Fig. 6B). In two separate experiments, we found that
photoaffinity labeling enhanced by 1 mM carbachol
was completely blocked by concentrations of atropine of
0.1 ␮M or higher (not shown). To assess the contribution of muscarinic M1 receptors to carbachol-enhanced
photoaffinity labeling, we first treated sublingual
membranes with a 200-fold molar excess of m1-toxin.
Basal levels of photoaffinity labeling was unaffected by
m1-toxin, whereas carbachol-enhanced labeling was
reduced 55% (Fig. 7).
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Fig. 4. Effect of PTX (25 ␮g/ml) on inhibition of [3H]NMS binding to
sublingual membranes (50 ␮g protein) by carbachol in the absence
(control) or presence of 100 ␮M GTP␥S. Nonspecific binding was
determined in the presence of 10 ␮M atropine. Values are means
from single representative experiments performed in quadruplicate.
Similar results were obtained in an additional experiment with a
separate membrane preparation.
duced by carbachol in a concentration-dependent
manner, the effect of which was maximal at 1 mM with
an apparent EC50 of ⬃7 ␮M (Fig. 5A). We then assayed
high-affinity GTPase activity in response to 1 mM
carbachol after prior ADP ribosylation of membranes
with maximal concentrations of either PTX or CTX.
Basal GTPase activity was reduced after treatment of
membranes with each toxin (Fig. 5B), suggesting that
G proteins susceptible to either PTX or CTX contribute
to the total basal high-affinity GTPase activity. In
contrast, the net increase in carbachol-mediated
GTPase activity was unaffected by pretreatment of
membranes with either toxin (Fig. 5B).
Agonist-enhanced photoaffinity labeling of membrane proteins with [32P]GTP-azidoaniline is a valuable tool in identifying the coupling of receptors to G
proteins (9). In initial experiments with rat sublingual
gland membranes, we found that multiple bands were
radiolabeled with [32P]GTP-azidoaniline in the absence of agonist, including bands of 74, 51, 46, 36, and
23 kDa and a predominant band at 42 kDa (Fig. 6A,
lane 1). In the presence of carbachol, we consistently
(10 separate experiments) observed agonist-enhanced
photoaffinity labeling of only 42-kDa proteins (Fig. 6A,
lanes 2 and 3). Photoaffinity labeling of all proteins was
unaffected by 100 ␮M ATP but inhibited completely by
either 30 ␮M guanosine 5⬘-O-(2-thiodiphosphate) (not
shown) or 100 ␮M GTP␥S (Fig. 6A, lanes 4 and 5).
Furthermore, carbachol-enhanced photoaffinity labeling was also insensitive to pretreatment of sublingual
membranes with either 25 ␮g/ml PTX or 10 ␮g/ml CTX
(not shown). Enhancement of photoaffinity labeling of
the 42-kDa band in the presence of carbachol was
concentration dependent, readily detectable at 0.1 ␮M,
and maximal at ⬃100 ␮M carbachol (Fig. 6B). The
Fig. 5. Carbachol-stimulated high-affinity GTPase activity in sublingual membranes. A: carbachol concentration dependence of highaffinity GTPase activity. Values are means ⫾ SE of a single experiment performed in triplicate. B: stimulation of high-affinity GTPase
activity by 1 mM carbachol in sublingual membranes either with or
without prior PTX (25 ␮g/ml) or CTX (10 ␮g/ml) treatment. Values
are means ⫾ SE of 3 separate experiments, each from a separate
membrane preparation. Mean activity in the presence of 1 mM
carbachol and 1 ␮M atropine was 35.6 ⫾ 2.1 pmol GTP/mg protein.
In all cases, activity in the presence of carbachol is greater statistically (P ⬍ 0.05, paired Student’s t-test) than in the absence of
carbachol. In addition, carbachol-stimulated activities for all 3 conditions are equivalent (P ⬎ 0.10, paired Student’s t-test).
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RECEPTOR-G PROTEIN COUPLING IN SUBLINGUAL GLANDS
At this stage, our combined results indicated that
both M1 and M3 receptors couple to G proteins with
␣-subunits of ⬃42 kDa and are insensitive to both PTX
and CTX. We therefore conducted Western blot analy-
Fig. 7. Inhibition of carbachol-stimulated photoaffinity labeling by m1toxin. Sublingual membranes were incubated with m1-toxin for 30 min
at room temperature before photoaffinity labeling with or without
carbachol (1 mM) or atropine (100 ␮M). Integrative intensity of each
42-kDa band was obtained by digital imaging. Values are means ⫾ SE
relative to basal labeling (without carbachol and atropine) for each set
of membranes treated with and without m1-toxin from 4 experiments.
For each set of conditions: *P ⬍ 0.05 vs. basal; †P ⬍ 0.05 vs. carbachol ⫹
atropine; §P ⬍ 0.05 vs. carbachol without m1-toxin.
sis to identify candidate ␣-subunits within sublingual
membranes that may couple to muscarinic receptors.
Possible candidates of known G protein ␣-subunits
were considered that satisfied the following criteria: 1)
insensitive to both PTX and CTX; 2) ⬃42 kDa in electrophoretic mobility; and 3) previously demonstrated
to be expressed in epithelial tissues. Candidates include a member (or members) of the Gq family, the
G␣12 family, and the PTX-insensitive Gi family member G␣z. Previous studies demonstrated that G␣16 and
its mouse homologue, G␣15, are restricted to hematopoietic tissues (28), whereas G␣z is distributed primarily to platelets and neurons (17). Thus we did not probe
for G␣16 or G␣z. We did probe with antibodies selective
for G␣q, G␣11, G␣14, G␣12, and G␣13. Results are shown
in Fig. 8. Antibody Z811, raised against the carboxyterminal region common to both G␣q and G␣11 (22),
reacted strongly to proteins of ⬃42 kDa in sublingual
membranes (Fig. 8A, lane 1). Similar results (Fig. 8A,
lane 3) were obtained with the G␣q-specific antisera
W082 (22) and E17 (not shown). To probe for G␣11, we
used antibody D17, raised to a peptide equivalent to
amino acids 13–29 of the amino-terminal domain
unique to G␣11. As shown in Fig. 8A, lane 5, this
antibody reacted with 42-kDa proteins as well as several lower and higher mass proteins in sublingual
membranes. Reactivity was blocked by preabsorption
with the antigen peptide (Fig. 8A, lane 6). Similar
results were obtained with membranes from rat
brains, except that the 42-kDa band was by far the
predominant band (not shown). We also used antibody
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Fig. 6. Photoaffinity labeling of sublingual membranes by [␣-32P]GTP-azidoaniline. Membranes were subjected to
photoaffinity labeling, followed by SDS-PAGE and autoradiography, as described in MATERIALS AND METHODS. A:
carbachol-stimulated photoaffinity labeling of 42-kDa proteins (arrowhead) in the absence or presence of GTP␥S or
ATP. Arrows indicate the positions of 205-, 97.4-, 66-, 45-, and 29-kDa molecular mass markers. B: photoaffinity labeling
of 42-kDa proteins with increasing concentrations of carbachol and inhibition by 100 ␮M atropine (lane 7). Relative
integrated intensity values are means ⫾ SE of 5 independent experiments and are the percentages of basal labeling (no
carbachol added) as determined by laser scanning. Arrows indicate the positions of molecular mass markers (kDa).
RECEPTOR-G PROTEIN COUPLING IN SUBLINGUAL GLANDS
C891
B825, raised against a peptide equivalent to amino
acids 114–133 specific for G␣11. This antibody is selective for G␣11, because it binds weakly to recombinant
G␣q (13). Reactivity of B825 against 42-kDa proteins
was barely detectable in sublingual membranes but
very intense in brain membranes (not shown). Antisera
raised against carboxy-terminal sequences specific for
G␣12 (AS233) and G␣13 (AS343) each reacted with
proteins in sublingual membranes that displayed a
relative mobility of ⬃43 kDa (Fig. 8B, lanes 1 and 3,
respectively). Similar results were obtained with antibodies S20 and A20, which were raised against aminoterminal domains of G␣12 and G␣13, respectively (not
shown). Antisera CT92 (1), specific for G␣14, recognized
a protein in brain but displayed no immunoreactivity
to sublingual membrane proteins of appropriate mass
(Fig. 8C, lanes 1 and 2, respectively).
Our results from Western analyses demonstrate the
presence of G␣q, G␣11, G␣12, and G␣13 in sublingual
membranes and, thus, further suggest that one or more
of these proteins may function in coupling to muscarinic receptors. As an approach to distinguish G proteins coupled directly to sublingual muscarinic receptors, we attempted to immunoprecipitate specific G
protein ␣-subunits from membranes after photoaffinity
labeling with [32P]GTP-azidoaniline in either the presence or the absence of carbachol. As shown in Fig. 9A,
lane 2, radiolabeled proteins of ⬃42 kDa were detected
in membranes challenged with carbachol, and proteins
were subsequently immunoprecipitated with antibody
Z811. No radiolabeled proteins were detected in membranes not exposed to carbachol and/or in membranes
immunoprecipitated in the presence of antigen peptide
(Fig. 9A, lanes 1, 3, and 4). These results suggest that
G␣q and/or G␣11 couple to sublingual muscarinic receptors. To discriminate between these two G proteins, we
used antibodies E17 (G␣q specific) and D17 (G␣11 specific) in further experiments. Antibody E17 was used
rather than antiserum WO82; the latter was raised
against an internal sequence of G␣q and was previously demonstrated not to immunoprecipitate native
G␣q proteins (12). Furthermore, we confirmed the specificity of each antibody by Western analysis in a preliminary experiment. No signal was detected for either
antibody when tested against 50 ng of the alternative
recombinant ␣-subunit protein, although positive controls (5 ng of appropriate recombinant protein, 50 ␮g
each of sublingual membrane and brain membrane
proteins) displayed strong signals (not shown). In subsequent immunoprecipitation experiments, antibody
E17 produced results similar to those of antibody Z811
(Fig. 9B). When antibody D17 was used with membranes treated without carbachol (Fig. 9C, lane 1),
there was a diffuse band barely apparent at ⬃42 kDa,
as were bands at positions of higher and lower mass. In
the presence of carbachol, only the 42-kDa protein
band displayed enhanced radiolabeling (Fig. 9C, lane
2). Radiolabeling of 42-kDa proteins and most of the
higher and lower mass proteins was blocked by inclusion of the G␣11 antigen peptide whether membranes
were challenged with carbachol or not (Fig. 9C, lanes 3
and 4). These results indicated both G␣q and G␣11
couple to muscarinic receptors in sublingual membranes. The proteins recognized by antibody D17 of
mass greater and less than 42 kDa likely represent
unknown cross-reactive guanine nucleotide binding
proteins, because similar proteins were detected in
Western analysis (Fig. 8A, lane 5) and after photoaf-
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Fig. 8. Western blot analysis of G protein ␣-subunits in membranes from rat sublingual glands or brains. A:
sublingual membranes (60 ␮g protein/lane). Lane 1, antibody Z811 (12 ␮g/ml); lane 2, Z811 plus blocking antigen
peptide; lane 3, antiserum W082 (1:1,000); lane 4, W082 preimmune serum (1:1,000); lane 5, antibody D17 (200
ng/ml); lane 6, D17 plus blocking antigen peptide. B: sublingual membranes (50 ␮g protein/lane). Lane 1,
antiserum AS233 (1:200); lane 2, normal rabbit serum (1:200); lane 3, antiserum AS343 (1:150), lane 4, normal
rabbit serum (1:150). C: brain and sublingual membranes (odd and even lanes, respectively; 50 ␮g protein/lane).
Lanes 1 and 2, antiserum CT92 (1:200); lanes 3 and 4, normal rabbit serum (1:200). Blocking antigen peptides were
used at a 10:1 molar ratio with respect to antibody IgG in the solution. Arrows indicate positions of 97.4-, 66-, 45-,
and 29-kDa 14C-labeled molecular mass markers. Arrowheads indicate the calculated mobility of 42- (A and C) and
43-kDa (B) proteins.
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RECEPTOR-G PROTEIN COUPLING IN SUBLINGUAL GLANDS
finity labeling with [32P]GTP-azidoaniline (Fig. 6A,
lanes 1–4). Using antibodies A20 (G␣13 specific) or S20
(G␣12 specific), we were unable to detect radiolabeled
proteins in immunoprecipitates from membranes incubated either with or without carbachol (not shown). As
controls for these experiments, we demonstrated carbachol-enhanced radiolabeling of 42-kDa proteins in
separate membrane aliquots that were subjected to
SDS-PAGE and autoradiography immediately after radiolabeling (not shown). In addition, we confirmed that
antibodies A20 and S20 immunoprecipitated G␣13 and
G␣12, respectively, by performing Western analysis of
immunoprecipitates using the same antibodies as
probes. Thus muscarinic receptors in sublingual membranes appear to couple primarily to G␣q and G␣11. We
considered conducting further experiments that would
incorporate m1-toxin to distinguish the relative coupling of G␣q and G␣11 to muscarinic M1 and M3 receptors in sublingual membranes, but such experiments
were determined to be unfeasible because the immunoprecipitation experiments described above were
near the limits of detection; autoradiographic film required 4–6 wk of exposure to indicate immunoprecipitated proteins.
To gain insight into the capacity of G␣q relative to
that of G␣11 to couple to sublingual muscarinic receptors, we determined the relative abundance of
each G protein ␣-subunit in sublingual membranes.
We used Western analysis to correlate the band
intensities obtained with increasing amounts of recombinant G␣q (15–40 ng) and G␣11 (8–24 ng) proteins with those obtained for two different amounts
of sublingual membrane proteins. Antibody D17 (200
ng/ml) was used to probe for G␣11. We used antiserum WO82 (1:1,000) rather than antibody E17 to
probe for G␣q because WO82 gave a stronger signal
and was in greater supply. As shown in Fig. 10,
increasing amounts of G␣q and G␣11 displayed linear
distributions of band intensities with respect to the
amount of protein applied. The average (⫾SE, n ⫽ 4)
amount of G␣q in sublingual membranes obtained in
two separate experiments in which 15- and 25-␮g
membrane proteins were used was 1.2 ⫾ 0.2 ng/␮g
membrane protein. The value derived for G␣11 in two
separate experiments in which 50- and 75-␮g membrane proteins were used was 0.21 ⫾ 0.03 ng/␮g
membrane protein (mean ⫾ SE, n ⫽ 4). The abundance of G␣q in sublingual membranes is therefore
nearly sixfold greater than that of G␣11.
Biochemical studies with cell lines have documented
the efficient coupling of M1 and M3 receptors to both
G␣q and G␣11 with the resultant activation of PLC-␤
isoforms (8, 13). Accordingly, we have found that inhibition of PLC blocks muscarinic activation of exocrine
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Fig. 9. Immunoprecipitation of G protein ␣-subunits from sublingual membranes after photoaffinity labeling with
[␣-32P]GTP-azidoaniline. Sublingual membranes were incubated for 30 min at room temperature before photoaffinity labeling without (lanes 1 and 3) or with 1 mM carbachol (lanes 2 and 4). Membranes were then solubilized
and immunoprecipitated with antibody Z811 (G␣q/11 specific; A), E17 (G␣q specific; B), or D17 (G␣11 specific; C) in
the absence (lanes 1 and 2) or presence (lanes 3 and 4) of specific blocking antigen peptide. Immunoprecipitates
were then subjected to SDS-PAGE and autoradiography. Blocking antigen peptides were used at a 10:1 molar ratio
with respect to antibody IgG. Arrows indicate positions of 97.4-, 66-, 45-, and 29-kDa 14C-labeled molecular mass
markers. Results are representative of 2 or more separate experiments.
RECEPTOR-G PROTEIN COUPLING IN SUBLINGUAL GLANDS
C893
GTP␥S or carbachol plus GTP␥S (Fig. 11A). In further
experiments, we found that specific inhibition of M1
receptors attenuates carbachol-induced PIP2 hydrolysis. As shown in Fig. 11B, PIP2 hydrolysis induced by 1
mM carbachol was reduced nearly 60% by m1-toxin
compared with control.
DISCUSSION
We determined the coupling of muscarinic receptors
in rat sublingual gland membranes to specific G protein ␣-subunits. Coupling was initially verified by the
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Fig. 10. Relative abundance of G␣q and G␣11 in rat sublingual
membranes. Two different amounts of sublingual membrane proteins and increasing amounts of either recombinant G␣q or G␣11
were subjected to SDS-PAGE and Western blot analysis as described
in MATERIALS AND METHODS. Transferred proteins were probed with
either antiserum WO82 (1:1,000) for G␣q or antibody D17 (200
ng/ml) for G␣11. After subsequent binding of [125I]-Protein A, blots
were exposed to Kodak Biomax MS film for 2–4 days. Autoradiograms were scanned (ScanMaker 9600XL), and the densities of
42-kDa bands determined using NIH Image 1.61. A: autoradiograph
of 42-kDa bands after probing with antiserum WO82. Results are
representative of 2 experiments. B: standard curves of 42-kDa band
densities vs. amounts of recombinant G␣q or G␣11. Results are
representative of 2 experiments for each ␣-subunit. Lines represent
linear regressions (r2 ⬎ 0.95 in both cases).
secretion by sublingual mucous acinar cells (unpublished observations). Given that muscarinic receptors
in sublingual membranes couple predominantly to G␣q
and G␣11, we used antibody Z811 to evaluate the direct
role of these ␣-subunits in the muscarinic activated
hydrolysis of sublingual membrane PIP2. In preliminary experiments, PIP2 hydrolysis was activated by
GTP␥S in a dose-dependent manner with near-maximal activity (200% above basal level) at 100 ␮M GTP␥S
(not shown). Carbachol (1 mM) alone had no effect on
PIP2 hydrolysis (basal: 80.6 ⫾ 5.2 pmol; carbachol:
82.6 ⫾ 6.5 pmol; n ⫽ 7, P ⫽ 0.544). GTP␥S was
required for carbachol activation of PIP2 hydrolysis,
with optimal activation at 1 ␮M GTP␥S. Under these
conditions, carbachol increased PIP2 hydrolysis an average of 28% above that observed by 1 ␮M GTP␥S
alone (carbachol ⫹ GTP␥S: 127 ⫾ 9.5 pmol; GTP␥S:
99.2 ⫾ 7.1 pmol; n ⫽ 7, P ⫽ 0.002). In subsequent
experiments incorporating antibody Z811 (Fig. 11A),
we found the antibody blocked stimulation of PIP2
hydrolysis by 1 ␮M GTP␥S, either with or without
carbachol. The antibody was without these affects if
first preincubated with its antigen peptide (Fig. 11A).
As an additional control, nonimmune rabbit IgG had
no affects on PIP2 hydrolysis stimulated by either
Fig. 11. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by
sublingual membranes. A: inhibition by antibody Z811 (G␣q/11 specific).
Values are means ⫾ SE of 4 experiments, each performed in duplicate.
Four equal membrane aliquots were preincubated in either the absence
(control) or presence of Z811 (1 ␮M IgG), Z811 premixed with 10 ␮M of
its antigen peptide, or 1 ␮M affinity-purified rabbit IgG. Each aliquot
was then assayed in the absence or presence of GTP␥S (1 ␮M) either
with or without carbachol (1 mM). For each set of conditions: *P ⬍ 0.05
vs. basal; †P ⬍ 0.05 vs. GTP␥S. B: inhibition of carbachol-stimulated
PIP2 hydrolysis by m1-toxin. Values are means ⫾ SE of 3 experiments,
each performed in duplicate. Two equal aliquots of sublingual membranes were incubated with or without m1-toxin as described in MATERIALS AND METHODS. For each set of conditions: *P ⬍ 0.05 vs. GTP␥S;
†P ⬍ 0.05 vs. atropine ⫹ carbachol ⫹ GTP␥S. §P ⬍ 0.05 vs. carbachol ⫹
GTP␥S without m1-toxin.
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RECEPTOR-G PROTEIN COUPLING IN SUBLINGUAL GLANDS
(30) and calcium-dependent fluid secretion (20). Furthermore, recent results from our laboratory indicate
that muscarinic-induced exocrine secretion is also calcium dependent and is mediated by activation of PLC
(unpublished observations). In the present investigation, we confirm the muscarinic stimulation of PIP2
hydrolysis in sublingual membranes. More importantly, Z811 (G␣q/11 specific antibody) completely
blocked this response, suggesting that activation of
PLC activity in sublingual membranes by muscarinic
receptors is mediated predominantly, if not totally, by
G␣q and G␣11. The absence of muscarinic receptor
coupling to PTX-sensitive G proteins (i.e., Gi proteins)
in sublingual glands further argues against a role for G
protein ␤␥-subunits in the muscarinic activation of
PLC.
A confounding factor in elucidation of muscarinic
regulation of exocrine secretion by sublingual mucous
acini is the expression of approximately equivalent
amounts of M1 and M3 receptor subtypes (4). Moreover,
immunolocalization experiments indicate the coexpression of both M1 and M3 receptor subtypes on sublingual mucous acinar cells (unpublished observations). Blockade of M1 receptors by m1-toxin results in
a 40–50% decrease in the maximal muscarinic receptor-induced exocrine response (5). Although m1-toxin
reduces the maximal secretory response, there is no
appreciable change in the apparent EC50 for agonistinduced secretion (5). In the present study, we found
that m1-toxin inhibits approximately one-half of the
carbachol-enhanced photoaffinity labeling of 42-kDa G
protein ␣-subunits as well as the muscarinic activation
of PLC activity. Collectively, these results are consistent with equivalent amounts of M1 and M3 receptors
expressed by acinar mucous cells that function separately to regulate exocrine secretion through G␣q/11
activation of PLC. A maximal muscarinic receptorinduced secretory response requires activation of both
receptor subtypes.
Carbachol-enhanced photoaffinity labeling of 42-kDa
proteins was half-maximal (EC50) between 1 and 10
␮M carbachol (Fig. 6B), similar to the EC50 (⬃7 ␮M) for
carbachol-stimulated GTPase activity. The high-affinity agonist binding site of sublingual muscarinic receptors (Ki of carbachol ⬃5 ␮M) thus mediates receptor
coupling to G proteins. In contrast, the EC50 reported
previously for carbachol-stimulated exocrine secretion
by acinar cells is 0.3 ␮M (3), ⬃10-fold lower than for
receptor-G protein coupling. This discrepancy in agonist affinities may reflect either an abundance of muscarinic receptors or their associated G proteins linked
to exocrine secretion within acinar mucous cells. M3
receptors are not in excess, because secretion induced
by these receptors alone (after m1-toxin blockade of M1
receptors) are insufficient to induce a maximal secretory response. Because the number of M1 receptors
expressed in isolated acini is similar to that for M3
receptors, and M1 receptors are required for at least
half of the maximal muscarinic secretory response, we
speculate they are also not in great excess. On the
other hand, the density of muscarinic receptors in
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GTP-induced shift in carbachol high-affinity binding
and by high-affinity GTPase activity. In both cases, the
effects of carbachol were insensitive to prior treatment
of membranes with PTX, although PTX catalyzed the
ADP ribosylation of membrane proteins of molecular
mass consistent with that of G␣i and G␣o subunits. In
a similar manner, CTX catalyzed the ADP ribosylation
of 44-kDa membrane proteins (presumably G␣s) but
had no effect on subsequent carbachol-induced GTPase
activity. Therefore, ␣-subunits susceptible to ADP ribosylation by either PTX or CTX do not appear to
regulate directly muscarinic receptor-induced cellular
functions in sublingual glands. Toxin-sensitive G proteins in sublingual membranes may instead mediate
other receptors within sublingual glands such as vasoactive intestinal peptide receptors and ␣2-adrenergic
receptors, presumably coupled to CTX-sensitive G␣s
and PTX-sensitive G␣i, respectively (3).
Photoaffinity labeling of sublingual membrane proteins with [32P]GTP-azidoaniline detected coupling of
muscarinic receptors to 42-kDa G protein ␣-subunits.
As determined by Western analysis, G␣q and G␣11 in
sublingual membranes displayed the same electrophoretic mobility as the 42-kDa proteins identified by
carbachol-enhanced photoaffinity labeling. In addition,
antibodies specific for G␣q or G␣11 immunoprecipitated
42-kDa proteins with enhanced radioactivity after
membranes were photoaffinity labeled in the presence
of carbachol. These results suggest sublingual muscarinic receptors are coupled predominately to Gq and
G11.
The absence of G␣14, as assessed by Western analysis of sublingual membranes, was not anticipated because G␣14 is expressed in other epithelial and glandular tissues, such as lung, kidney, thymus, and testis
(28). Both G␣12 and G␣13 were detected in sublingual
membranes, although the electrophoretic mobility of
each ␣-subunit was slower than the that of the 42-kDa
proteins recognized by photoaffinity labeling. Moreover, we were unable to immunoprecipitate detectable
amounts of radiolabeled G␣12 and G␣13 after photoaffinity labeling of membranes in the presence or absence
of carbachol. Although we cannot rule out the possibility that these latter negative results reflect the limited
sensitivity of the immunoprecipitation assay with native tissue, it appears that neither G␣12 nor G␣13
couples significantly to sublingual muscarinic receptors. Correspondingly, both G␣12 and G␣13 have only
been shown to couple to nonmuscarinic receptors including thrombin, thromboxane A2, bradykinin, and
lysophosphatidic acid receptors. In addition, both
␣-subunits regulate cellular events other than the PLC
or adenylyl cyclase pathways, such as Na⫹/H⫹ exchange and the c-Jun mitogenic pathway (7). The direct effector(s) of G␣12 and G␣13 signaling have yet to
be established, although recent evidence suggests a
role for guanine nucleotide exchange factors (18). Further studies of sublingual acinar cells are thus needed
to elucidate the functional role of G␣12 and G␣13.
In sublingual glands, muscarinic receptor activation
results in the generation of inositol 1,4,5-trisphosphate
RECEPTOR-G PROTEIN COUPLING IN SUBLINGUAL GLANDS
G␣q/11 (24), whereas in rat sublingual glands, ␣1-adrenergic receptors are not expressed (19). Signaling
events regulating specific cell functions are therefore
not necessarily ubiquitous for the discrete exocrine cell
types that may be present, either together or independently, within the numerous and diverse salivary
glands lining the oral cavity. An understanding of such
differences is required if fully effective therapeutic
treatments are to be developed to treat those patients
suffering from hyposalivary function.
We thank Dr. Alan V. Smrcka for assistance in setting up the
PIP2-hydrolysis assays and for helpful discussions.
This study was supported by National Institute of Dental Research Grant DE-10480.
Present address of W. Luo: Department of Cell and Cancer Biology, Division of Clinical Sciences, National Cancer Institute/National Institutes of Health, Bldg. 10, Rm. 3B47, 9000 Rockville Pike,
Bethesda, MD 20892.
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