Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48105
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ABSTRACT |
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Stimulus-secretion
coupling in the pancreatic acinar cell is initiated by the
secretagogues CCK and ACh and results in the secretion by exocytosis of
the contents of zymogen granules. A key event in this pathway is the G
protein-activated production of second messengers and the subsequent
elevation of cytosolic-free Ca2+.
The aim of this study was therefore to define the heterotrimeric G
protein -subunits present and participating in this pathway in rat
pancreatic acinar cells. RT-PCR products were amplified from pancreatic
acinar cell mRNA with primers specific for
G
q, G
11, and
G
14 but were not amplified with
primers specific for G
15. The sequences
of these PCR products confirmed them to be portions of the rat
homologues of G
q,
G
11, and
G
14. The pancreatic-derived cell line AR42J similarly expressed
G
q,
G
11, and
G
14; however, the Chinese
hamster ovary (CHO) cell line only expressed
G
11 and
G
q. These data indicate that
caution should be exercised when comparing signal transduction pathways
between different cell types. The expression of these proteins in
acinar cells was confirmed by immunoblotting samples of acinar membrane
protein using specific antisera to the individual G protein
-subunits. The role of these proteins in
Ca2+ signaling events was
investigated by microinjecting a neutralizing antibody directed against
a homologous sequence in G
q,
G
11, and
G
14 into acinar cells and CHO
cells. Ca2+ signaling was
inhibited in acinar cells and receptor-bearing CHO cells in response to
both physiological and supermaximal concentrations of agonists. The
inhibition was >75% in both cell types. These data indicate a role
for G
q and/or
G
11 in intracellular
Ca2+ concentration signaling in
CHO cells, and in addition to
G
q and
G
11,
G
14 may also fulfill this role
in rat pancreatic acinar cells.
Gq proteins; microinjection
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INTRODUCTION |
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THE MAJOR PHYSIOLOGICAL role of the pancreatic acinar
cells is to synthesize, store in zymogen granules, and secrete by
regulated exocytosis digestive enzymes (26). Secretion is
stimulated physiologically, primarily by the hormone CCK and the
neurotransmitter ACh. A key event in acinar stimulus-secretion coupling
is the elevation of intracellular
Ca2+ as a consequence of the
action of the second-messenger inositol 1,4,5-trisphosphate
(IP3).
IP3 is formed as a result of the
heterotrimeric G protein-activated hydrolysis of phosphatidylinositol
4,5-bisphosphate by phospholipase C (PLC). Heterotrimeric G proteins
are composed of three subunits (,
,
) and are usually defined
on the basis of their
-subunit. In pancreatic acinar cells, the
heterotrimeric G protein
-subunits believed responsible for
stimulation of PLC activity are members of the
Gq family. This contention is
based on the observations (13, 24) that the production of
IP3 and Ca2+ signaling events are not
sensitive to pertussis or cholera toxin, which modify the effects of
many other
-subunits. In addition, G
q-directed antisera, when
introduced through a patch pipette, inhibits the agonist stimulation of
a Ca2+-activated current in acinar
cells (31).
Genes for four members of the Gq
family have been cloned and have been named
G11,
G
q,
G
14, and
G
15. Although the four proteins
appear to have widespread distribution in neonatal tissue, the
expression of G
14 and
G
15 is restricted in adult
tissue (1, 23). Both proteins have been shown to be abundant in hematopoietic tissue, and, in addition,
G
14 is highly expressed in
kidney and testis. In contrast,
G
q and
G
11 are thought to be expressed
almost ubiquitously, although the level of expression can vary widely
(14). An exception to this rule is that
G
q but not
G
11 is expressed in platelets
(14). Presumably, as a result of the high sequence identity of
G
q and
G
11 (>85% amino acid
identity), it appears, at least in overexpression systems, that
receptors coupled to this pathway cannot distinguish between these
proteins and the extent of activation of PLC-
is not markedly different (17, 18). In contrast to
G
q and
G
11, evidence (2, 9) suggests
that differences exist in the ability of agonist receptors to couple to
G
14 and
G
15 and the efficacy with which
these G proteins activate PLC-
differs from
G
11 and G
q. While it is known that
individual members of a G protein family can have a restricted
distribution, it is not clear whether in an individual cell type
multiple forms of a particular protein are expressed. In this study,
the Gq family
-subunit
complement of acinar cells and the pancreatic-derived cell line AR42J
is defined and compared with the Chinese hamster ovary (CHO) cell line,
which is often used for the study of signal transduction pathways (5,
22, 30). Using immunoneutralization, we demonstrate that expression of
G protein heterotrimers containing these
-subunits can account for
Ca2+ signaling events in
pancreatic acinar cells.
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METHODS AND MATERIALS |
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Materials.
Fura 2-AM and sulforhodamine B (Lissamine rhodamine B 200) were
purchased from Molecular Probes (Eugene, OR), collagenase (CLSPA grade)
from Worthington Biochemicals (Freehold, NJ), BSA (fraction V) from ICN
Immunobiologicals (Lisle, IL), and minimal essential amino acid
supplement from GIBCO (Grand Island, NY). All other
materials were obtained from Sigma Chemical (St. Louis, MO).
Gq-COOH terminus
(G
q-CT) and internal sequence
G
11-specific antisera were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The
Gq-specific antiserum was obtained
from Calbiochem. Molecular biology reagents are from sources indicated.
Preparation of pancreatic acini. Acini were prepared by methods previously described (12, 25, 30). In brief, pancreata were excised from freely fed adult male Sprague-Dawley rats (200-250 g). Acini were prepared by enzymatic digestion with purified collagenase, followed by mechanical shearing. Acini were then filtered through 150-µm Nitex mesh, purified by sedimentation through 4% BSA in HEPES-Ringer, and then suspended in a physiological saline solution (PSS) containing 10 mg/ml BSA, 0.1 mg/ml soybean trypsin inhibitor, and (in mM) 137 NaCl, 4.7 KCl, 0.56 MgCl2, 1.28 CaCl2, 1.0 Na2HPO4, 10 HEPES, 2 L-glutamine, and 5.5 D-glucose and essential amino acids. The pH was adjusted to 7.4 and equilibrated with 100% O2. Enriched plasma membrane fractions were prepared as previously described (3).
Western blotting. Membrane samples (8-50 µg) were subjected to electrophoresis on 12% SDS-PAGE gels. The resolved proteins were transferred to nitrocellulose (Schleicher & Schuell, Keene, NH). Immunoreactivity was visualized using peroxidase-conjugated secondary antibodies followed by detection using the enhanced chemiluminescence (ECL) detection system exposed on ECL Hyperfilm (Amersham Life Science, Arlington Heights, IL), as previously described (22). Immunoprecipitations were performed as follows: enriched membranes were sonicated in 0.5 ml of ice-cold lysis buffer (100 mM NaF, 50 mm Tris, 150 mM NaCl, 10 mM EDTA, 1 mM benzamidine, 1% Triton X-100, 1% 2-mercaptoethanol, 10 µg/ml leupeptin, and 10 µg/ml pepstatin, at pH 7.4) and then sonicated. After 30 min on ice, the samples were centrifuged for 30 min at 100,000 g in a Beckman bench top ultracentrifuge. The supernatant was assayed for protein concentration. Samples of equal protein concentration were then incubated with antiserum overnight at 4°C, after which immobilized protein A beads (Pierce) were added to each sample. As a control, a sample was processed with no cellular lysate, which would eventually be used to ascertain specific bands on the immunoblot. After the samples were rotated for 2 h, they were then microcentrifuged and the supernatant was discarded. The beads were washed four times (50 mM Tris, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100), suspended in SDS-PAGE sample buffer, and boiled for 3 min. The proteins were then separated by SDS-PAGE and transferred to nitrocellulose before immunoblotting.
RT-PCR analysis of Gq family
-subunits.
Total RNA was isolated after disruption of acini, cultured cells, or
whole pancreas with guanidinium thiocyanate and centrifugation through
a CsCl gradient, according to the method developed by Chomczynski and
Sacchi (4), using a total RNA isolation kit (Invitrogen, San Diego,
CA). We reverse transcribed ~1 µg of RNA at 42°C for 50 min
with 200 units of Superscript II RNase
H
RT and 50 ng random
hexamers (Superscript preamplification system, GIBCO). PCR reactions
were performed in 100-µl vol containing 10 mM
Tris · HCl (pH 8.3), 50 mM KCl, 1.5 mM
MgCl2, 200 µM of each dNTP, 0.2 µM of each primer, and 2.5 units of AmpliTaq DNA polymerase (GeneAmp
PCR core reagents, Perkin-Elmer, Foster City, CA). The temperature
parameters for 40 cycles were 2 min at 94°C and 2 min at the
annealing temperature followed by another 3 min at 72°C. The cDNA
product was used as a template in PCR with primers specific for the
-subunits (G
q,
G
11,
G
14,
G
15), using mouse pancreas
cDNA as a positive control and including a negative control with no
template. The RT-PCR also included a negative control with no RT to
rule out amplification of DNA from contaminating sources. The
amplification primers were designed against the known mouse mRNA
sequences with appropriate restriction sites engineered onto the
5' ends. Primers for G
q
were designed to amplify a 997-bp region from bases 68 to 1065 of the
mouse sequence (GQ:
5'-CTGAGCGAGGAGGCCAAGGAAG-3' and
5'-TTGTAGGCGAAGCAGAAACGTC-3'; annealing temperature,
55°C). Primers for G
11 were
designed to amplify a 346-bp region from bases 339 to 685 of the mouse
sequence (G11:
5'-AGACGCTCAAGATCCTCTACAAGT-3' and
5'-CACCTACACCCGCCCGTCTC-3'; annealing temperature,
57°C). A primer pair was also designed to amplify bases
216-791, which is a region common to mouse
G
q,
G
11, and
G
14
(GQF:
5'-GATCATCCACGGGTCGGGCTACT-3' and
5'-TCTTGGCGTACCTCCTCCTCTCGTT-3'; annealing
temperature, 55°C). Primers for
G
14 were designed to amplify a
424-bp insert spanning bases 641-1065 of the mouse sequence
(G14:
5'-GTATCGCCATGCCCTCTTTCGTG-3' and
5'-TTCTACAGTTTCGCCGGTCCCT-3'; annealing temperature,
63°C). Primers for G
15 were
designed to amplify bases 188-898 of mouse G
15
(G15:
5'-GGCCTGGTGAGAGCGGGAAGAGTA-3' and
5'-TAAGTGTGGAGGGTGGACCGGTGT-3'; annealing temperature,
67°C) and against a 253-bp region of the rat spleen PCR product
(rG15:
5'-CGCCGAGGACGACTACATC-3' and
5'-GTCCTCTTGGCATACTTCCTCTCA-3'; annealing temperature,
67°C). PCR products were analyzed by electrophoresis in 1% agarose
gels containing ethidium bromide. The PCR products were purified
(Geneclean II kit, BIO 101, Vista, CA) in preparation for cloning and
sequencing. The purified double-stranded fragments were restriction
digested and ligated into pBluescript II
(KS
) (Stratagene, La
Jolla, CA) that had been likewise cut. The plasmids containing inserts
were sequenced using Sequenase quick-denature plasmid sequencing kit
(Amersham). Both strands of the double-stranded templates were
sequenced. The rat sequences were identified as homologues of mouse
Gq family
-subunit mRNA
sequences using the BLAST service of the National Center for
Biotechnology Information (NCBI) database. These sequences were then
translated, aligned, and compared with available mouse, human, and
bovine counterparts (retrieved from the NCBI database) using DNA-STAR
LaserGene programs (Madison, WI).
Measurement of intracellular Ca2+ concentration. Isolated acini were incubated with 2.5 µM fura 2-AM at ambient temperature for 30 min and then washed and resuspended in fresh PSS without BSA. For measurement of intracellular Ca2+ concentration ([Ca2+]i), fura 2-loaded acini were transferred to a chamber, mounted on the stage of a Zeiss Axiovert 35 microscope, and continuously superfused at 1 ml/min with PSS without BSA. CHO cells were grown on glass coverslips, loaded for 45 min with 4 µM fura 2-AM, and similarly superfused. Solution changes were rapidly accomplished by means of a valve attached to an eight-chambered superfusion reservoir, which was maintained at 37°C. Determination of [Ca2+]i was performed using digital imaging microscopy with an Attofluor (Rockville, MD) ratiovision system, as previously described (7, 29). Briefly, the 340 nm-to-380 nm excitation ratio was alternately achieved by a computer-controlled filter and shutter system, and the resultant emission at 505 nm was recorded at the rates indicated in the figures, by an intensified charge-coupled device (CCD) camera, and subsequently digitized. Mean gray values obtained by excitation at 340 and 380 nm in user-defined areas of interest were used to compute 340 nm-to-380 nm ratios. Calibration of fluorescence ratio signals was accomplished as previously described according to the equation of Grynkiewicz et al. (7) by comparing the fluorescence of known standard Ca2+ buffers containing fura 2.
Microinjection of antisera. Microinjection of antibodies was performed using a Burleigh Piezoelectric cell penetrator system (Fishers, NY) mounted on the stage of an Axiovert 35 microscope/Attofluor imaging system, which was itself positioned on a vibration isolation platform. Briefly, pipettes of tip diameter of <0.1 µm were pulled on a micropipette forge (Sutter Instruments, Novato, CA) from 1.0-mm glass tubing containing a filament (World Precision Instruments, Sarasota, FL). Pipettes were filled with test agents together with 0.25 µM sulforhodamine B as an indicator of the efficacy of injection in a buffer solution containing 120 mM potassium glutamate and 20 mM HEPES adjusted to pH 7.2. The pipette solution was filtered through a 0.22-µm filter. The pipettes were then mounted in a Perspex holder connected to a pressure injector (Medical Systems, Greenvale, NY). Aliquots of acinar cells were seeded onto coverslips that had been coated with Cell-Tak (Collaborative Research, Bedford, MA) to aid firm adherence of the cells. The final approach and penetration of a single acinar cell was accomplished by making multiple 3-µm jumps toward the cell using the piezoelectric actuator. After penetration, the pipette was rapidly withdrawn by making a single reverse jump of 15 µm. During this procedure and 10 min postinjection, the cells were maintained in PSS containing no added Ca2+. After this 10-min period, the cells were incubated for a further 5 min in a similar solution but containing 10% FCS before they were returned to the normal PSS containing Ca2+ before the monitoring of [Ca2+]i. The success of the injection was assessed by monitoring the morphology of the cell pre- and postinjection, together with the capacity of the cell to retain the injected dye or previously loaded indicator and the ability to maintain an appropriate basal [Ca2+]i. When dye loss from the injected cell was rapid or the cell suffered obvious insult from the injection, the experiment was discarded. The excitation and emission characteristics of sulforhodamine B (excitation, 560 nm; emission, 630 nm) and fura 2 (excitation, 340 nm-to-380 nm ratio; emission, 505 nm) allowed simultaneous monitoring of injection efficacy and [Ca2+]i measurement by virtue of a four-position filter and shutter system. Images at the respective wavelengths were captured by intensified CCD camera and subsequently digitized for analysis.
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RESULTS |
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Characterization of Gq family
-subunits by RT-PCR in pancreatic acini
The expression of
-subunits of the
Gq family in rat pancreatic acini
was investigated by PCR analysis of reverse-transcribed RNA prepared
from the rat exocrine pancreas. As the exact nucleotide sequence of
each of the Gq family
-subunits
is known for murine tissue, oligonucleotide primers were designed
against conserved regions in these genes to use RT-PCR to amplify from
reverse-transcribed rat acinar RNA the cDNA for the
Gq homologues that are expressed in rat pancreatic acinar cells. RNA prepared from rat spleen together with that from the cell line AR42J, which is derived from pancreatic tissue, were included for comparison. Initially, primers were designed
to recognize a sequence that is unique to
G
11. RT-PCR resulted in
products of the expected size amplified from RNA derived from rat
pancreatic acinar cells, whole pancreas, and AR42J cells (Fig.
1A).
As controls, PCR reactions were performed routinely in which no RT was
included to exclude the possibility that products had been amplified
from contaminating DNA. In addition, reactions were performed in which
no template was added. In both of these cases, no product was ever
amplified. The products amplified from rat pancreatic acinar cells were
purified, ligated into pBluescript, cloned, and sequenced. The products
from multiple clones were completely sequenced in both directions, and,
compared with sequences in GenBank, the sequence amplified most closely
resembled mouse G
11. In all,
530 bases were sequenced from clones amplified utilizing the
G11 and
GQF primer pairs. The translated
region of the mouse gene consists of 1364 bases. At the nucleotide
level, the portion of the rat
G
11 homologue was 95%
identical to that of the mouse and the predicted amino acid identity
between mouse and rat protein was >99%. In a similar fashion, a
primer pair was designed to target a 997-bp region of
G
q. As shown in Fig.
1B, products of appropriate size were
amplified from rat pancreatic acini, whole rat pancreas, and AR42J
cells. The product amplified from acini was cloned and then sequenced
as described. The nucleotide sequence was most highly homologous to the
GenBank sequence corresponding to mouse
G
q and thus in all probability
represents the rat G
q homologue, with the portion of the rat sequence being 95%
identical at the nucleotide and 98% identical at the amino
acid level (953 bases were sequenced, from a predicted total of
1460, based on the mouse translated region).
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RT-PCR analysis of Gq family
-subunits expressed in CHO cells.
Because CHO cells are often used for expression studies defining
signaling pathways activated by G protein-coupled receptors, including
studies of pancreatic secretagogues (5, 22, 30), the
G
q family complement of this
cell type was investigated. Using the primer pairs designed to amplify
-subunits from pancreatic tissue, we performed similar PCR reactions
utilizing reverse-transcribed RNA prepared from CHO cells, transfected
with the CCK-A receptor (CHO-CCK-A). PCR products of appropriate size
were amplified using primers designed against both
G
q and
G
11 but not amplified using primers against G
14 and G
15 (Fig.
2). These data would imply that the CHO
cell expresses G
q and
G
11 but not
G
14 and
G
15 and thus expresses a more
restricted complement of G
q
family
-subunits compared with the pancreas.
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Western analysis of Gq
family proteins.
Although preparation of pancreatic acinar cells by these methods is
known to de-enrich nonacinar cells significantly (25), PCR has the
capability of amplifying message from a very small number of copies of
a particular mRNA. It is therefore possible that the
G
q family fragments amplified
by PCR could have been derived from a contaminating cell type, other
than the predominating acinar cell. To confirm that
G
11,
G
q, and
G
14 proteins are expressed in
acinar cells, we performed immunoblots of acinar membrane proteins,
utilizing an antiserum directed against individual G
q family members. Initially,
acinar proteins separated by electrophoresis and transferred to
nitrocellulose were probed with an antiserum (G
q-CT) directed against a
19-amino-acid region that is identical in the COOH terminus of
G
11 and
G
q and highly conserved in
G
14 (2 amino acid
substitutions). As shown in Fig. 3, a
single strong band of ~42 kDa was resolved in acinar cells,
indicating the presence of G
q
family proteins. Similar procedures using membranes prepared from CHO
cells and AR42J cells also resolved a band of 42 kDa, which in all
probability represents G
q
family proteins (data not shown). Similar samples were probed with
antisera specific to G
11 and
G
q. Because these antisera
failed to unequivocally resolve
G
11 and
G
q by conventional immunoblots,
experiments were performed to enrich these proteins by first
immunoprecipitating G
q family
proteins using the G
q-CT
antiserum before electrophoresis and transfer to nitrocellulose and
subsequent probing with specific antisera. In this case, bands of
appropriate molecular mass were resolved, confirming the presence of
G
11 and
G
q (Fig. 3). Immunoblots utilizing an antiserum specific for
G
14 (23) (kindly provided by
Dr. Danquing Wu, University of Rochester) also resolved a protein of
~42 kDa, indicating the expression of
G
14 in acinar cells. The
specificity of these antisera was confirmed, as they did not cross-react inappropriately with recombinant
G
11 and
G
q standards. These data
confirm the expression of G
11,
G
q, and G
14 in pancreatic acinar cells.
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Functional characterization of
Gq subunits.
To demonstrate a role for G
q
subunits in stimulating PLC activity and the resulting signaling
cascade in acinar cells, we performed experiments to monitor the effect
of functionally ablating G
q
proteins, while monitoring intracellular
Ca2+ signaling events. Antiserum
G
q-CT is directed against a
region of the G
q subunit that
is thought to be involved in interaction between the
-subunit and
intracellular domains of cell surface receptors. Moreover this region
of the
-subunit is thought to be exposed in the intact
heterotrimeric complex (21). For these reasons, it is a good candidate
for a neutralizing antibody, and indeed antibodies directed against
this general region of the
-subunit have proved to be effective in
reducing PLC-mediated responses in other cell types (8, 22, 27, 31).
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DISCUSSION |
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Activation of intracellular Ca2+
signaling events in nonexcitable cells, such as pancreatic acinar
cells, is believed to be predominately mediated through a cascade that
involves the Gq family of
heterotrimeric GTP binding proteins. Because this family consists of
four related proteins, the aim of this study was to define which
members of the G
q family are
expressed in pancreatic acinar cells and to implicate expression of
these individual proteins in mediating
Ca2+ signaling events on
stimulation by secretagogues. As a secondary goal, the complement of
G
q proteins expressed in acinar
cells was compared with that expressed in two model cell lines, the pancreas-derived AR42J cell and the CHO cell transfected with pancreatic secretagogue receptors.
In pancreatic acinar cells, RT-PCR and immunoblotting techniques
demonstrated that G11,
G
q, and
G
14 are expressed, both at the
level of mRNA and protein. The expression of both
G
11 and
G
q is consistent with the
almost ubiquitous expression of these proteins, while the expression of
G
14 is somewhat more surprising
based on its limited reported distribution.
G
15 does not appear to be
expressed in pancreatic tissue, and its expression appears limited to
blood cells or tissue involved in the genesis of, or rich in, blood
cells (1, 23). The expression of
G
q family proteins was also
defined in AR42J cells and CHO cells. In a manner similar to that of
the native pancreatic acinar cells, AR42J cells expressed
G
q,
G
11, and
G
14 but not
G
15. In contrast, by PCR
techniques, CHO-CCK-A cells expressed
G
q and
G
11 but not
G
14 or
G
15. These data have relevance
to the use of these cell lines for study of acinar signal transduction.
Although it appears that use of the AR42J cell line for such studies
would seem appropriate based on the expression of
G
q family members, studies
utilizing CHO cells should be treated with some caution since the cell
line does not express the full complement of acinar G
q family members and thus the
potential for altered downstream signaling events is a possibility.
The physiological impetus underlying expression of multiple members of
this G protein family in a particular cell type is unclear. It has been
postulated that molecular diversity expressed in a particular cell type
either may be related to achieving signal specificity or alternatively
may simply be related to redundancy. Because
G11 and
G
q do not differ markedly in
their distribution, recognition by receptors, or activation of PLC-
,
it would appear that their coexpression in the majority of cells may
represent evolutionary redundancy. Levels of
G
q and
G
11 can, however, vary widely
in particular cell types; for example, in the cerebellum, levels of
G
q are up to five times higher
than G
11. In this particular case, cerebellar function is disrupted in transgenic knockouts of
G
q but not of
G
11, indicating that the level
of a particular protein can be critical for coupling effectively (15).
Because the present study did not address the relative abundance of
G
q vs.
G
11 in the acinar cell, further
study will be necessary to determine if these proteins serve an
identical functional role in this cell type.
G14 and
G
15, in contrast, appear to
couple effectively to only a subset of cell-surface receptors and
differ in their efficacy for activating PLC (2, 9, 18). For example, in
RBL-1 cells M1 receptors fail to
mount a Ca2+ response in cells
injected with antisense oligonucleotides directed against
G
q, but the
Ca2+ response is unaffected in
cells injected with antisense RNA directed against
G
14 (9). These
data can be interpreted as indicating that coupling in cells expressing
G
11/G
q
and G
14 cannot be regarded as
promiscuous and that expression of
G
14 allows a cell type
expressing numerous PLC-coupled receptors to gain specificity. This is
indeed an attractive possibility in the pancreatic acinar cell, since
this cell type expresses multiple PLC-coupled receptors. In addition,
these receptors appear not to be coupled identically, since activation
of individual receptors results in markedly different patterns of
Ca2+ signaling events (16, 28).
The coupling of individual receptors to specific members of the
G
q family when multiple
proteins are present in a particular cell type has not been reported.
Precedent exists, however, for specificity of interaction between
members of a G
family and an effector. For example, the coupling of
receptors to Ca2+ channels by
individual Go
family members,
presumably to achieve signal specificity, is well documented in
GH3 pituitary cells. In this case,
inhibition of Ca2+ channels
through muscarinic receptor stimulation is mediated through a
heterotrimer that includes a
Go
1
subunit, while somatostatin-induced inhibition is mediated through a
Go
2
subunit-containing complex (10).
Ablation of protein function by immunoneutralization by specific
antisera is a useful technique for probing the function of a particular
protein. Using this technique, it has been demonstrated that
Ca2+ signaling stimulated by
agonists, such as thyrotropin-releasing hormone in
GH3 cells, thromboxane
A2 in platelets, and angiotensin in rat liver, is a result of receptors for these agonists interacting with Gq family proteins (8, 20,
27). In a similar fashion, this study has demonstrated that both CCh
and CCK-induced Ca2+ signaling in
single pancreatic acinar cells is markedly inhibited by injection of
antiserum directed against G
q
family members. This inhibition is evident even at physiological
concentrations of agonist where it is impossible to detect changes in
IP3 concentration. It has been
presumed that signaling at physiological concentrations of agonists is
the result of the activity of
G
q proteins since Ca2+ signaling events can be
inhibited by high concentrations of heparin, an antagonist of the
IP3 receptor (6, 29). This study
thus provides direct evidence that physiological concentrations of agonist result in the activation of
G
q family proteins.
As a consequence of the epitope that the antiserum is directed against,
the mechanism of immunoneutralization of this particular antiserum-protein interaction is likely to be a result of the antiserum
"masking" the region of the protein that is essential for
liganded-receptor recognition (21). Because the extreme COOH terminus
of Gq proteins is predicted to
be exposed in the intact (nonactivated) heterotrimer, the most
plausible mechanism for inhibition of
Ca2+ signaling would be that
binding of the antiserum to the intact heterotrimer inhibits the
dissociation of heterotrimer, thus preventing activation of the
effector. Alternatively, it is possible that binding of antiserum to
the activated
-subunit sterically hinders its interaction with
PLC-
. The lack of inhibition of responses in cells injected with
G
q and
G
11 (or combination)-specific antisera is likely due to the antiserum either not recognizing the
protein in its native form or binding to a region not important in its action.
In this study, it was also demonstrated that the
Gq-CT antiserum would inhibit
CCK-induced signaling in CHO cells to a similar marked extent (>75%)
compared with acini. This is of interest since this study has
demonstrated that acini but not CHO cells express
G
14 in addition to
G
q/G
11.
The similarity in the degrees of inhibition argues that the antiserum
also recognizes and neutralizes G
14 in acini, despite the two
amino acid substitutions in this region between
G
q/G
11
and G
14. In support of the
contention that the G
q-CT
antiserum recognizes G
14,
Milligan and colleagues (14) in a study designed to separate
G
q/G
11
on urea gradient gels showed that a similar antiserum would reveal
three bands in some cells, which the authors speculated probably
represented G
q,
G
11, and
G
14.
In conclusion, this study has demonstrated the expression of individual
members of the Gq
heterotrimeric G protein family and shown that activation of these
proteins is a key step in the generation of a
Ca2+ signal in pancreatic acinar
cells, stimulated by both physiological and pathophysiological
concentrations of agonists. Further studies are needed to determine if
G
q,
G
11, and
G
14 contribute equally and
interchangeably to the response to each pancreatic secretagogue or
whether specificity exists in signaling through a particular secretagogue receptor.
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ACKNOWLEDGEMENTS |
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We thank Nellie Park for technical support during the project. We also thank Dr. Craig Logsdon and Dr. Linda Samuelson for helpful discussion throughout the project.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-41122 (J. A. Williams) and DK-54568 (D. I. Yule), Michigan Diabetes Research and Training Center Grant P60-DK-20572, and Michigan Gastrointestinal Peptide Center Grant P30-DK-34933.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: D. I. Yule, Dept. of Pharmacology and Physiology, School of Medicine and Dentistry, Univ. of Rochester, Box 711, Rochester, NY 14642-8711.
Received 10 August 1998; accepted in final form 26 September 1998.
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