Departments of Molecular Pharmacology and
§ Physiology and Biophysics, Diabetes and Metabolic Diseases
Research Program, University Medical Center, SUNY/Stony Brook,
Stony Brook, New York 11794
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gs regulates the differentiation of 3T3-L1
mouse embryonic fibroblasts to adipocytes, a process termed
adipogenesis. Through the expression of chimera created by substituting
regions of Gs
with corresponding regions of the G protein Gi
2,
the domain of Gs
involved in repression of adipogenesis was
localized to sequence 146-235 of the molecule (Wang, H-y., Johnson,
G. L., Liu, X., Malbon, C. C. (1996) J. Biol.
Chem. 271, 22022-22029). As a prelude to alanine-scanning
mutagenesis, chimeras in Gs
constructed from trisection of the
sequence 125-213 of Gi
2 were expressed stably, and clones were
evaluated for the ability of the chimera to repress adipogenesis in
response to the inducers, dexamethasone and methylisobutylxanthine, in
combination. The chimera containing sequence 150-177 of Gi
2 repressed adipogenesis, whereas the chimeras with either sequence 125-149 or 178-213 of Gi
2 failed to repress induction of
adipogenesis. Alanine-scanning mutagenesis of these two critical
domains was performed first in clusters and then confirmed by analysis
of single mutations. Six residues unique to Gs
were identified as critical to repression of adipogenesis, Asn167,
Cys200, Leu203, Ser205,
Val214, and Lys216. Leu203 and
Ser205 are required in tandem, as mutagenesis to alanine of
either one alone was without effect on repressor activity. The
remaining four residues are required for repressor activity; mutation
of any one of these abolishes the ability of Gs
to repress
adipogenesis, although not affecting the ability of the mutant form of
Gs
to regulate adenylylcyclase. Using conserved landmarks found in
the crystal structures of Gi
1 and Gs
, the Leu203 and
Ser205 cluster appears to be exposed, closely aligned and
located in switch I region. Asn167, Val214, and
Lys216 project to regions on Gs
that are exposed in the
GTP
S-liganded state of the
subunit. We speculate that these
residues constitute an important contact domain between Gs
and the
effector controlling adipogenesis, which is yet to be identified.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Swiss mouse 3T3-L1 cells originally isolated by Green and Meuth
provide a unique model for insulin-sensitive primary fat cells (2-4).
The cell line rapidly differentiates to an adipocyte phenotype when
treated with inducers, such as insulin (5) or dexamethasone and
methylisobutylxanthine (MIX)1
in combination (see Wang et al. (6), and Refs. therein). In addition to mediating signaling from a populous class of plasma membrane receptors to a less populous group of effectors that includes
adenylylcyclases, phospholipase C, and various ion channels (7-9),
heterotrimeric G proteins participate in more complex biological
responses, including oncogenesis (9), early (10) and neonatal (11)
mouse development, as well as cellular differentiation (6, 12).
The present study focuses on the ability of the G protein Gs to
control adipogenic conversion of the 3T3-L1 fibroblasts to adipocytes.
Gs
has been shown to play a key role in the differentiation of
3T3-L1 cells, as evidenced by the following observations. Gs
expression declines dramatically within 48 h of induction of
differentiation; constitutive expression of Gs
in 3T3-L1 cells
blocks induction of cell differentiation by known inducers; suppression
of Gs
expression by antisense oligodeoxynucleotides (mimicking the
inducer-driven decline in Gs
) accelerates the cell differentiation
from a 10-day to a 3-day process in the presence of inducers; and
treatment with oligodeoxynucleotides antisense to Gs
alone provokes
adipogenesis in the absence of the classical inducers (1, 6, 13-15).
Overexpression of the G protein that antagonizes many Gs
effects,
Gi
2, provokes adipogenesis in either the absence or the presence of
the inducers (16).
The central question remains how Gs controls cell differentiation.
The ability of Gs
to repress adipogenesis is not thought to involve
adenylylcyclase based upon the following observations. Elevation of
intracellular cAMP concentrations by treating cells with either the
diterpene forskolin or pertussis toxin does not affect the
differentiation process, direct addition of dibutyryl cAMP itself to
the cultures does not alter differentiation, and treatment of cells
with 2',5'-dideoxyadenosine to reduce intracellular cyclic AMP
concentrations likewise does not alter differentiation. Treatment of
cells with cholera toxin does block adipogenesis, through activation of
Gs
, much like expression of the constitutively active mutant form of
Gs
(G225T). Although both cholera and pertussis toxins elevate
intracellular cAMP, only cholera toxin blocks adipogenesis (6).
Recently, we found that expression of the chimeric G protein in which
the sequence 145-235 of Gs
is substituted for the corresponding region of Gi
2 (Gi
2 1-122/Gs
145-235/Gi
2 236-394)
inhibited cell differentiation as effectively as wild-type Gs
(1).
These data indicate that the sequence harboring residues 146-235 of Gs
, which is not the region interacting with adenylate cyclase (17-19), is critical in controlling cell differentiation. The region 146-235 of Gs
includes Switch I and Switch II (20), which are involved both in contact with
complex, binding of guanine
nucleotides, as well as the Gap region (20, 21).
In the present study, we sought to define more precisely the domain and
amino acid residues of Gs that are responsible for the control of
adipogenesis, the differentiation of 3T3-L1 embryonic fibroblasts
to adipocytes. The repressor domain of Gs
was trisected into smaller
sequences that were substituted with the corresponding domains of
Gi
2 and the chimeras stably expressed in 3T3-L1 cells. Sequences
147-171 and 200-235, but not 172-199 of Gs
are critical in
control of cell differentiation. Alanine scanning mutagenesis of
sequences 147-171 and 200-235 identified four amino acids
(Asn167, Cys200, Val214, and
Lys216) and one cluster (Leu203 and
Ser205) that are critical to the ability of Gs
to
repress differentiation of 3T3-L1 cells.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction of Chimeras--
Chimeras were constructed from
pCW1Gs, pCW1GsQ227L, pCW1Gi
2Q205L, and pCW1G
i/sBam
(1, 17). The small HindIII fragment of pCW1Gs
and
pCW1Gi
2Q205L was inserted into the HindIII site of
pAlter-1 (Promega Corporation, Madison, WI), respectively. A
recombinant with the 5' of the insert toward the ClaI site
of pAlter-1 was selected. The BglII-ClaI fragment
of the pAlterGs
was transferred into the
BgllI-ClaI site of pAlterGi
2Q205L to avoid two
AflII sites at the 3' end of the Gs
cDNA (22). Using site-directed mutagenesis (Promega Corporation, Madison, WI), an
Mfe3I site and an AflII site were introduced at
690-695 and 773-779 of Gs
, where
690CAGCTG695 and
773GCTTCGC779 of DNA sequences were changed to
690CAATTG695 and 773 CTTAAGA779, respectively. The codons for both Gs
and
Gi
2 were not changed. The plasmid is designated pAlterGs
MA. To
construct chimera G
s1-146/i125-149/s (sisEM), pAlterGi
2Q205L
was amplified as using T7 primers and Gi
2EcoRI sense oligonucleotide
5'-AAGAATTCTCGGGCGTCATCCGGAGG. The fragment was digested with
EcoRI and inserted into the EcoRI sites of
pAlterGs
Q227L (the 5' of the insert toward the ClaI site
of pAlter-1). A recombinant displaying the correct orientation was
amplified using primers ClaI 21 sense oligonucleotide
5'-GCTAATCGATGATAAGCTGTC and MfeI 26 antisense
oligonucleotide 5'-CATTCAATTGATATTCCCGTGAGCGG. The fragment was
digested with ClaI and MfeI and inserted into the
ClaI-MfeI site of pAlterGs
MA. To generate
chimera G
s1-171/i150-177/s (sisMA), pCW1-Gi
2Q205L was amplified
using as primers, MfeI 31 sense oligonucleotide
5'-ATATCAATTGAATGACTCAGCCGCTTACTAC and AflII 29 antisense
oligonucleotide 5'-GGTCCTTAAGACATCCTGCTGTGTAGGGA. The fragment was
digested with MfeI and AflII, and inserted into the MfeI-AflII site of pAlterGs
MA. To generate
chimera G
s1-199/i178-213/s (sisAB), pCW1G
i/sBamHI
was amplified using primers as AflII 28 sense
oligonucleotide 5'-ATGTCTTAAGGACCCGTGTGAAGACCAC and BglII 21 antisense oligonucleotide 5'-CCCAGCGAGGACCTTCTCAGC. The fragment was
digested with AflII and BgllI, and inserted into
the AflII-BgllI site of pAlterGs
MA.
Site-directed Mutagenesis--
Mutations were introduced into
pAlterGs by oligonucletide-directed in vitro mutagenesis
using a kit purchased from Promega. All mutations and chimeras were
verified by restriction enzyme digestion and DNA sequencing using
Sequenase Version 2 Kit (U. S. Biochemical, Cleveland, OH). To
subclone all the chimeras and mutants, pCW1Gs
Q227L was partially
digested with HindIII, filled in by Klenow fragment, and
then ligated. Plasmids lacking the HindIII site at the 3'
end of Gs
were selected. All chimeras and mutants were digested with
HindIII and BglII and the isolated fragments of
interest inserted into the HindIII-BglII site of the plasmid. Direct dideoxy sequencing was employed to verify the
sequence of the chimeras and mutations. All mutants and chimera were
constructed from wild-type versions of Gs
and Gi
2 with normal
intrinsic GTPase activity.
Stable Expression of Chimeras and Mutants in 3T3-L1 Cells-- Mouse embryo fibroblast 3T3-L1 cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were maintained in culture in 100-mm Petri dishes in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. The protocols for stable transfection of 3T3-L1 cells employed in these studies were described previously (6, 16). Stably transfected clones were selected (400 µg/ml) and then maintained (100 µg/ml) in the presence of the active form of the gentamicin analogue, G418 sulfate (Life Technologies, Inc.).
Immunoblotting--
Aliquots of crude membrane fractions (50 µg of protein/SDS-polyacrylamide gel electrophoresis/lane) from
aliquots of each subclone were subjected to SDS-polyacrylamide gel
electrophoresis. The separated proteins were transferred to
nitrocellulose and the blots were stained with a rabbit polyclonal
antibody specific for Gs (CM129). The immune complexes were made
visible by staining with a second antibody (goat anti-rabbit IgG)
coupled to calf alkaline phosphatase (6).
Cyclic AMP Accumulation--
Aliquots (0.5 × 105 cells) of 3T3-L1 cells were washed and incubated in
Kreb's phosphate buffer (pH 7.5, 106 cells/ml) containing
the cyclic AMP phosphodiesterase inhibitor Ro 20-1724 (0.1 mM) in either the absence or the presence of the -adrenergic agonist (
) isoproterenol (10 µM) for 15 min at 37 °C. The reaction was terminated by the addition of
ethanol. Measurements of cyclic AMP accumulation were made in
triplicate from separate aliquots of cells. Cyclic AMP accumulation was
determined by the competitive protein binding assay, using the bovine
adrenal cyclic AMP-binding protein (23).
Determination of Adipogenesis--
Clones transfected with
vector, wild type Gs, chimeras or mutants were maintained in 24-well
plates for propagation. The differentiation protocol was described
previously (1). Protocols for histochemical staining techniques are
described in detail elsewhere (6, 16). Adipogenesis was established via
staining of accumulated lipid with oil red O.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The strategy adopted to the analysis of the sequence of Gs
responsible for the repression of adipogenesis was based upon our
previous analysis in which the sequence of 145-235 of Gs
was
identified as critical (1). Gs
sequence 145-235 embedded into the
corresponding region of Gi
2 retains the full capacity to block
adipogenesis (1). The primary goal was to analyze by alanine-scanning
mutagenesis the fine detail of the region of Gs
repressing
adipogenesis. To make the task manageable, the region(s) of Gs
to be
subjected to alanine-scanning mutagenesis had to be smaller than the
parent sequence 145-235. To accomplish the task, the "repressor"
region of Gs
was trisected and regions 125-149, 150-177, and
178-213 of Gi
2 substituted individually for the corresponding
region of Gs
. The chimeras constructed in this fashion are displayed
in Fig. 1. The inability of a chimera with the Gi
2 region embedded in Gs
to repress adipogenesis would identify region(s) as candidate(s) for further analysis by
alanine-scanning mutagenesis.
|
The cDNAs encoding each of the chimeras (Fig. 1) were inserted
individually in the pCW1 mammalian expression vector. The pCW1 vector
is driven by the SV40 early promoter and harbors a selectable marker,
the neomycin resistance gene (neor). Mouse embryonic
fibroblasts were stably transfected and the derivative clones were
selected in the presence of the gentamicin analogue G418. Each of the
clones selected displays expression of Gs well in excess of that
observed in the cells stably transfected with the empty vector alone
(Fig. 2A). Immunoblots of cell
membranes prepared from the stably transfected clones were stained with a rabbit polyclonal antibody raised against the decapeptide C terminus
of Gs
(CM129). Expression of Gs
in clones harboring the
expression vector for a chimera minimally was approximately 1-fold
greater than the endogenous levels of Gs
expressed by 3T3-L1 clones
stably transfected with the empty vector pCW1 alone. Constitutive
expression of Gs
at a level 50% greater or more that endogenous
Gs
is readily able to repress the ability of dexamethasone and MIX
to induce adipogenesis (1). Chimeras of Gs
in which a smaller region
of Gi
2 (125-149, 150-177, or 178-213) was substituted for the
corresponding region in Gs
had little effect on the cAMP response of
the clones to stimulation by isoproterenol, when compared with cells
transfected with wild-type Gs
alone. Basal cAMP accumulation of
these clones in comparison to that of the transfectants expressing
wild-type Gs
alone was reduced slightly, perhaps reflecting the
predominant Gi
2 nature of the expressed protein (Fig.
2B). Earlier studies have demonstrated that Gs
-Gi
2
chimera with these types of substitutions retain their capacity to bind
GTP and transduce signaling (17, 24).
|
Adipogenesis is readily detected by staining the cultures for lipid
accumulation with oil red O (Fig. 3). The
cellular nuclei were made visible by counterstaining with hematoxylin.
For clones stably transfected with the empty expression vector, no
differentiation was observed in the absence of the inducers
dexamethasone + MIX (Fig. 3, panel A). When exposed to
dexamethasone + MIX, cultures harboring the empty expression vector
pCW1 display robust differentiation (panel B). Marked lipid
accumulation, the hallmark of adipocytes, was evidenced throughout the
cultures, as shown earlier (1, 6). Clones constitutively expressing
wild-type Gs, in contrast, do not respond to induction by
dexamethasone + MIX, failing to differentiate into adipocytes
(panel D). Clones expressing either chimera sisEM
(panel F) or chimera sisAB (panel J) displayed
phenotypes identical to clones transfected with empty vector, fully
differentiating in response to dexamethasone + MIX and replete with
accumulated lipid stained by the oil red O. Chimera sisMA, in sharp
contrast, continues to repress dexamethasone + MIX-induced adipogenesis (panel H) much like expression of wild-type Gs
(panel D). These data demonstrate that regions 147-171
(sisEM) and 200-235 (sisAB) of Gs
are critical in the control of
adipogenesis, when substituted with the corresponding region of Gi
2
the chimera lose the ability to repress adipogenesis. Region 172-199
of Gs
, to the contrary, appears to be dispensable and can be
replaced by the corresponding region of Gi
2 without altering the
ability of chimera to repress adipogenesis.
|
Having identified two smaller regions of Gs critical for expression
of its ability to repress adipogenesis, we developed a strategy to
perform alanine-scanning mutagenesis within the sisEM domain and
analyze the influence of these mutations on the repressor function of
the expressed molecule. Initial mutagenesis created both single
(Gs
M3, Gs
M5, and Gs
M6) and multiple (Gs
M1, Gs
M2,
Gs
M4, and Gs
M7-M10) alanine substitutions in protein sequences
147-171 and 200-235 of Gs
(Fig. 4,
A and B), presuming that alanine substitution of a critical residue would abolish the
ability of the mutant Gs
to repress adipogenesis. Clones stably
transfected with pCW1 harboring the cDNA of Gs
with one or more
mutations displayed increased immunoreactive Gs
, reflecting the
expression of the mutant forms in excess of the endogenous level of
Gs
. Clones were selected that expressed the mutant Gs
molecules
at levels approximately 1-fold greater than the staining observed in
clones harboring empty vector alone (Fig.
5A). All of the clones
expressing Gs
mutants display elevated levels of basal cAMP
accumulation compared with clones harboring empty expression vector
alone and each displayed isoproterenol-stimulated cAMP accumulation
(Fig. 5B).
|
|
In the context of region 147-171 (sisEM domain), alanine-scanning
mutations of GsM1 through Gs
M4 displayed no effect on the ability
of the mutant form of Gs
to repress adipogenesis in response to the
inducers dexamethasone + MIX (Fig. 6).
Clones constitutively expressing Gs
M1, 2, 3, and 4 (panels
C, D, E, and F, respectively)
were refractory to induction of adipogenesis. In contrast, expression
of Gs
M5, a single alanine substitution for asparagine at position
167 abolishes the ability of the mutant form of Gs
to repress
adipogenesis (panel G). Clones expressing N167A Gs
no
longer repressed adipogenesis, displaying robust lipid accumulation in
response to inducers (panel G), much like the wild-type
cultures of 3T3-L1 or the clones stably transfected with empty
expression vector alone (panel A).
|
Analysis of the sisAB region (Gs residues 200-235) initially
focused upon aspartic acid residue 229 which was the only residue within the region of 221-235 of Gs
that varied from that of Gi
2 (Fig. 4B). The D229S mutant form of Gs
retains its
ability to repress adipogenesis in clones challenged with dexamethasone + MIX (Fig. 3, panel L). With the C-terminal region of the
AB region shown to be unimportant in the repressor activity of Gs
,
alanine-scanning mutagenesis was focused upon the N-terminal sequence
from residue 200-220 of this region (Fig. 4B). Five
mutations, one a single alanine substitution (Gs
M6) and the others
multiple substitutions (Gs
M7-M10), were constructed and shown to be
expressed in stably transfected clones at >1-fold over endogenous
Gs
(Fig. 5A). These clones displayed increased basal cAMP
accumulation over that observed for clones expressing the endogenous
Gs
(Fig. 5B) and were examined further for their ability
to respond to induction with dexamethasone + MIX.
Expression of GsM6 (C200A) abolished the ability of Gs
to repress
adipogenesis in the clones (Fig. 6, panel H). Clones
expressing C200A Gs
now stain prominently for accumulated lipid
following treatment with dexamethasone + MIX. Expression of Gs
M7
(L203A and S205A), likewise, abolished the ability of Gs
to repress adipogenesis in response to dexamethasone + MIX, resulting in robust lipid accumulation in the clones (Fig. 6, panel I).
In contrast, alanine substitution for Phe208 and
Lys211 of Gs
in the clones expressing Gs
M8 had no
discernible effect on the ability of Gs
to repress adipogenesis, as
demonstrated by the absence of oil red O staining of lipid in these
clones (Fig. 6, panel J). The triple mutation of
Gln213, Val214, and Asp215 to
alanine resulted in a loss of repressor activity for the mutant Gs
M9
(Fig. 6, panel K). In the presence of the inducers
dexamethasone + MIX, clones expressing the Gs
M9 mutant form of Gs
fully differentiated into adipocytes, staining prominently by oil red
O. Substitution of Lys216, Val217,
Asn218, and His220 with alanine also abolished
the ability of the mutant Gs
to repress adipogenesis in response to
dexamethasone + MIX (Fig. 6, panel L). Thus, the mutations
found in Gs
M6, 7, 9, and 10 render the Gs
unable to repress
adipogenesis as does the wild-type Gs
(Fig. 6, panel
B).
The alanine-scanning mutagenesis of single residues had revealed that
N167A Gs and C200A Gs
mutants were devoid of repressor activity.
Mutagenesis of multiple residues as a single cassette identified
regions critical for repressor activity, whereas establishing the
precise residue(s) necessary for repressor activity of Gs
would
require finer detailed analysis. Single point alanine substitutions were created for each of nine residues implicated as critical to the
repressor activity of Gs
(Fig. 4C). Mutant forms of Gs
were stably transfected at levels approximately 1-fold greater than
that of endogenous Gs
(Fig. 7).
Although Gs
M7 with L203A and S205A double mutation has lost the
ability of Gs
to repress adipogenesis in response to dexamethasone + MIX (Fig. 6, panel I, and Fig.
8, row A, panel c),
single point mutations of L203A (Fig. 8, row A, panel
d) or S205A (Fig. 8, row A, panel e) were without effect on the repressor activity. These data argue persuasively that Leu203 and Ser205 together play a critical
role in the control of adipogenesis exerted by Gs
.
|
|
The expression of GsM9, a mutant form of Gs
with alanine
substitution of Gln213, Val214, and
Asp215, resulted in the loss of repressor activity (Fig. 6,
panel K, and Fig. 8, row B, panels
b-d). The single point mutation of G213A had no discernable
effect on the ability of the mutant Gs
to repress adipogenesis.
Similarly, mutation of D215A resulted in a mutant form of Gs
that
fully repressed the ability of dexamethasone + MIX to induce
differentiation, just as observed with constitutive expression of
wild-type Gs
. In sharp contrast to the G213A and D215A mutations,
alanine substitution of Val 214 abolished the ability of Gs
to
repress adipogenesis of the clones in response to dexamethasone + MIX.
These data reveal the basis of the Gs
M9 cassette of mutations to
abolish the repressor activity of Gs
resides solely in the V214A
mutation.
The GsM10 cassette of mutations includes K216A, V217A, N218A, and
H220A (Fig. 4B). This cluster of alanine substitutions was
analyzed further by creation of stably transfected clones of 3T3-L1
cells that expression of each of the individual mutations of the sisAB
region of Gs
(Fig. 4, panel C). Although expression of
the Gs
M10 cassette eliminates the ability of Gs
to repress adipogenesis in the clones, single alanine substitutions of V217A, N218A, and H220A were without effect, i.e. constitutive
expression of each of the single mutant forms of Gs
repressed the
ability of dexamethasone + MIX to induce adipogenesis equally well
(Fig. 8, row C, panels b-e). Only one of the
mutations, K216A, was found to mimic the effects of the Gs
M10
cassette on loss of repressor activity by Gs
.
Single mutations of N167A, C200A, V214A, and K216A as well as the
double mutation L203A,S205A abolished the ability of expressed Gs to
repress adipogenesis in 3T3-L1 cells. Mutant forms of Gs
were
expressed at levels approximately 1-fold greater than that of
endogenous Gs
(Fig. 9A) and
retained the ability to elevate both basal cyclic AMP accumulation and
mediate stimulation of cyclic AMP accumulation in response to
isoproterenol (Fig. 9B).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Comparisons of the sequences of the domains in Gs
(Tyr147-Leu171 and
Cys200-Ile235) that repress adipogenesis in
3T3-L1 cells with the corresponding sequences of other G protein
subunits reveals significant levels of non-identity. Many residues
within these two protein sequences, however, such as
Ile180, Glu182, Arg193,
Gly199, Gln200, Arg201,
Glu203, Lys206, and Trp207 involved
in binding of
complex by Gt
are conserved in Gs
also (21).
The protein sequence Met221-Ile235 of Gs
is
highly conserved in virtually all of the heterotrimeric G protein
subunits (21). In an effort to define more precisely the residues
within the putative repressor domains that are required for the control
of adipogenesis, the amino acid sequences of Gs
and Gi
2 were
compared (22), as displayed in Fig. 4 (panels A-C). The
displays identify 35 identical residues within these domains. Of the 35 residues noted, 28 are conserved in all heterotrimeric G protein
subunits (21). Of the 26 nonidentical residues, 21 were mutated to
alanine in clusters targeting the regions of interest in chimeras sisEM
and sisAB (Fig. 4). The remaining five amino acids (Ala150,
Lys151, Ala152, Asp229, and
Arg232) include naturally occurring alanine at positions
150 and 152, and therefore were not studied further. Asp 229 is the
only non-conserved residue of Gs
found in the sequence from 221 to
235, and D229S mutant form of Gs
is shown in the present work to
retain the ability to repress adipogenesis (Fig. 3). Mutagenesis of
either Lys151 or Arg232 was not undertaken,
simply because other mammalian heterotrimeric G protein
subunits
have the same amino acids at these positions (21), and the lysine for
arginine substitution is considered a conservative substitution.
The focus of the analysis was to evaluate the structural basis for
repression of adipogenesis by Gs, exploiting our knowledge of the
repressor domain by alanine-scanning mutagenesis. Repressor activity of
protein sequence 146-235 of Gs
was identified (6) through the
construction and then expression of various chimeras between Gs
(repressor) and corresponding regions of Gi
2 (activator). The
ability of these Gs
/Gi
2 chimeras to block adipogenesis in 3T3-L1
clones challenged with well-known inducers of differentiation was
evaluated (1). Since Gi
2 and Gi
1 display more than 95% identify
in their primary sequence (22), the crystal structure of Gi
1-GTP
S
was adapted for a first-approximation description of the regions of
Gi
2 analyzed in the present work (Fig.
10). The structure displayed is that of
Gi
1 in which domains EM (147-171), MA (172-199), and AB (200-235)
are projected as regions I, II, and
III, respectively. The major region implicated in control of
adenylylcyclase (AC) is displayed in yellow (9), while that for regions I (EM), II (MA), and III (AB) are rendered cyan,
green, and blue, respectively (Fig. 10,
panels A and B). Maintaining the same landmarks
of conserved regions, the projection of Gs
residues upon the
corresponding structure of Gi
2 would place Leu203 and
Ser205 at positions of Gi
1 residues Lys180
and Thr182, respectively (Fig. 10, panel C). In
Gi
1, Lys180 and Thr182 appear as exposed
residues, available for protein-protein contact. It is of interest that
Lys180 and Thr182 of Gi
1 have been shown to
participate in the binding of the GTPase activator for Gi
1, RGS4
(24). Both Leu203 and Ser205 are essential
residues to the repressor activity of Gs
, substitution of both to
alanine abolishes the ability of the mutant Gs
to block
adipogenesis. The results predict that this domain (Fig. 10,
panel D) is an important contact site for Gs
with the
effector controlling differentiation in these cells or that these
residues, when altered via mutagenesis, interrupt some extended
conformation of Gs
critical to the repressor activity of the
molecule.
|
The Cys200 residue is a unique feature of Gs, not shared
by any other heterotrimeric G protein
subunit (Fig. 4).
Cys200 is a critical residue for Gs
function with
respect to repression of adipogenesis. Alanine substitution of
Cys200 effectively abolished the ability of Gs
to exert
its repressor activity on adipogenesis. Projection of this information
on the structure of Gi
1 identifies Thr 177 (Fig. 10, panels
C and D), a residue located in the C-terminal end of
Switch I region near the junction with linker 2 (19, 20).
Cys200 of Gs
is embedded in a sequence highly conserved
among G protein
subunits Leu Arg Cys Arg Val Xaa Thr,
including Gi
1, Gi
2, and Go
. The projections suggest that
Leu203 and Ser205 (both in Switch I region) as
well as Cys200 are in close proximity and likely exposed
residues important in Gs
repressor function (Fig. 10, panel
D).
Three of the remaining residues critical to repressor activity,
Asn167, Val214, and Lys216, appear
to be exposed when projected upon the corresponding area of the Gi1
structure (Fig. 10, panels C and D).
Asn167, located at the flex region between
helices D
and E (19, 20), is embedded in the sequence Arg Ser Asn Glu
Tyr Gln Leu that is highly conserved among G protein
subunits
including Gs
, Gi
1, Gi
2, and Go
. Val214 and
Lys216, located in the C-terminal reach of Switch I region
(19, 20), are embedded in a unique region of six residues in Gs
flanked both by 10 residues N-terminal as well as 20 residues
C-terminal that display a high degree of conservation among several G
protein
subunits, including Gi
1, Gi
2, and Go
. The protein
sequence 213-218 of Gs
would appear to play some unique role(s) in
its function, including a critical role in repressor activity, since alanine substitution of either Val214 or Lys216
abolishes the ability of Gs
to block adipogenesis.
The recent elucidation of the crystal structure of Gs at 2.5 A in a
complex with GTP
S (25) affords the opportunity to relate the
mutagenesis data to the structure of Gs
(Fig.
11). To facilitate the discussion, the
domain of Gs
implicated in the control of adenylylcyclase
(AC) is displayed in yellow, while the GTP organizing
elements Switch 1 (Sw1), Switch 2 (Sw2), and Switch 3 (Sw3) are rendered in cyan,
blue, and green, respectively (Fig. 11,
panel A). The Leu203, Ser205
cluster, essential for represser activity of Gs
, is displayed in
white (Fig. 11, panel A), as are Asn167,
Val214, and Lys216 residues (Fig. 11,
panel B). Cys200 is far less visible in the
Gs
-GTP
S structure than in the Gi
1-GTP
S structure (Fig. 11,
panel A). A stick model of Gs
, in which the GTP molecule
has been purposely deleted from the structure, illuminates all of the
residues essential for the repressor activity of Gs
(Fig. 11,
panel C). Inspection of the ribbon and coil diagram of Gs
highlights the critical placement of the Leu203,
Ser205 cluster in Switch 1 (Sw1), the proximity
of Asn167 to Switch 3 (Sw3), and the exposure of
Val214 and Lys216 (Fig. 11, panel
D).
|
The current study adds to our expanding understanding of the
structure-activity relationships in the Gs molecule. Constitutive expression of Gs
blocks adipogenesis and induction of adipogenesis occurs through a rapid loss of Gs
in 3T3-L1 adipocytes. Using construction of chimeric
subunits as well as alanine-scanning mutagenesis we provide an insight into the regions of Gs
that are
required for repressor activity. The regions do not overlap with those
implicated in the control of adenylylcyclase (Figs. 10 and 11). In
concert, alterations in intracellular cyclic AMP accumulation fail to
influence either the induction or the course of adipogenesis (6). The
nature of the effector through which Gs
repressed adipogenesis
remains unsolved. The structural information garnered from the current
analysis will assist in efforts aimed at identifying this additional
and novel effector for Gs
.
![]() |
ACKNOWLEDGEMENTS |
---|
We express our thanks to Erich Bremer for
expert assistance in molecular modeling and to Dr. Stephen Sprang
(Department of Pharmacology, University of Texas Southwestern Medical
School, Dallas, TX) for providing to us the coordinates for the crystal structure of GTPS-bound form of Gs
.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Service, National Institutes of Health NIDDK Grant DK30111.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.
¶ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, DMDRP, Health Sciences Center, SUNY/Stony Brook, Stony Brook, NY 11794-8631. Tel.: 516-444-7873; Fax: 516-444-7696.
1
The abbreviations used are: MIX,
methylisobutylxanthine; DMEM, Dulbecco's modified Eagle's
medium; GTPS, adenosine-5'-O(thiotriphosphate).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|