A Novel Mutation Adjacent to the Switch III Domain of Gs
in a Patient with Pseudohypoparathyroidism
Dennis R. Warner,
Pablo V. Gejman,
Regina M. Collins and
Lee S. Weinstein
Membrane Biochemistry Section (D.R.W.), Laboratory of Molecular
and Cellular Neurobiology, National Institute of Neurological
Disorders and Stroke, Clinical Neurogenetics Branch (P.V.G.),
National Institute of Mental Health, Metabolic Diseases Branch
(R.M.C., L.S.W.), National Institute of Diabetes and Digestive and
Kidney Diseases, National Institutes of Health, Bethesda,
Maryland 20892
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ABSTRACT
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A novel Gs
mutation
encoding the substitution of arginine for serine 250
(Gs
S250R) was identified in a patient with
pseudohypoparathyroidism type Ia. Both Gs
activity and Gs
expression were decreased by
about 50% in erythrocyte membranes from the affected patient. The cDNA
of this Gs
mutant, as well as one in which
the S250 residue is deleted (Gs
-
S250),
was generated, and the biochemical properties of the products of
in vitro transcription/translation were examined. Both
mutants had a sedimentation coefficient similar to that of wild type
Gs
(
3.7S) when kept at 0 C after
synthesis. However when maintained for 12 h at 3037 C, both mutants
aggregated to a material sedimenting at
6.3S or greater
(Gs
-S250R to a greater extent than
Gs
-
S250), while wild type
Gs
sedimented at
3.7S, suggesting that
the mutants were thermolabile. Incubation in the presence of high doses
of guanine nucleotide partially prevented heat denaturation of
Gs
S250 but had no protective effect on
Gs
-S250R. Sucrose density gradient
centrifugation at 0 C in the presence and absence of ß
-dimers
demonstrated that, in contrast to wild type
Gs
, neither mutant could interact with
ß
. Trypsin protection assays revealed no protection of
Gs
-S250R by GTP
S or
AlF4- at any temperature.
GTP
S conferred modest protection of
Gs
-
S250 (
50% of wild-type
Gs
) at 30 C but none at 37 C, while
AlF4- conferred slight
protection at 20 C but none at 30 C or above. Consistent with this
result, Gs
-
S250 was able to stimulate
adenylyl cyclase at 30 C when reconstituted with
cyc- membranes in the presence of GTP
S but
not in the presence of
AlF4-.
Gs
-S250R showed no ability to stimulate
adenylyl cyclase in the presence of either agent. Stable transfection
of mutant and wild-type Gs
into
cyc- S49 lymphoma cells revealed that the
majority of wild type Gs
localized to
membranes, while little or no membrane localization occurred for either
mutant. Modeling of Gs
based upon the
crystal structure of Gt
or
Gi
suggests that
Ser250 interacts with several residues within
and around the conserved NKXD motif, which directly interacts with the
guanine ring of bound GDP or GTP. It is therefore possible that
substitution or deletion of this residue may alter guanine nucleotide
binding, which could lead to thermolability and impaired function.
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INTRODUCTION
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Heterotrimeric G
proteins1 couple heptahelical
receptors (receptors with seven membrane-spanning regions) to
intracellular effectors and are composed of three subunits,
, ß,
and
(reviewed in Refs. 13). In the inactive state the
-subunit
is associated with a tightly but noncovalently bound ß
-dimer.
Binding of hormone to receptor effects a conformational change in the G
protein
-subunit causing it to release GDP with concomitant binding
of the activator GTP and dissociation from ß
. The GTP-bound
-subunit can then interact with specific enzymes or ion channels.
For the stimulatory G protein, GS, these include the
stimulation of adenylyl cyclase and modulation of ion channels (4, 5).
In addition, the ß
-subunit itself can modulate effectors (6).
Hydrolysis of bound GTP by an intrinsic GTPase activity returns the
-subunit to the basal, GDP-bound, state. Recently, x-ray
crystallography has revealed details of the structure of heterotrimeric
G-proteins (specifically, Gt
and Gi
1) and
allowed prediction of a GTPase mechanism by comparing the activated
(GTP
S- or AlF4- bound) and unactivated
(GDP-bound) species (7, 8, 9, 10, 11, 12). Gt
and Gi
1
have two domains, one with a ras-like conformation encoding
the structural elements necessary for guanine nucleotide
binding/hydrolysis and effector interaction. The other domain is
predominantly helical and may function to inhibit the release of GDP in
the inactive state (11). Upon GTP binding, an active conformation is
attained by the movement of three regions (named switch I, II, and III)
to perform the functions associated with activation (10). Switches I
and II contain the residues that position the catalytic water molecule,
while switch II contains residues that, along with the amino terminus,
interact with the ß
-subunit. Switch III appears to play no direct
role in GTP hydrolysis, but stabilizes switch II while in the GTP-bound
state and also provides contact points with the helical domain,
important for the high affinity binding of GTP (7).
Heterozygous inactivating mutations of the gene encoding
Gs
(GNAS1) are the molecular basis for Albright
hereditary osteodystrophy (AHO, Refs. 13 and 14), a syndrome
characterized by short stature, obesity, and in some cases,
subcutaneous ossifications and mental retardation. Within AHO kindreds,
patients may have the somatic features of AHO alone
(pseudopseudohypoparathyroidism, PPHP) or AHO in association with
resistance to multiple hormones (e.g. PTH or TSH) that
activate Gs-coupled pathways in their target tissues
[pseudohypoparathyroidism (PHP) type Ia] (1). Gs
mutations that lead to PHP type Ia and PPHP are heterogeneous and may
affect GNAS1 mRNA processing or produce inactive or altered proteins
(15). One example of the latter includes a C-terminal mutation of
Gs
(Arg385
His
substitution)2 found in PHP
type Ia that uncouples it from receptors (16). A Gs
mutation that causes both testotoxicosis, AHO, and resistance to PTH
and TSH encodes an Ala366
Ser substitution and interferes
with guanine nucleotide binding (17), resulting in thermolability at
normal body temperature but activation at the slightly lower testicular
temperature. Recently, an Arg231
His substitution of
Gs
was reported by Farfel et al. (18), which
disturbs the interaction with ß
or alters the receptor-induced
conformational change, but leaves the remaining functions intact (18).
We have identified a novel Gs
missense mutant from a
patient with PHP type Ia and AHO where Ser250 is
substituted with an Arg residue. This mutant Gs
does not
bind ß
or stimulate adenylyl cyclase and is thermolabile. Based on
sequence homology with Gt
and Gi
1,
Ser250 in Gs
is predicted to be positioned
between switches II and III, at the juxtaposition of three ß-sheets,
ß4, ß5, and ß6, which come together at the GDP-binding pocket
(10). Modeling suggests that the mutation may alter the tertiary
structure of the guanine nucleotide-binding pocket, resulting in
defective guanine nucleotide binding, manifested as an enhanced
thermolability.
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RESULTS
|
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Examination of erythrocyte membranes from the affected
patient with PHP type Ia revealed an approximate 50% deficiency in
both Gs bioactivity, as measured by the cyc-
reconstitution assay, and expression of Gs
protein, as
determined by immunoblot (see Fig. 1
).
The patients mother, who does not have clinical evidence of AHO or
hormone resistance and who does not have a GNAS1 mutation (see below),
showed no decrease in Gs bioactivity or expression. The
GNAS1 gene was screened for mutations by denaturing gradient gel
electrophoresis (DGGE) analysis of PCR-amplified genomic DNA fragments
encompassing GNAS1 exons 213 and their intron-exon splice junctions
(14, 19). DGGE analysis of a genomic fragment encompassing GNAS1 exons
10 and 11, which was amplified from the patients genomic DNA,
revealed a wild type band and two slower migrating bands that were not
present in the mothers sample or in numerous other normal control or
patient samples (see Fig. 2
). By direct
sequencing of the genomic DNA fragment (see Fig. 3
), the patient was shown to have a
heterozygous single base substitution (C to G) within the coding region
of exon 10 that encodes the substitution of Arg for Ser at codon 250
(Gs
-S250R). This mutation creates an EcoRII
restriction site. Digestion of PCR-amplified genomic DNA fragments with
EcoRII confirmed the presence of the mutation in the patient
and its absence in her unaffected mother (data not shown). The
biochemical and genetic results confirm the diagnosis of PHP type
Ia.

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Figure 2. DGGE Analysis of the GNAS1 Exon 1011 Genomic
Fragment with a GC Clamp Attached to the 3'-End
Genomic DNA fragments encompassing GNAS1 exons 10 and 11 were
PCR-amplified with a GC clamp attached to the 3'-end to increase the
sensitivity of DGGE. The oligonucleotide primers used for PCR were
5'-AAGAATTCTTAGGGATCAGGGTCGCTGCTC-3' (upstream primer) and
5'CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCCATGAACAGCCAGCAAGAGTGGA
3' (downstream primer with GC clamp) with the underlined
sequences complementary to the GNAS1 gene (14, 19). DNA fragments were
analyzed by DGGE as previously described (14). The patient (PHP) sample
had a wild type band present in normals as well as two slowly migrating
bands. The patients unaffected mother (M) showed only the wild type
band. Electrophoresis of DNA samples in a nondenaturing 5% acrylamide
gel revealed only a single band (data not shown).
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Figure 3. Direct Sequencing of the Exon 1011 Genomic
Fragment
Genomic DNA was amplified by PCR using the same oligonucleotides as for
DGGE and directly sequenced. Whereas the direct sequence of genomic DNA
amplified from a normal subject (left) reveals only an
AGC triplet at codon 250 (Ser), the corresponding sequence in the
affected patient (PHP, right) reveals a G and C at the
third position of the codon. This indicates a heterozygous single base
substitution that encodes the substitution of Arg (AGG) for Ser (AGC)
at codon 250 (19). The mutation creates a new EcoRII
restriction site, and the presence of the mutation in the PHP patient
was confirmed with an EcoRII restriction digest (data
not shown).
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RNA from whole blood was isolated, and the relative expression of the
mutant and wild type allele in the patient was assessed by analysis of
RT-PCR products. Direct sequencing of RT-PCR product amplified from RNA
of the patient showed that the mutant and wild type allele were
equivalently expressed (data not shown). EcoRII digests of
cloned RT-PCR products showed eight of 18 clones to be mutant,
consistent with equal expression of both alleles. In contrast, as noted
above, the expression of Gs
protein in membranes is only
50%. Results in stably transfected cyc- cells (see below)
suggests that this is not likely due to accumulation of the mutant
protein in the cytosol.
We cloned Gs
-S250R into the transcription vector
pBluescript for in vitro transcription/translation and into
the transfection vector, pZEM228c for expression in S49
cyc- cells. We also created another Gs
Ser250 mutant in which this residue was deleted
(Gs
-
S250). We characterized both this mutant and
Gs
-S250R after in vitro translation and after
expression in S49 cyc- cells. Gs
-S250R was
refractory toward stimulation of adenylyl cyclase, both after in
vitro translation/cyc- reconstitution and after
stable expression in cyc- (Table 1
). Like Gs
-S250R,
Gs
-
S250 was unable to stimulate adenylyl cyclase in
cyc- transfectants. However, Gs
-
S250 did
exhibit low but reproducible activity after in vitro
translation/cyc- reconstitution in response to GTP
S,
but not AlF4- (Table 1
). Additionally,
accumulation of cAMP in
Gs
-
S250/cyc- cells after
stimulation with either isoproterenol or cholera toxin was similar to
mock-transfected cyc- cells (data not presented).
Immunoblotting of stable cyc- transfectant homogenates
after centrifugation at 100,000 x g for 1 h shows
that only small amounts of both wild type and mutant Gs
accumulated in the cytosolic fraction (Fig. 4
). Only wild-type Gs
showed significant accumulation in the membrane fraction.
In our initial experiments with in vitro translated
Gs
-
S250 and Gs
-S250R, we observed that
they exhibited an unusual gradient profile in response to mild heat
treatment (Fig. 5
, top two
panels). When in vitro translates of
Gs
-
S250 or Gs
-S250R were held on ice
for 12 h, the gradient profile was virtually the same as that
obtained for wild type Gs
, with a peak migrating with a
sedimentation coefficient of
3.7S.3 However, after 12
h at either 30 C or 37 C, most of the protein was present in a
6.3S
or higher peak of material. Wild type Gs
was stable for
at least 2 h at 37 C, giving a profile similar to that for wild
type held on ice for the same length of time. In the presence of excess
GDP or GTP
S, a small proportion of Gs
-
S250 was
able to maintain the 3.7S conformation. However, guanine nucleotides
had no effect on Gs
-S250R (not shown). To determine
whether the stable 3.7S conformation of Gs
-
S250 and
Gs
-S250R observed on ice could bind ß
, in
vitro translates were mixed with purified bovine brain ß
and
held on ice for 12 h and then centrifuged through a 520% sucrose
gradient (Fig. 5
, bottom panel). Although wild type
Gs
shifted completely into a peak of
5.0S
heterotrimer, there was no influence by ß
on the gradient profile
of either Gs
-
S250 or Gs
-S250R,
suggesting that neither mutant is capable of interacting with
ß
.

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Figure 5. Sucrose Density Gradient Analysis of in
Vitro Translated Gs -S250R,
Gs - S250, and Wild Type Gs
Mutant or wild type Gs translation products (2.5 µl)
were prepared as described in Materials and Methods. The
top two panels show the gradient profiles of each
-subunit after incubation at 0 C ( ), 30 C ( ), or 37 C ( )
for 1 h (Gs -S250R) or 2 h
(Gs - S250 and wild type Gs ). The
samples were centrifuged through a 200 µl 520% (wt/vol) sucrose
gradient for 1 h at 436,000 x g. Gradients
were fractionated from the top at 6 µl per fraction, and every other
fraction was analyzed by SDS-PAGE and PhosphorImaging. Each data point
is the amount of Gs expressed as a percent of total
Gs fractionated from that gradient. The autoradiogram in
the top panel is representative of the data obtained for
Gs -S250R held at 0 C or 37 C for 1 h. In the
bottom panel, in vitro translated
Gs was mixed with 20 µg/ml ß and incubated on ice
for 12 h before centrifugation through sucrose gradients as described
above. Values along the top are the position and
sedimentation coefficients
(s020,w) of standard proteins
(carbonic anhydrase, 2.8; ovalbumin, 3.7; and globulin, 7.2).
The arrows along the right side of the autoradiograms
mark the position of full-length Gs (52 kDa). The
smaller molecular mass products result from initiation of protein
synthesis at downstream methionine codons as these are immune
precipitated with a C-terminal antibody (data not shown).
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We examined the ability of AlF4- or GTP
S to
protect the mutant and wild type Gs
from trypsin
digestion, which is a function of the ability of each agent to bind to
Gs
and induce the active conformation (20).
Gs
-
S250, Gs
-S250R, or
Gs
-wild-type in vitro translates were
equilibrated with AlF4- or 100
µM GTP
S for 2 h at various temperatures and then
digested with trypsin (Fig. 6
). Wild-type
Gs
was well protected by AlF4-
and GTP
S. Gs
-
S250 exhibited low protection in the
presence of AlF4- up to 20 C, dropping to
<1% at higher temperatures (3037 C). GTP
S was a better ligand,
approaching 50% of the level seen with wild type Gs
,
when equilibrated at 30 C. However, at 37 C protection fell off,
consistent with the complete denaturation observed at higher
temperatures by sucrose density gradient centrifugation.
Gs
-S250R was protected at a level of less than 1% with
either AlF4- or GTP
S, suggesting that this
mutant is more severely affected than Gs
-
S250.

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Figure 6. Trypsinolysis of in Vitro Translated
Gs -S250R, Gs - S250, and Wild Type
Gs in the Presence of AlF4- or
GTP S as a Function of Temperature
Gs -S250R, Gs - S250, and wild type
Gs were translated in vitro as described
in Materials and Methods. Samples of in
vitro translates (12.5 µl) were equilibrated for 2 h
with AlF4- + 100 µM GDP or 100
µM GTP S at the indicated temperature in 20
mM HEPES, pH 8.0, 10 mM MgCl2, 1
mM EDTA, and 1 mM DTT. Trypsin digestion was
initiated with 200 µg/ml tosyl-L-phenylalanine
chloromethyl ketone (TPCK)-trypsin, incubated for 5 min at 20 C, and
terminated by boiling in sample buffer. The digestion products were
separated by SDS-PAGE, after which dried gels were exposed first to a
phosphor storage plate for quantification by PhosphorImaging and then
to x-ray film for autoradiography. A representative autoradiogram is
presented showing protection from trypsinolysis of wild type
Gs and Gs - S250 in the presence of
AlF4-. The bottom panel shows
graphically the protection from trypsinolysis of wild type
Gs , Gs -S250R, and
Gs - S250 conferred by AlF4-
( ) or GTP S () as a function of temperature. Protection is
defined as the amount of 38 kDa trypsin-resistant core expressed as a
percent of full-length, undigested Gs . The graphic data
are presented as an average ± range for two experiments.
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DISCUSSION
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We identified a heterozygous missense mutation
(Gs
-S250R) in a severely affected patient with PHP type
Ia. This mutation, as well as a mutation that deletes serine 250
(Gs
-
S250), encodes a Gs
protein with
severely impaired function. Gs
-S250R was unable to
attain the active conformation or stimulate adenylyl cyclase in the
presence of AlF4- or GTP
S. In contrast,
Gs
-
S250 was capable of stimulating adenylyl cyclase
to some extent at 30 C in the presence of GTP
S but not in the
presence of AlF4-. Consistent with this
result, GTP
S was able to partially protect Gs
-
S250
from trypsin digestion at 30 C but AlF4- could
not.
The results of sucrose gradient centrifugation experiments suggest that
the gross conformation of both mutants is similar to that of wild type
Gs
at 0 C. Both, however, appear to be thermolabile at
higher temperatures (Gs
-S250R to a greater extent than
Gs
-
S250). Preliminary characterization of another
Gs
mutant (Gs
-R258W) shows it to be
thermolabile at 37 C. However, high concentrations of guanine
nucleotide protect Gs
-R258W from heat denaturation
(D. R. Warner and L. S. Weinstein, manuscript in
preparation). In contrast, high concentrations of guanine nucleotide
did not protect Gs
-S250R from heat denaturation and
protected Gs
-
S250 only slightly (data not shown),
suggesting that the capacity of these mutants to bind guanine
nucleotide is impaired. Impaired guanine nucleotide binding has been
previously shown to be associated with thermolability in a
Gs
mutant (Gs
-A366S, 17 . The
results of sucrose density gradient experiments are consistent with the
results of the trypsin protection assay, which suggests that
Gs
-S250R is unable to bind guanine nucleotide at all
while Gs
-
S250 can bind guanine nucleotide with low
affinity and undergo the conformational switch. The decrease in
protection by AlF4- at 30 C suggests that GDP
binds to Gs
-
S250 less well than GTP
S at this
temperature. However, based upon these results, we cannot rule out that
the Ser250 mutants are thermolabile and cannot switch to
the active conformation despite normal guanine nucleotide binding.
Neither mutant was able to interact with ß
dimers, even at low
temperatures. Impaired guanine nucleotide binding is one possible
explanation for the inability to form heterotrimer. It is also possible
that mutation of serine 250 has a more direct effect on the switch II
region, which directly interacts with ß
(8, 9). The inability of
the S250 mutants to localize to membranes when stably transfected into
cyc- cells may at least partially be the direct result of
their inability to interact with ß
. The lack of interaction with
ß
may also alter palmitoylation (21), although some experiments
suggest that Gs
can interact with membranes in the
absence of palmitoylation (22, 23). It also appears that
Gs
-S250R does not localize to membranes in cells of the
affected patient since the level of Gs
protein in
erythrocyte membranes from the patient is only 50% of that from normal
subjects.
A molecular model of Gs
was generated based upon the
crystal structure of the
-subunit of the heterotrimeric G protein
transducin (Gt
) to determine how these mutations may
lead to abnormal function. This residue is located at the C terminus of
the ß4 sheet adjacent to a highly conserved serine residue
(Ser251) between the switch II and III regions of the
GTPase domain (10, 12). Analysis of the Gs
model using
the computer program Look, version 2.0 (Molecular Applications Group,
Palo Alto, CA; see Fig. 7
) reveals direct
contacts between Ser250 and Leu266 in the
3-helix, Leu291 and Asn292 in the
ß5-sheet, Leu297 in the
G-helix, and
Ile341 in the
4-helix. Asn292 is the first
residue of the conserved NKXD motif present in the GTPase superfamily
members that interacts with the guanine nucleotide ring (24), where
Asn, Lys, and Asp are strictly conserved, and X denotes a nonconserved
residue. Residues 291 through 297 form a loop around the side chain of
Ser250. These interactions of Ser250 with
Leu291, Asn292, and Leu297 may
function to stabilize contacts of the conserved NKXD residues
(Asn292, Lys293, and Asp295) to the
guanine ring or provide rigidity to this portion of the guanine
nucleotide-binding pocket. Analysis of a Gs
model based
upon Gi
1 shows similar results. Also, analysis of the
Gt
crystal structure showed that the residue in the
position corresponding to Gs
Ser250
(Leu223) made similar interactions with residues in the
NKXD sequence except for an additional interaction with
Lys267. Lys267 is the nonconserved residue in
the NKXD motif of Gt
. It is interesting to note that the
majority of heterotrimeric G
-subunits have Leu in the
position analogous to Gs
Ser250, and in
virtually all of these
-subunits the nonconserved residue in the
NKXD motif is Lys (Lys267 in Gt
). In
contrast, in Gs
, Gln is the nonconserved residue, and
our model does not predict a direct interaction of this residue with
Ser250.
Examination of the sequence of 82 heterotrimeric G protein
-subunits
(release 33 of the Swiss protein database) reveals that in all
sequences the residue analogous to Ser250 is restricted to
one of five uncharged residues (Ile, Ser, Met, Leu, and Cys).
Substitution of a charged residue in this position, as in the case of
Gs
-S250R, might completely disrupt interactions of this
residue with the
3- and
4-helices and the NKXD region. In
Gs
-
S250, since the next residue in the sequence is
also a serine, the deletion may only result in an improper alignment of
the remaining residues with preservation of the integrity of the
guanine nucleotide-binding pocket. Therefore this mutant still
demonstrates some activity under certain conditions.
The
4-helix is thought to comprise part of the receptor contact site
and transmit conformational changes to residues that interact directly
with GDP and induce guanine nucleotide exchange (25). It is tempting to
speculate that the S250 mutants may result in an overall conformation
that is attained during formation of the ternary complex with receptor,
resulting in decreased guanine nucleotide affinity and consequently
thermolability. In conclusion, the mutation of Ser250 in
this PHP type Ia patient highlights a residue that is likely to be
important in maintaining the integrity of the guanine nucleotide
binding pocket.
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MATERIALS AND METHODS
|
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Patient
The patient is a 44-yr-old black female with a diagnosis of PHP
type Ia. She was born prematurely at 8 months after an otherwise normal
gestation at a weight of 4 lb, 11 oz. She was critically ill, requiring
hospitalization for 1 month after birth. She required two more
subsequent admissions over the first 6 months for regurgitation and
pneumonia with episodic apnea. At age 6 months she weighed only 8 lb
but began to grow slowly. In early childhood she developed gradual
obesity and was delayed in her developmental milestones. At age 10,
subcutaneous calcifications were noted and hypocalcemia,
hyperphosphatemia, and hypothyroidism were diagnosed. Lack of a
calcemic or phosphaturic response to exogenous infusion of bovine
parathyroid extract confirmed the diagnosis of PHP. At age 12 she was
referred to the NIH. On physical examination, notable features included
brachydactyly of multiple bones in the hands and feet, short stature,
obesity, depressed nasal bridge, and palpable subcutaneous
calcifications. She suffers from moderately severe mental retardation.
Baseline and provocative endocrine testing confirmed resistance to PTH
and TSH. Although the response of GH to provocative testing was normal,
somatomedin C levels were below the normal limits on two occasions. The
patient also had primary amenorrhea with mildly deficient sexual
development. Gonadotropin responses to GnRH were normal. The patient
also had a decreased plasma cAMP response to glucagon infusion,
although the glycemic response was normal. Karyotype was 46 XX (t
13;15). The patient has three siblings who are clinically normal and
are presently unavailable for testing. Neither parent (nor any of their
ancestors) have clinical or biochemical features of PHP. The patients
mother has primary hyperparathyroidism and a thyroid nodule. The
patients father is presently unavailable for
testing.
Preparation of Erythrocyte Membranes and Determination of
Gs Activity
Erythrocyte membranes were prepared from the patient, her
mother, and four normal subjects, as previously described (26), and
frozen in aliquots at -70 C. Erythrocyte membranes were reconstituted
with membranes from mutant S49 mouse lymphomas cells
(cyc-, which lack Gs
expression) and
adenylyl cyclase activity in the presence of isoproterenol (10
µM), and GTP (10 µM) was determined as
previously described (26). Protein was determined by the method of
Bradford (Bio-Rad Laboratories, Hercules, CA).
Immunoblots
Erythrocyte membranes were heat-denatured in SDS-mercaptoethanol
loading buffer. Samples (equal amounts of protein and sample volume per
lane) were electrophoresed in 10% tricine-SDS gels (Novex, San Diego,
CA) and transferred to polyvinylidene difluoride (PVDF) filters
(Immobilon-P) by electroblotting. Filters were blocked in
Tris-buffered-saline (TBS) with 0.5% Tween-20 (TBST) and 1% BSA at 25
C for 1 h. Filters were then incubated with affinity-purified RM
antibody (2 µg/ml), directed to the carboxy-terminal decapeptide of
Gs
(27), in TBST, 1% BSA overnight at 25 C. After three
washes in TBST, filters were incubated for 2 h in TBST, 1% BSA
with [125I]protein A (NEN/DuPont, Boston, MA), 0.1
µCi/ml, and then rewashed three times with TBST. Filters were exposed
to x-ray film, and bands were quantified with a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA).
DNA Isolation and Genetic Analysis
Genomic DNA was isolated from whole blood and screened for
mutations within GNAS1 using PCR and denaturing gradient gel
electrophoresis (DGGE) as previously described (14). PCR fragments were
purified using Centricon 100 filters (Amicon, Beverly, MA) and directly
sequenced using the Sequenase kit (U.S. Biochemical, Cleveland,
OH).
RT-PCR
RNA was isolated from 100-µl aliquots of whole blood using the
Catrimox-lithium chloride method (Iowa Biotechnology Corp., Coralville,
IA). Crude RNA pellets were digested with DNAse I (amplification grade,
BRL, Rockville, MD) in a 10-µl reaction for 15 min at 25 C. Samples
were heated at 65 C for 15 min after addition of 20 mM EDTA
(1 µl). Downstream oligonucleotide primer (50 pmol) was added, and
the samples were heated at 65 C for 2 min and placed on ice. RNA
samples (6 µl of template-primer mixture) were reverse transcribed at
45 C for 1 h in 25 µl reaction mixtures containing 1 µl AMV
reverse transcriptase (SuperScript II, BRL), deoxynucleotide
triphosphates (0.8 mM each), 1 µl RNAse inhibitor (BRL),
10 mM dithiothreitol (DTT), and reaction buffer supplied by
the manufacturer. Negative control reactions without RT were also run
for each sample. RT reactions were heated to 72 C for 5 min and 5 µl
added to 100 µl PCR reactions containing deoxynucleotide
triphosphates, 200 µM each, upstream and downstream
oligonucleotide primers, 0.5 mM each, 0.01% (wt/vol)
gelatin, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5
mM MgCl2, and 2.5 U Taq polymerase
(Perkin Elmer, Norwalk, CT). Amplification consisted of denaturation at
94 C for 5 min followed by 30 cycles consisting of annealing at 58 C
for 45 sec, primer extension at 72 C for 1 min, and denaturation at 94
C for 1 min and 1 final cycle with a 3-min primer extension. For
amplification of the GNAS1 exon 10 coding region the primers were:
upstream, 5'-ATGTTTGACGTGGGTGGCCAGC-3'; downstream,
5'-CACAGAGAT-GGTGCGCAGCCAT-3' (19).
Cloning of Gs
Mutants
RT-PCR fragments encompassing exon 10 of Gs
were
cloned by digestion with HincII and Sse8387I and ligated
into the transcription vector pBluescript II (Stratagene, La Jolla, CA)
that contained wild type human Gs
with the same fragment
removed. Mutations were verified by DNA sequencing and synthesis of
full-length Gs
tested by in vitro translation
and immune precipitation with RM antibody. For transfection into
cyc-, the cDNA was subcloned into pZEM228c under control
of the metallothionein promotor (28).
Transfection of Cyc- Cells with
Gs
Constructs
The cyc- variant of S49 murine lymphoma cells was
grown in DMEM supplemented with 10% heat-inactivated horse serum and,
for transfected cells, 125 µg/ml (active) G-418 (Geneticin,
GIBCO/BRL, Gaithersburg, MD). Before transfection,
Gs
-pZEM228c was linearized with AatII and
sterilized by ethanol precipitation. Cyc- cells were
prepared for transfection by two washes with ice-cold Dulbeccos PBS
(Ca+2 and Mg+2 free) and suspended at 1 x
108 cells/ml in the same buffer. Cells, (4 x
107 cells in 400 µl) were placed in an ice-cold
electroporation cuvette (BTX, Inc., San Diego, CA; P/N 620), mixed with
2025 µg vector DNA, and immediately electroporated using a BTX
model 600 Electro Cell Manipulator with the following settings:
charging voltage, 500 V; capacitance, 200 microfarads; and resistance,
R10 (720 ohms). Using these conditions, the average field strength for
each sample was
800 V/cm, and the pulse length was
2.6 msec.
After electroporation, cells were held on ice for 10 min and then
suspended in 6 ml DMEM and placed in an incubator with a 5%
CO2 atmosphere. The next day, 14 ml of fresh media,
supplemented with 400 µg/ml active G-418, and 20% conditioned media
were added. Every 35 days, 50% of the medium was replaced with fresh
medium. After approximately 1 month in culture, G-418-resistant cells
were either tested without further manipulation or cloned in soft agar
as described previously (29). After inducing Gs
expression with 75 µM ZnSO4 for 1216 h,
postnuclear supernatant fractions were prepared according to Salomon
and Bourne (30). The postnuclear supernatant fraction was centrifuged
for 1 h at 50,000 rpm (108,920 x g at rmax) in a
Beckman TL-100 centrifuge with a TLA100.2 rotor (Beckman Instruments,
Fullerton, CA). The supernatant was removed and the pellet was
suspended in 20 mM HEPES, pH 8.0, 2 mM
MgCl2, 1 mM EDTA, 0.5 µg/ml leupeptin, 2
µg/ml aprotinin, and 100 µg/ml soybean trypsin inhibitor, and both
fractions were adjusted to 10% glycerol and stored at -80 C until
needed. Supernatant and membrane proteins were separated on 10% gels
by SDS-PAGE and transferred to Immobilon-P membranes that were probed
with RM antiserum as described above.
Adenylyl Cyclase Assays
Postnuclear supernatants fractions from zinc-induced transfected
cyc- cells were assayed for adenylyl cyclase activity
according to Kassis et al. (31). Reactions were for 15 min
at 30 C, and [32P]cAMP was purified and measured as
described previously (32). For in vitro translated
Gs
subunits, stimulation of adenylyl cyclase was
measured after reconstitution into purified cyc- plasma
membranes (33). Stimulation and [32P]cAMP quantification
were the same as described above.
In Vitro Translation and Gradient Centrifugation
[35S]methionine-labeled Gs
was
prepared by coupled in vitro transcription and translation
as described previously (34). Rate zonal centrifugation was performed
on 520% linear sucrose gradients (200 µl volume) made up in 20
mM HEPES, pH 8.0, 1 mM EDTA, 1 mM
DTT, 50 mM GDP, 100 mM NaCl, 1 mM
MgCl2, and 0.1% Lubrol-PX. Sample volume was 10 µl,
which included 2.54 µl of the translation reaction, 0.1%
Lubrol-PX, 1 mM GDP, and, when present, 20 µg/ml ß
.
The samples were centrifuged for 1 h at 100,000 rpm (436,000
x g at rmax) at 4 C in a Beckman TL-100
centrifuge with a TLA100.2 fixed angle rotor. The gradients were
fractionated with a Brandel microfractionator, model FR-115X (Brandel,
Gaithersburg, MD), at 6 µl per fraction. After an equal volume of
twice concentrated Laemmli sample buffer (35) was added, the samples
were boiled for 5 min and analyzed by 10% SDS-PAGE. Gels were fixed in
10% acetic acid/25% methanol/1% glycerol for 1 h, dried, and
then exposed to a storage phosphor screen (Molecular Dynamics,
Sunnyvale, CA). After 12 days, data were collected with a Molecular
Dynamics PhosphorImager, model 445 S1, and analyzed with Image Quant
software (Molecular Dynamics).
Trypsin Digests
In vitro translates
([35S]methionine-labeled, 12.5 µl) were incubated for
12 h in 20 mM HEPES, pH 8.0, 10 mM
MgCl2, 1 mM EDTA, and 1 mM DTT,
with or without 10 mM NaF/10 µM
AlCl3/100 µM GDP or 100 µM
GTP
S. Tosyl-L-phenylalanine chloromethyl ketone-trypsin
was then added to a final concentration of 200 µg/ml. Digestions were
carried out for 5 min at 20 C before addition of sample buffer (30)
followed by boiling for 5 min. Samples were analyzed by SDS-PAGE and
phosphorimaging as described above for the sucrose gradient samples.
For visualization of the data an autoradiogram also was made.
Construction of Gs
Model
The primary sequence of Gs
-1 (19) was aligned
with that of Gt
from the Brookhaven National Laboratory
(ID code, 1TAG) and modeled with the SegMod module of Look (Molecular
Applications Group, Palo Alto, CA) using 500 rounds of energy
minimization. The root mean square deviation of the
-carbons of the
modeled Gs
from Gt
was 0.94 Å.
 |
ACKNOWLEDGMENTS
|
---|
The authors acknowledge the excellent technical assistance
provided by Kenny C. Lai, Nadine American, Randy Lee, Shuhua Yu, and
Dawen Yu. The authors also thank Dr. Robert Pearlstein for helpful
advice with Look.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dennis R. Warner, Membrane Biochemistry Section, Laboratory of Molecular and Cellular Neurobiology, National Institute of Neurological Disorders and Stroke, Building 49, Room 2A28, National Institutes of Health, Bethesda, Maryland 20892-4440.
1 The abbreviations used are: G protein, guanine
nucleotide binding protein; Gs, stimulatory G protein;
Gs
-S250R, mutant of Gs with
serine250 to arginine substitution;
Gs
-
S250, mutant of Gs in
which serine-250 was deleted; GNAS1, human gene encoding
Gs
; AlF4-, mixture of 10
µM AlCl3 and 10 mM NaF; GTP
S,
guanosine-5'-O-(3-thiotriphosphate). 
2 All numbering is based on the
Gs
-1 sequence reported by Kozasa et al.
(19 ). 
3 The actual value for Gs
is
2.8S-3.15S and 4.55S-4.7S for the heterotrimer (36 37 ). The resolving
power of a sucrose gradient in a fixed angle rotor is inferior to that
centrifuged in a swinging bucket rotor. Speed was traded for resolving
power and, therefore, the measured sedimentation coefficients are less
accurate. 
Received for publication April 29, 1997.
Revision received July 24, 1997.
Accepted for publication July 25, 1997.
 |
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