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Endocrinology and Reproduction Research Branch (S.J., R.D.S., A.J.B., K.J.C.), National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; Division of Cardio-Renal Products, Center for Drug Evaluation and Research, Food and Drug Administration, Rockville, Maryland 20857 (G.J.); and Department of Physiology, Semmelweis University of Medicine, H-1088, Budapest, Hungary (L.H.)
Address all correspondence and requests for reprints to: Dr. K. J. Catt, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 49, Room 6A-36, 49 Convent Drive, Bethesda, Maryland 20892-4510. E-mail: catt{at}helix.nih.gov
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the signaling events activated by the AT1-R have been well characterized (2), several other aspects of the receptor’s structure and function are still poorly understood. For example, although many GPCRs are known to be subject to post-translational modifications such as lipidation (3), phosphorylation (4), and glycosylation (5), the lipidation status of the AT1-R is unknown, and phosphorylation of endogenous (6) and expressed (7) AT1-Rs has only recently been demonstrated. Similarly, although the glycosylation status of several GPCRs has been investigated (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19), there have been no systematic studies of the nature and role of glycosylation in AT1-R function.
Each of the cloned mammalian AT1-Rs contains three putative Asn-Xaa-Ser/Thr consensus sequences (20) for glycosylation on asparagine residues. One potential site (Asn4) is situated near the N terminus of the receptor, while the other two sites (Asn176 and Asn188) are located on the second extracellular loop. However, it is not known which (if any) of these sites are glycosylated in vivo, and what role (if any) such putative glycosylation plays in AT1-R function. We therefore mutated the Asn4, Asn176, and Asn188 residues of an influenza hemagglutinin (HA) epitope-tagged rat AT1a-R (HA-AT1a-R) in various combinations (to lysine, glutamine, and glutamine, respectively) to generate three single-point mutant receptors (N4K, N176Q, and N188Q), three double-point mutant receptors (N4K/N176Q, N4K/N188Q, and N176Q/N188Q), and a triple-point mutant receptor (N4K/N176Q/N188Q). We then investigated the binding parameters, signaling capability, migration pattern, and immunoblotting signal in SDS-PAGE of each receptor transiently expressed in COS-7 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mutagenesis and transient expression of the rat AT1a
receptor
The influenza hemagglutinin (HA) epitope (YPYDVPDYA) was
inserted after the codons of the amino-terminal first two amino acids
(MA) into the complementary DNA (cDNA) of the rat AT1a
receptor subcloned into pcDNA3.1(+) (Invitrogen, San
Diego, CA) as previously described (6). Mutant HA-AT1a-Rs
were created using the Mutagene kit (Bio-Rad Laboratories, Inc., Hercules, CA) and verified by dideoxy sequencing using
Thermosequenase (Amersham Pharmacia Biotech).
COS-7 cells were seeded at 106 cells per 10 cm dish or 5 x 104 cells per 24-well culture plate in DMEM containing 10% (vol/vol) FBS, 100 µg/ml streptomycin, and 100 IU/ml penicillin (COS-7 cell medium) and maintained in a humidified atmosphere of 5% CO2 in air at 37 C for 3 days before use. Cells were transfected using 0.5 ml (24-well plate) or 5 ml (10-cm dish) of OptiMEM containing 10 µg/ml of LipofectAMINE and the required DNA (1 µg/ml) for 6 h at 37 C. After changing to fresh COS-7 cell medium, the cells were cultured for an additional 2 days before use.
Photoaffinity labeling of AT1-Rs
Cells in 10-cm dishes were incubated overnight at 4 C with the
high-affinity photoaffinity ligand,
125I-[Sar1(4-N3)Phe8]Ang
II (22) (
107 cpm/dish). After washing, cells were
exposed to UV light for 10 sec. Noncovalently bound
125I-[Sar1(4-N3)Phe8]Ang
II was removed by incubating the cells for 10 min in ice-cold 150
mM NaCl containing 50 mM acetic acid. After
additional washes in ice-cold PBS, dishes were drained and the cells
were scraped into lysis buffer (LB-: 50 mM Tris, pH 8.0,
100 mM NaCl, 5 mM EDTA, 10 µg/ml aprotinin,
10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml
pepstatin, 10 µg/ml benzamidine, 1 mM AEBSF) and
probe-sonicated (Sonifier Cell Disruptor: Heat Systems Ultrasonics,
Plainview, NY) for 45 sec. After removal of nuclei by centrifugation
for 10 min at 750 x g, membranes were collected by
centrifugation for 45 min at 200,000 x g.
Immunoprecipitation of AT1-Rs
Membrane pellets from BAG cells or
HA-AT1a-R-expressing COS-7 cells were solubilized in
ice-cold LB+ [LB- supplemented with 1% (vol/vol) NP 40, 1% (wt/vol)
Na deoxycholate, and 0.1% (wt/vol) SDS] by Dounce homogenization.
After clarification for 10 min at 10,000 x g, 1 µl
of the HA.11 antibody (for HA-AT1a-Rs) or 5 µl of an
anti-AT1-R antibody (6) (for endogenous
AT1-Rs), together with 2% (vol/vol) protein A Sepharose,
were added to the supernatants overnight at 4 C with tumbling. Immune
complexes were collected by centrifugation and washed three times with
ice-cold LB+ lacking protease inhibitors. After the final wash, immune
complexes were eluted into sample buffer (23) for 1 h at 48 C.
After resolution by SDS-PAGE, photoaffinity-labeled AT1-Rs
were visualized using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
Immunoblotting of HA-AT1a-Rs
Membrane pellets from HA-AT1a-R-expressing COS-7
cells were solubilized into sample buffer (23) and heated to 48 C for
1 h, and constituent proteins were resolved by SDS-PAGE. After
transfer, polyvinylidene fluoride (PVDF) membranes were incubated for
1 h at room temperature in Tris-buffered saline (TBS)
containing 0.05% (vol/vol) Tween-20 and 5% (wt/vol) dried milk
protein (TBST/5% milk). Blocked membranes were then incubated with the
HA.11 antibody (1 in 1000) in TBST/5% milk for 1 h, washed
for 30 min in TBST, and then incubated in TBST/5% milk containing
peroxidase-conjugated goat antimouse antibody (Kirkegaard & Perry Laboratories, Gaithersburg, MD) at 1 in 5000 for 30 min.
After a final 30-min wash in TBST, immune complexes were visualized
using enhanced chemiluminescence (Kirkegaard & Perry Laboratories).
Radioligand binding assays
HA-AT1a-R-expressing COS-7 cells in 24-well plates
were incubated in DMEM/25 mM HEPES (pH 7.4) containing
125I-[Sar1,Ile8]Ang II for 3
h at room temperature. After two washes with ice-cold PBS, cells were
harvested in 0.5 N NaOH/0.05% SDS, and radioactivity was
measured by 
-spectrometry. Scatchard analysis of binding competition
data was performed using the LIGAND program. Membranes prepared
from HA-AT1a-R-expressing COS-7 cells were subjected to
kinetic 125I-Ang II binding assays as previously described
(24). Kinetic data were analyzed by the PRISM program (Graphpad Software, Inc., San Diego, CA).
Inositol phosphate production
Transfected COS-7 cells in 24-well plates were labeled by
overnight incubation in inositol-free DMEM containing 0.1% (wt/vol)
BSA, 2.5% (vol/vol) FBS, antibiotics, and 20 µCi/ml
myo-[2-3H]inositol. After washing and
preincubation at 37 C with 10 mM LiCl for 30 min, 100
nM Ang II was added for an additional 20 min. Inositol
phosphates were extracted as described (24) and applied to AG 1-X8
columns (Bio-Rad Laboratories, Inc.). After washing three
times with water and twice with 0.2 M ammonium formate, the
combined inositol bisphosphate + inositol trisphosphate
fractions were eluted with 1 M ammonium formate in 0.1
M formic acid, and radioactivities were determined by
liquid scintillation counting.
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Results |
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Immunoprecipitation of HA-AT1a-Rs (Figs. 1
and 2)
gave better resolution than was achieved when solubilized membranes
were resolved by SDS-PAGE (Figs. 2 and 4A). It is apparent from Fig. 1
that the photoaffinity-labeled wild-type HA-AT1a-R migrates
as a very broad band with an apparent Mr of 100,000 to
200,000. Since the deduced molecular mass of the rat
AT1a-R protein is only 41 kDa (25), the additional apparent
Mr of the receptor is presumably attributable to
post-translational glycosylation. This was confirmed when the
photoaffinity-labeled triple-point mutant receptor, N4K/N176Q/N188Q
(which lacks any N-linked glycosylation sites), was found to migrate as
a doublet with an approximate Mr of 40,000 (Fig. 1), which
is consistent with the deduced size (41 kDa) (25) of the
AT1a-R protein. Similarly, enzymatic deglycosylation of the
photoaffinity-labeled HA-AT1a-R with
peptide-N-glycosidase-F [PNGase F; which cleaves N-linked
carbohydrate from protein (26)] also gave rise to a doublet with an
Mr of approximately 40,000. A similar doublet was also
present in immunoblots of the deglycosylated receptor (Fig. 2).
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-N-acetylgalactosaminidase
[which cleaves O-linked carbohydrate moieties from protein (27)], on
the migration of the photoaffinity-labeled HA-AT1a-R in
SDS-PAGE (data not shown). However, since the endogenous
AT1-R from BAG cells migrates as a relatively discrete band
of Mr 60,000–65,000, but (like the expressed
HA-AT1a-R) also runs as a doublet with an Mr of
approximately 40,000 after deglycosylation (Figs. 2
and 3), it is also apparent that the
expressed receptor is glycosylated to a greater extent in COS-7 cells
than is the endogenous receptor in BAG cells. Furthermore, the very
broad migration pattern of the expressed (compared with the endogenous)
receptor indicates a greater degree of variability in the nature and/or
extent of glycosylation of the former (compared with the latter)
receptor.
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), this smearing probably resulted from
the presence of similarly migrating (and overlapping) partially
deglycosylated intermediates.
We therefore attempted to demonstrate partially deglycosylated
intermediates using the endogenous AT1-R in BAG cells,
since this receptor runs more discretely in SDS-PAGE (Figs. 2 and 3).
Membranes prepared from photoaffinity-labeled BAG cells were incubated
for 1–40 min with 10 U/ml PNGase F, or for 40 min with 0.1–10 U/ml of
PNGase F. Like the nonglycosylated N4K/N176Q/N188Q receptor in COS-7
cells, complete deglycosylation of the BAG cell AT1-R gave
rise to a doublet with an Mr of approximately 40,000,
although the use of a 10% linear resolving gel in the experiment shown
in Fig. 3 produced greater separation of the doublet’s component bands
than was seen for the expressed receptor using 8–16% gradient gels
(Figs. 1 and 2). Incomplete deglycosylation of the BAG cell
AT1-R with PNGase F revealed two (slow and fast) migrating
partially deglycosylated intermediate species with approximate
Mrs of 50,000–55,000 and 43,000–45,000, respectively.
Since PNGase F completely removes the entire carbohydrate moiety from
glycosylated asparagine residues (27), the time- and
concentration-dependent progression from the fully glycosylated
receptor, through slow and (subsequently) fast migrating intermediates,
to the fully deglycosylated doublet (Fig. 3) indicates the sequential
removal of carbohydrate from each of three sites. All three of the
potential sites therefore appear to be glycosylated in BAG cell
AT1-Rs. The relatively broad migration pattern of the
faster intermediate (which, presumably, contains receptors glycosylated
at only one site) also indicates some heterogeneity in the nature
and/or extent of glycosylation at individual sites of the endogenous
AT1-R in BAG cells, although to a lesser extent than for
the expressed HA-AT1a-R in COS-7 cells (Fig. 1).
Immunoblotting of mutant HA-AT1a-Rs
We evaluated the relative amounts of each mutant
HA-AT1a-R by employing the HA epitope tag to detect total
cell receptor in immunoblots of membranes prepared from
photoaffinity-labeled COS-7 cells. After detection of cell surface
photoaffinity-labeled HA-AT1a-Rs in the PhosphorImager, the
membrane was probed with the anti-HA antibody to detect total (cell
surface + intracellular) receptor. No receptor was detected in cytosol
prepared from HA-AT1a-R-expressing COS-7 cells (data not
shown). It is apparent from Fig. 4A that
the amounts of photoaffinity-labeled receptor varied widely between the
mutant receptors.
Immunoblotting with the anti-HA antibody (Fig. 4B) confirmed that the
very broad migration pattern of the HA-AT1a-R and its
mutants in SDS-PAGE was due to the presence of multiple bands that
comigrated with the photoaffinity-labeled receptor (Fig. 4A). The
identity of these bands as the receptor was confirmed by the absence of
immunoreactive bands in membranes prepared from (non-HA-tagged)
AT1a-R-expressing COS-7 cells (Fig. 4B). In addition,
treatment of solubilized membranes with PNGase F before immunoblotting
resulted in the disappearance the multiple bands and the appearance of
a doublet with an Mr of approximately 40,000 (Fig. 2).
There was, in general, a correlation between the relative intensities
of immunoreactive receptors and their relative intensities of
photoaffinity labeling. In addition, an increasing number of mutated
glycosylation sites correlated (in general) with decreasing expression,
such that the triple-point mutant receptor gave no discernible signal
in either immunoblotting or photoaffinity labeling.
However, there was a marked discrepancy between the intensity of
photoaffinity labeling for the N4K and N176Q single-point mutant
receptors and their signal intensities in immunoblotting. Whereas the
intensity of photoaffinity labeling for the N4K receptor was greater
than for the N176Q receptor (Fig. 4A), the immunoblotting signal of the
N176Q receptor was greater than that of the N4K receptor (Fig. 4B).
This unexpected finding prompted us to investigate the binding
characteristics of the mutant HA-AT1a-Rs.
Binding characteristics of mutant HA-AT1a-Rs
The specific binding of the peptide antagonist,
125I-[Sar1,Ile8]Ang II, to each
of the mutant HA-AT1a-Rs was less than to the wild-type
receptor, and its binding to the double-point mutant receptors (31%,
21%, and 36% of wild-type for the N176Q/N188Q, N4K/N176Q, and
N4K/N188Q receptors, respectively) was generally lower than to the
single-point mutant receptors (30%, 47%, and 60% of wild-type for
the N4K, N176Q, and N188Q receptors, respectively) (Fig. 5). Specific binding to the triple-point
mutant receptor, N4K/N176Q/N188Q, was very low (4% of wild-type). The
magnitude of specific
125I-[Sar1,Ile8]Ang II binding
generally correlated with the amount of immunoreactive receptor and was
consistent with the results of Scatchard analysis (not shown) of
125I-[Sar1,Ile8]Ang II binding
competition data, which showed a lower number of receptors [but no
significant differences in dissociation constant (KD)
values] for the N176Q and N188Q receptors compared with wild-type.
However, the binding data for the N4K mutant receptor did not transform
to linearity in Scatchard analysis (data not shown). Therefore, to
compare the binding affinity of the N4K mutant receptor with that of
the wild-type receptor, we measured the rates of association and
dissociation of 125I-Ang II to membranes prepared from
COS-7 cells expressing each receptor.
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), but also
that it dissociated more slowly from the N4K mutant receptor than it
did from the wild-type receptor (Fig. 6B). Further analysis of this
kinetic binding data revealed that the rates of
125I-Ang II association and dissociation at the N4K mutant
receptor were approximately twice and half, respectively, of those at
the wild-type receptor. These data suggest that the binding affinity of
the N4K mutant receptor for agonist ligands is approximately 4-fold
greater than that of the wild-type receptor. Similar results were
obtained when an S6A mutant HA-AT1a-R, containing a
mutation to alanine at Ser6 (which forms part of the
consensus sequence for glycosylation at Asn4), was used in
place of the N4K mutant receptor (data not shown). Hence, the
apparently higher affinity of the N4K mutant receptor probably explains
its relatively greater photoaffinity labeling compared with the
wild-type receptor (Fig. 3A), despite its lower expression level (Fig. 3B). Conversely, in view of its unchanged binding affinity compared
with the wild-type receptor, the relatively low photoaffinity labeling
of the N176Q receptor compared with its signal intensity in
immunoblotting suggests that glycosylation at this site may play a
permissive role in the delivery of HA-AT1a-Rs to the plasma
membrane.
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), the magnitude of the maximal response
for the single- and double-point mutant receptors was, in general, less
than that of the wild-type receptor, and the maximal response of the
triple-point mutant receptor was small (Fig. 7B). However, when data
were normalized to B0 values derived from
125I-[Sar1,Ile8]AngII binding to
replicate cells, it was apparent that each mutant receptor stimulated
similar or greater inositol phosphate production than the wild-type
receptor (Fig. 7C). When the concentration dependency of Ang
II-stimulated inositol production was examined, the EC50
values for inositol phosphates production were unaffected by mutation
of the putative glycosylation sites (Fig. 7A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of N-linked glycosylation has been investigated for a number of GPCRs including the H2 histamine (8), LH (9), ß-adrenergic (10), m2 muscarinic (11), calcium (12), V2 vasopressin (13), TSH (14), GnRH (15), PTH (16), platelet-activating factor (17), vasoactive intestinal peptide (18), and glucagon-like peptide 1 (GLP-1) (19) receptors. In most cases, impaired glycosylation (by site-directed mutagenesis or treatment of cells with tunicamycin) caused a reduction in cell surface receptor expression but had little effect on ligand binding or signaling ability. Notable exceptions were the glycosylation-deficient receptors for calcium (12) and the glycoprotein hormones, GLP-1 (19), LH (9), and TSH (14), which, in addition to reduced surface expression, also displayed impaired ligand binding and signaling abilities. However, for most GPCRs, impaired glycosylation is associated with reduced cell surface expression of otherwise normal receptors.
In only two studies did the authors compare the reduced cell surface expression of receptor with the total amount of receptor in the cell, or with the rate of receptor synthesis. Thus, reduced surface expression of the vasoactive intestinal peptide receptor was associated with its sequestration in the perinuclear endoplasmic reticulum (ER) (18), and the reduced surface expression of the platelet-activating factor receptor was associated with a normal rate of receptor synthesis (17). Our use of the HA epitope tag to detect total (cell surface + intracellular) receptor in immunoblots, which enabled us to make a comparison between the amount of total and cell surface receptor, indicated that reduced cell surface expression of glycosylation-deficient HA-AT1a-Rs correlates with a reduction in the total amount of receptor in the cell and is not due to any intracellular sequestration of receptors. However, our attempts to assess the rate of receptor synthesis by immunoprecipitation of mutant HA-AT1a-Rs from 35S-methionine pulse-labeled cells were unsuccessful, largely due to low incorporation of the radiolabel. Reduced expression of glycosylation-deficient HA-AT1a-Rs must result either from reduced receptor synthesis and/or from enhanced receptor degradation. However, since nascent membrane proteins are glycosylated cotranslationally by enzymes located in the lumen of the ER (5, 20), whereas protein synthesis occurs on ribosomes tethered to the cytoplasmic side of the ER, it is difficult to envisage how impaired glycosylation of nascent HA-AT1a-Rs in the lumen could exert an inhibitory effect on receptor translation on the ribosome.
In view of the known protection from degradation that glycosylation confers on circulating glycoproteins (20), it seems reasonable to assume that glycosylation protects nascent and/or mature HA-AT1a-Rs from proteolytic degradation in the ER. Although single-site glycosylation at Asn4, Asn176, or Asn188 is sufficient to confer some protection from degradation (compared with the nonglycosylated N4K/N176Q/N188Q receptor), further protection is provided by glycosylation at one or two additional sites. The mechanism(s) of such putative degradation of glycosylation-deficient HA-AT1a-Rs, and the exact site(s) at which it may occur, are unknown. However, since no N4K/N176Q/N188Q receptors (and little of the double-point mutant receptors) were detectable in immunoblots, it is likely that proteolysis occurs rapidly after receptor synthesis. In contrast, our findings also indicate that glycosylation is not required for the normal membrane insertion and folding of the HA-AT1a-R, for its trafficking and delivery to the plasma membrane, or for ligand binding and signaling.
The very broad migration pattern of the expressed HA-AT1a-R in SDS-PAGE (compared with the endogenous BAG cell AT1-R) was due to a greater degree of glycosylation of the former (compared with the latter) receptor, although the reason(s) for this difference is unclear. Although only 5–10% of cells in a COS-7 culture express the HA-AT1a-R (6), those that do so express the receptor at a high level that is probably 10- to 30-fold greater than that of the endogenous AT1-R in individual BAG cells. It is possible, therefore, that overexpression of the receptor in individual COS-7 cells results in the saturation of one or more of the enzymes involved in the glycosylation of HA-AT1a-Rs. After their initial N-linked attachment to nascent membrane proteins in the ER, carbohydrates are subsequently modified and trimmed to produce mature glycoproteins (5, 20). Saturation of the trimming enzymes by overexpressed HA-AT1a-Rs in COS-7 cells might therefore result in retention of the larger immature carbohydrates on the expressed receptor compared with the smaller, fully mature carbohydrates on the endogenous receptor.
For the same reason, a mixed population of HA-AT1a-Rs bearing either modified or unmodified carbohydrate moieties at one, two, or three sites might account for the greater heterogeneity (and broader migration pattern) of the expressed, compared with the endogenous, receptor. In support of this hypothesis, each double-point mutant receptor migrated as a broad band with an approximate Mr of 55,000–110,000, indicating considerable variability in the nature and/or extent of glycosylation at each site. However, the broad migration patterns of the partially deglcosylated BAG cell AT1-R intermediates also indicate some heterogeneity in the nature and/or extent of glycosylation of the endogenous receptor.
Interestingly, the deglycosylated photoaffinity-labeled HA-AT1a-R ran as a doublet in SDS-PAGE, as did the deglycosylated receptor in immunoblotting. Since cells were transfected with a single DNA species, it is unclear why the deglycosylated HA-AT1a-R migrates as a doublet, although it is possible that the two bands result from a (nonglycosylation) post-translational processing event, such as lipidation, of a subset of receptors. However, this seems unlikely since the only potential attachment site of a lipid anchor to the HA-AT1a-R (at Cys355) is absent from a mutant receptor truncated at Ser335, yet this receptor also runs as a doublet in SDS-PAGE (data not shown).
In conclusion, we have demonstrated that the HA-AT1a-R can be glycosylated at all three of its putative N-linked glycosylation sites when expressed in COS-7 cells, and that all three sites appear to be glycosylated on the endogenous AT1-R in BAG cells. Heterogeneity in the nature and/or extent of glycosylation, which is greater for the expressed receptor compared with the endogenous receptor, accounts for the broad migration patterns of AT1-Rs in SDS-PAGE. An increasing number of mutated glycosylation sites was associated with decreasing cell surface receptor expression, which was very low for the unglycosylated N4K/N176Q/N188Q receptor. In general, the degree of cell surface receptor expression was correlated with the total amount of cell receptor, suggesting that glycosylation may protect nascent receptors from intracellular proteolytic degradation. However, those glycosylation-deficient receptors that appeared on the plasma membrane functioned normally, indicating that glycosylation is not necessary for normal receptor-ligand binding and signaling and is not a prerequisite for the normal delivery of HA-AT1a-Rs to the plasma membrane. Future studies will endeavor to determine the mechanism(s) and site(s) of degradation of glycosylation-deficient HA-AT1a-Rs.
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Acknowledgments |
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Footnotes |
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2 Supported in part by an International Research Scholar’s Award
(HHMI 75195–541702) from the Howard Hughes Medical Institute.
Received October 9, 1998.
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) or N4K mutant (•) HA-AT1a-R-expressing
COS-7 cells were incubated with 125I-Ang II in the presence
or absence of an excess (10 µM) of unlabeled
[Sar1,Ile8]Ang II for the indicated times at
25 C. At each time point, membranes were collected and specific
125I-Ang II binding was determined. In panel B, membranes
were incubated with 125I-Ang II for 60 min at 25 C before
the addition of an excess (10 µM) of unlabeled
[Sar1,Ile8]Ang II. Specific
125I-Ang II binding to wild-type (
), N176Q (
),
and N188Q (•) mutant HA-AT1a-Rs is shown in panel A.
Maximal production of [3H]inositol phosphates for the
double- and triple-point mutant HA-AT1a-Rs is shown in
panel B. Maximal data for [3H]inositol phosphates
normalized to an equal number of receptors are shown in panel C. Data
represent the mean (± SEM) from three independent
experiments.