From the Margaret M. Dyson Vision Research Institute, Department of Ophthalmology, Cornell University Medical College, New York, New York 10021
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ABSTRACT |
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On the basis of sequence homology and structural
similarities, metabotropic glutamate receptors (mGluRs), extracellular
Ca2+-sensing receptor, -aminobutyric acid type B
receptor, and pheromone receptors are enlisted in a distinct family
within the larger G protein-coupled receptor superfamily. When
expressed in heterologous systems, group I mGluRs can activate dual
signal transduction pathways, phosphoinositides turnover and cAMP
production. To investigate the structural basis of these coupling
properties, we introduced single amino acid substitutions within the
second and third intracellular loops (i2 and i3) of mGluR1
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Wild-type and mutant receptors were expressed in human embryonic kidney
293 cells and analyzed for their capacity to stimulate both signaling
cascades. Each domain appeared to be critical for the coupling to
phospholipase C and adenylyl cyclase. Within i2, Thr695,
Lys697, and Ser702 were found to be selectively
involved in the interaction with Gq class
subunit(s),
whereas mutation of Pro698 and the deletion
Cys694-Thr695 affected only Gs
coupling. Furthermore, the mutation K690A profoundly altered mGluR1
signaling properties and imparted to the receptor the ability to couple
to the inhibitory cAMP pathway. Within i3, we uncovered two residues,
Arg775 and Phe781, that are crucial for
coupling to both pathways, since their substitution leads to receptor
inactivation.
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INTRODUCTION |
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Metabotropic glutamate receptors
(mGluRs)1 are coupled to
heterotrimeric G proteins, and through this interaction they regulate the intracellular level of second messenger molecules such as inositol
trisphosphate and cyclic AMP (cAMP). Furthermore, they have been shown
to modulate the activity of voltage-sensitive Ca2+ and
K+ channels, G protein-regulated inward rectifier
K+ channels, as well as GABAA,
-amino-3-hydroxy-5-methylisoxazole-4-propionate, and
N-methyl-D-aspartate receptors (1-2). Thus far,
eight mGluRs (mGluR1 through mGluR8; see Refs. 3-10) have been cloned.
They have been categorized into three groups according to their
sequence homology, agonist selectivity, and main signal transduction
pathway activated in heterologous systems (1, 11-12). Group I mGluRs (mGluR1 and -5) mobilize intracellular Ca2+ by stimulating
phosphoinositide (PI) turnover and promote cAMP accumulation; group II
(mGluR2 and 3) and group III (mGluR4, -6-8) receptors inhibit adenylyl
cyclase.
mGluRs, together with the parathyroid Ca2+-sensing
receptors (PCaR1 (13)) and the GABAB receptor (14), form a
separate family within the seven transmembrane domain G protein-coupled
receptor (GPCR) superfamily (15-17). mGluRs are characterized by a
large amino-terminal extracellular domain that comprises the glutamate binding site (18-19). The intracellular loops connecting the putative membrane-spanning helices are also distinctive. In mGluRs, they are
relatively small compared with those of seven transmembrane domain
receptors belonging to the rhodopsin/-adrenergic family. Most
importantly, there is no significant sequence homology between members
of the mGluR family and other cloned GPCRs.
The structure-function relationships supporting the coupling to G
proteins have been well investigated for members of the rhodopsin/-adrenergic family. Although all the cytoplasmic domains of these receptors take part in G protein activation to some degree, i3
appears to harbor the structural elements that impart specificity to
the interaction (20-21).
However, much less is known about the structural determinants of the coupling of mGluRs to G proteins. Recently, the analysis of chimeric receptors derived from the Gi-coupled mGluR3 bearing different portions of the cytoplasmic domains of the Gq-coupled mGluR1 has shown that i2 of group I receptors is necessary, but not sufficient, for the specific activation of phospholipase C (PLC) and that both i3 and the cytoplasmic tail of the receptor appear to be also necessary for efficient coupling to this pathway (22-23). The finding that a point mutation in i3 of the human PCaR1 is responsible for loss of receptor function (24) also suggests that this domain could be important for mGluR-G protein interaction.
In heterologous systems, group I mGluRs have been shown to stimulate cAMP accumulation (25-26). However, the domains of the receptor and the signaling partners involved in the activation of this pathway have not been characterized.
In this work, we introduced single amino acid substitutions within i2
and i3 of mGluR1 to identify specific residues that contribute to the
interaction with G proteins. To gain insights on the structural
elements involved in selective interaction with either pathway, we
tested the mutant receptors for their ability to induce PI turnover and
cAMP accumulation. Here we show that both i2 and i3 play a role in
determining selective coupling to Gs and Gq
class subunits and that mutations of single residues differentially
affect the activation of the two transduction pathways.
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EXPERIMENTAL PROCEDURES |
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Site-directed Mutagenesis--
Site-directed mutagenesis was
carried out by a modification of the polymerase chain reaction (PCR)
technique as described by Nelson and Long (27). The oligonucleotides
required for the mutagenesis (Ransom Hill Bioscience, Ramona, CA;
Table I) consisted of the following:
(a) forward mutagenic primers containing single/multiple base mismatches; (b) reverse hybrid primer serving as
3'-anchor, harboring at the 5'-end 20 nucleotides randomly selected and
unique and not complementary to the mGluR1 sequence and, at the
3'-end, 21 complementary nucleotides; (c) forward 5'-anchor
primer; (d) reverse complementary hybrid primer (c-primer)
identical to the random 20-mer. All polymerase chain reaction reactions
were carried out in a 50-µl reaction volume containing 200 µM deoxynucleotides and 2.5 units of Taq
polymerase (Boehringer Mannheim). In the Step 1 polymerase chain
reaction reaction, 50 pmol each of mutagenic and hybrid primer were
used with the template pRc-mGluR1
(pRc-CMV vector; Invitrogen, San
Diego, CA) to yield an amplified product comprising the mutation and
the unique sequence. Step 1 was 30 cycles of 1 min at 94 °C
denaturation, 3 min at 37 °C annealing, 3 min at 74 °C
elongation. The amplified DNA was fractionated on agarose gel,
purified, and used together with pRC-mGluR1
in the Step 2 reaction.
Step 2 was a single cycle of 5 min at 94 °C, 2 min at 37 °C, and
10 min at 74 °C. At Step 2 completion, 50 pmol each of the 5'-anchor
and c-hybrid primer was added, and 30 additional cycles were completed
(Step 3). Step 3 products were digested with BglII and
SphI for all mutants but i2* (Table I), which were digested
with NcoI and SphI and cloned into pBS-mGluR1
(pBlueScriptSK(
) vector; Stratagene, San Diego, CA). The sequence of
the amplified DNAs was determined by the Sanger method (Sequenase, U. S. Biochemical Corp./Amersham Life Science, Inc.). Mutant
receptors were subcloned from the pBlueScript construct into pRc-CMV by digestion with SacII and ApaI (i2 and i2*
mutants) or SacII and BspEI (i3 mutants).
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Expression in HEK 293 Cells--
HEK 293 cells were cultured in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal bovine serum (FBS). Cells were transfected by electroporation
with an Electro Cell Manipulator (BTX, San Diego, CA) using 4-mm gap
cuvettes. The DNA used in transfection experiments was purified with
Qiagen columns (Qiagen, Chatsworth, CA) according to the procedure
recommended by the manufacturer. In a standard experiment, cells were
harvested by trypsinization and washed 3 times with PBS; for each
electroporation 5 or 10 million cells were used with either 10 or 20 µg of DNA (according to the purpose of the experiment) in 0.3-ml
final volume. Electroporations were conducted at 260 V, 950 microfarads, 72 , yielding 20 ms as average time constant. The
transfection efficiency, about 30%, was determined by co-transfection
(1:1 ratio) with the pRc-lacZ vector and subsequent
chromogenic reaction. All the tissue culture reagents were purchased
from Life Technologies Inc. HEK 293 were generously provided by Dr. M. Chao at Cornell University Medical College.
Inositol Phosphates Accumulation--
Following electroporation,
cells were left to recover in DMEM supplemented with 10% dialyzed FBS
and 2 mM GlutaMAXTM (Life Technologies Inc.)
for 3-5 h and metabolically labeled for approximately 16 h with 1 µCi ml1 [3H]myo-inositol (NEN
Life Science Products) in inositol-free DMEM. Cells were washed with
PBS and incubated for 2 h with 2 mM pyruvate and 2 units/ml glutamic-pyruvic transaminase (Sigma) (28). After washing with
PBS, cells were incubated for 10 min in Hanks' balanced saline
solution containing 10 mM LiCl; 1 mM Glu
(Sigma) was then added for 20 min. Agonist stimulation was stopped by
placing the cells on ice and adding 5% ice-cold trichloroacetic acid.
Separation of total [3H]inositol phosphates was carried
out essentially as described (29) by chromatography on AG 1-X8
anion-exchange resin (Bio-Rad); total inositol phosphates were eluted
with 700 mM ammonium formate, 100 mM formic
acid.
In Vivo cAMP Accumulation--
Three hours after
electroporation, cells were metabolically labeled for approximately
16 h with 1 µCi ml1 [3H]adenine (NEN
Life Science Products) in DMEM supplemented with 10% dialyzed
FBS and 2 mM GlutaMAXTM. Cells were then washed
with PBS, treated with glutamic-pyruvic transaminase for 2 h (see
above), and preincubated for 30 min with Hanks' balanced saline
solution containing 1 mM 3-isobutyl-1-methylxanthine (Sigma). 1 mM Glu in the presence of
3-isobutyl-1-methylxanthine was then added for 20 min. Cells were lysed
with ice-cold 5% trichloroacetic acid, and adenine nucleotides were
fractionated according to the method of Salomon (30).
Immunofluorescence and Immunoblot Analysis--
For
immunofluorescence studies, electroporated cells were grown for up to
18 h in serum-containing medium on gelatin-coated coverslips to
facilitate adhesion. Cells were then washed with PBS containing 2 mM MgCl2, fixed for 2 min with ice-cold
methanol, and permeabilized with 0.4% Triton X-100. The primary
antibody M5-14 made in rabbit and directed to the carboxyl terminus of the rat mGluR1 (31) was diluted 1:400 and reacted with a fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody (Jackson
ImmunoResearch Laboratories, West Grove, PA). Coverslips were washed,
mounted on glass slides with Vectashield (Vector Laboratories,
Burlingame, CA), and analyzed using a Sarastro Phoibos 1000 confocal
laser scanning system attached to a Nikon microscope.
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RESULTS |
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Receptors belonging to the rhodopsin/-adrenergic family are
characterized by the presence of short amphipathic
-helices at the
amino and carboxyl termini of their third intracellular loop. Amino
acids within these microdomains play a key role in determining
selective coupling to heterotrimeric G proteins (20-21). Unlike these
GPCRs, mGluR1 i3 is small, non-homologous, and well conserved among all
members of the family, despite their different coupling properties
(Fig. 1a).
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To evaluate the contribution of single amino acid residues in i3 of
mGluR1 to G protein coupling, we mutagenized Arg775
(R775W and R775K), corresponding to Arg796 in PCaR1;
Pro778 (P778Q); Phe781 (F781P and F781S);
Asn782 (N782I); and Glu783 (E783Q). These
residues are conserved among all metabotropic and calcium-sensing
receptors and were substituted with amino acids designed to change the
charge distribution of the loop (Fig. 1b).
The only residue in i3 that distinguishes receptors coupled to different signal transduction pathways is Ala779, which is conserved in group I mGluRs, whereas Glu is present in groups II and III receptors. Since this seems to suggest that the amino acid at this position could participate in coupling selectivity, we replaced Ala779 with Glu (A779E).
Within i2 (the largest intracellular loop in mGluRs) only few amino
acids are in common between groups I and II/III receptors (Fig.
1c). The regions of homology are concentrated at the amino and carboxyl termini of the domain, which are predicted to adopt -amphipathic conformation. The intervening stretch of residues is
highly hydrophilic and therefore constitutes a potential protein interaction site. Previous studies conducted with chimeric receptors showed that the 16-amino acid region from Lys691 to
Gln706 is required to impart the ability to activate PLC to
Gi-coupled mGluRs (22-23). We mutagenized residues within
this region that, according to sequence alignment, are conserved among
all mGluRs (Lys690, Pro698, and
Ser702) and residues that are shared only by PLC-coupled
receptors (Lys692, Thr695, Arg696,
Lys697, and Ala705) (Fig. 1c). Among
them, the positively charged amino acids that are part of the central
hydrophilic region of i2 were substituted with hydrophobic residues to
perturb the charge distribution. Group I mGluRs also differ from group
II and III in the size of i2 (26 versus 24 amino acids). To
test whether such a difference is structurally relevant for G protein
coupling, we generated two deletion mutants. In the
Cys694-Thr695 mutant, Cys694 and
Thr695, which are shared only by group I receptors and are
located in the center of i2, were deleted. In the
K692G/Cys694-Thr695 mutant, the same deletion
was accompanied by the substitution of Lys692 to Gly; this
substitution reduces the number of positively charged residues located
in the amino terminus of i2, thus increasing the structural similarity
of the mutant mGluR1 with group II and III receptors.
Receptor-mediated Stimulation of PI Turnover--
To assess the
ability of the mutant receptors to activate the PLC signaling cascade,
we measured the accumulation of inositol phosphates (IPs) upon
stimulation with Glu in HEK 293 cells. Wild-type receptor activation
induces a 3-4-fold increase of IP production compared with basal level
(not-stimulated cells); the specificity of this response is confirmed
by the absence of effect of Glu in cells transfected with the reporter
construct pRc-lacZ alone (Fig.
2a). Consistent with recent
reports, mGluR1 expression generates a high basal level of inositol
phosphates; this effect, distinctive to the long splice variant of the
receptor, has been attributed by other investigators to intrinsic
(agonist-independent) activity (28).
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Receptor-mediated Stimulation of the cAMP Pathway--
Wild-type
and mutant receptors were tested for their ability to induce cAMP
accumulation in HEK 293 cells. The endogenous adenylyl cyclase isoform
in this cell line has not been identified yet; however, it is known to
be Gs and forskolin-activated but not sensitive to
Ca2+, protein kinase C, or stimulation (36). Upon
stimulation with Glu, mGluR1
induces a small but significant
increase in cAMP (Fig. 3, a
and b). The properties of the endogenous cyclase make it
likely that such effect is generated through direct activation of
Gs, although with low efficiency. This was ascertained by
co-transfecting the wild-type mGluR1
together with increasing amount
of the cDNA coding for Gs, which resulted in a
proportional increase of Glu-induced mGluR1
-mediated cAMP
accumulation (Fig. 3a).
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Properties of the K690A Mutant-- From the characterization of the K690A mutant activity profile, it became clear that Lys690 is critical for coupling to both Gq and Gs. In particular, in the K690A mutant the interaction with Gq is weakened, whereas that with Gs is strengthened compared with wild type.
It has been suggested that mGluR1
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Expression Level and Localization of mGluR1 and Mutant
Receptors--
To verify that the effects brought about by the amino
acid substitutions on receptor effector-function are not due to changes in the synthesis and/or localization of the protein, we analyzed the
expression level and distribution of wild-type and mutant receptors,
transfected in HEK 293 cells, by Western blot and immunofluorescence. In immunoblots, the polyclonal antibody M5-14 detects selectively the
receptor protein, thus yielding a single band of 145 kDa estimated molecular mass, as predicted for mGluR1
(Fig.
6a). The expression level of
the receptor increases in linear fashion with the amount of DNA
introduced into the cells. To detect variations in the syntheses of
wild-type and mutant receptors, we transfected HEK 293 cells with an
adequate amount of the corresponding DNAs (2.5 µg) to obtain a level
of protein expression within the linear range of increase (Fig.
6b). This analysis does not show any significant difference
in the level of synthesis of the mutant receptors tested compared with
the wild type.
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DISCUSSION |
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Physiological and pharmacological studies have shown that mGluRs
activate multiple second messenger cascades, inhibit neurotransmitter release, and regulate K+ and Ca2+ channel
activity (1, 38, 39). These multi-signaling effects are thought to be
mediated by multiple mGluRs. In addition, it is possible that some
receptors have the capacity to interact with several different subsets
of G protein subunits (3-4, 25). Furthermore, the associated
subunits could also contribute to specific signaling by
regulating channel opening (40), different isoforms of adenylyl cyclase
(36, 41), and PLC (42, 43). Hence, the final output of mGluR activation
in the central nervous system may also depend on the repertoire of
signaling molecules available in the vicinity of the receptor. This
could be particularly important in neurons, which can undergo
activity-dependent changes in dendritic and axonal
compartments.
In neurons, group I mGluRs have been shown to stimulate local intracellular calcium release via the PLC cascade and modulate other biochemical pathways and channel activities. This results from the stimulation of protein kinase C following the release of diacylglycerol. Although there is, thus far, no unequivocal demonstration for the direct involvement of group I mGluRs in the activation of the cAMP stimulatory pathway in vivo, evidence for the activation of the cAMP stimulatory pathway in cortical neurons following both group I and group II receptor activation has been presented (44-45). This discrepancy with studies conducted on receptors expressed in heterologous systems could be due to the paucity of pharmacological tools capable of discriminating selectively group I receptors.
Here, we focused on the signaling properties of mGluR1 in a
heterologous system, HEK 293 cells. In these cells we have demonstrated that the receptor activates at least two different signaling cascades, the PLC pathway (via interaction with Gq) and the
stimulatory cAMP pathway (via interaction with Gs).
Recently several other GPCRs, such as
2-adrenergic
receptors, muscarinic receptors, and the parathyroid hormone
(PTH)/PTH-related peptide receptor, have been shown to possess dual or
multiple effector functions. There is now accumulating evidence that
this multi-signaling activity is due to coupling to different G protein
subunits, sometimes leading to opposite effects, for example
Gs and Gi as in the case of the
2-adrenergic receptor (46). In several instances, the structural domains involved in differential coupling have been partially uncovered; however, it is not yet possible to draw general rules from these studies. For example, in the case of the
PTH/PTH-related peptide receptor, some residues within the amino
terminus of i3 appear to be independently involved either in PLC or
adenylyl cyclase stimulation, whereas mutation of other residues
affects both cascades (47). On the other hand, in the case of the
2-adrenergic receptor, both i2 and the amino terminus of
i3 are involved in stimulating cAMP production, and the determinants of
coupling to Gi are located in different regions of i3 (48,
49).
Since the mGluRs bear no sequence homology with the other GPCR families, their study, together with that of other GPCRs, will unravel conserved structural features involved in coupling to G proteins. By mutating amino acids that are shared by all mGluRs, we have identified two residues in i3, Arg775 and Phe781, that are required for productive coupling to both the PLC and stimulatory cAMP pathway. The finding that the R775W substitution can be "rescued" by replacing Arg775 with another positively charged residue points to a requirement for a positive charge at this position. A more dramatic phenotype is obtained by substituting Phe781 with either hydrophobic or polar residues; both mutations cause complete loss of function of the receptor. This finding suggests that Phe781 is a key residue at the surface of interaction between receptor and G proteins. Together these observations indicate that i3 could play a pivotal role in providing a site for interactions with both Gq and Gs. On the other hand, two other mutants in i3 appear to operate independently in their ability to couple to G proteins as follows: Pro778 seems to be important for a full Glu-dependent Gq stimulation, and Asn782 is required for a full activation of the stimulatory cAMP pathway.
Our study has uncovered several residues within i2 that are selectively
involved in either the PLC pathway or the stimulatory cAMP pathway,
suggesting the existence of two independent G protein-specific interaction domains. One example is the deletion of Cys694
and Thr695. These residues are shared only by group I
receptors and are located in the center of i2, between two predicted
-helical regions at the amino and carboxyl termini of this loop.
They appear to be necessary to support coupling to Gs but
not to Gq. Interestingly, the K690A substitution in i2
generates a receptor that is less effective than wild type in
activating Gq but more effective in activating
Gs. This suggests that Lys690 could be located
at a position critical for both Gs and Gq
coupling. Searching for common motifs shared by mGluR1
i2 and
Gs-coupled GPCRs, we found an interesting homology between
the amino terminus of i2 and the equivalent portion of the human
2A-adrenergic receptor (
2C10) i3, which
is known to be required for coupling to Gs (48). Sequence
alignment shows that the K690A substitution increases the sequence
similarity of mGluR1 with these receptors (Fig.
7).
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Remarkably, the K690A mutation also confers to mGluR1 the ability to
couple to Gi. Thus, a single amino acid residue appears to
act as a molecular switch for receptor-G protein coupling selectivity. A similar structural control of coupling specificity is mirrored in the
subunit of G proteins; in fact, the four carboxyl-terminal amino
acids of
q,
i2,
o,
z, and
s determine their coupling specificity (50), and only one of these four residues is different between
o and
i2. An alternative
explanation for the phenotype of the K690A mutant is that mGluR1
itself might be able to weakly stimulate Gi and that the
mutation enhances this interaction. This interpretation would be
consistent with the observation by Aramori and Nakanishi (25)
that the stimulation of cAMP accumulation induced by mGluR1
in CHO
cells is potentiated by PTx.
Several mutations introduced in i3 and i2 of mGluR1 appeared to
specifically affect either the constitutive or the
agonist-dependent activity of the receptor. As for several
GPCRs, mGluR1
has recently been shown to be able to stimulate IP
production in an agonist-independent fashion (28). This constitutive
activity is thought to result from the ability of the receptor to adopt
an active conformation in the absence of bound ligand. Our observations
suggest that by altering the conformation of the G protein-coupling
domains of the receptor, it is possible to specifically affect either the constitutive or the agonist-dependent activity. Similar
mutants have been described for several other GPCRs and have led to the formulation of the "allosteric ternary complex" model (51), and the
phenotype of the mutations described here can be accounted for by this
model. The extension of this model to mGluRs is particularly significant because mGluRs differ from other GPCRs in their mode of
ligand binding; indeed, the ligand binding site of mGluRs resides in
the large extracellular domain (18-19), whereas in most GPCRs it is
located in the transmembrane region. Because inverse agonists are not
yet available, the physiological relevance of the constitutive activity
of mGluR1
in neurons cannot be assessed.
Taken together, these findings suggest that for mGluRs, as for other
GPCRs, more than one domain is required to form the surface for the
interaction with G protein subunits. This improved understanding of
mGluR-G protein interaction will provide a valuable paradigm to analyze
the coupling properties of GABAB receptors, the recently discovered pheromone receptors (52-53), and possibly other related receptors yet to be discovered.
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ACKNOWLEDGEMENTS |
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We thank Drs. A. A. Hirano, L. R. Levin, J. A. Wagner, and J. G. Connolly for critical reading
of the manuscript; Dr. J.-P. Pin for having provided the mGluR1
clones and for sharing unpublished data. The mGluR2,
2-adrenergic receptor, and adenylyl cyclase type II
clones were kindly donated by Drs. S. Nakanishi and L. R. Levin.
We also thank Dr. X.-Y. Huang for providing the Gs clone. The mGluR1
antibody was a generous gift from Dr. C. Romano.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant EY09534 and by the Samuel and May Rudin Foundation.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. Section 1734 solely to indicate this fact.
Supported in part by a New York Academy of Medicine David Warfield
Fellowship in Ophthalmology and a Tolly Vinick Pilot Grant. To whom
correspondence should be addressed: Dyson Vision Research Institute,
P. O. Box 233, Cornell University Medical College, 1300 York Ave.,
New York, NY 10021. Tel.: 212-746-2209; Fax: 212-746-8101; E-mail:
afrance{at}med.cornell.edu.
§ Recipient of a Research to Prevent Blindness Career Development Award.
1
The abbreviations used are: mGluR, metabotropic
glutamate receptor; Glu, glutamate; PCaR1, parathyroid
Ca2+-sensing receptor; GPCR, G protein-coupled receptor;
GABAA or GABAB, -aminobutyric acid type A or
B; PLC, phospholipase C; i2/i3, second/third intracellular loop; HEK
293, human embryonic kidney 293 cells; DMEM, Dulbecco's modified
Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered
saline; IP, inositol phosphate; PI, phosphoinositides; PTx, pertussis
toxin; CHO, Chinese hamster ovary cells; PTH, parathyroid
hormone.
2 Developed at the National Institutes of Health and available by anonymous ftp from zippy.nimh.nih.gov.
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REFERENCES |
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