Identification of Amino Acid Residues of Gsalpha Critical to Repression of Adipogenesis*

Xunxian LiuDagger , Craig C. MalbonDagger , and Hsien-yu Wang§

Departments of Dagger  Molecular Pharmacology and § Physiology and Biophysics, Diabetes and Metabolic Diseases Research Program, University Medical Center, SUNY/Stony Brook, Stony Brook, New York 11794

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Gsalpha 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 Gsalpha with corresponding regions of the G protein Gialpha 2, the domain of Gsalpha 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 Gsalpha constructed from trisection of the sequence 125-213 of Gialpha 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 Gialpha 2 repressed adipogenesis, whereas the chimeras with either sequence 125-149 or 178-213 of Gialpha 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 Gsalpha 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 Gsalpha to repress adipogenesis, although not affecting the ability of the mutant form of Gsalpha to regulate adenylylcyclase. Using conserved landmarks found in the crystal structures of Gialpha 1 and Gsalpha , 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 Gsalpha that are exposed in the GTPgamma S-liganded state of the alpha  subunit. We speculate that these residues constitute an important contact domain between Gsalpha and the effector controlling adipogenesis, which is yet to be identified.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 Cbeta , 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 Gsalpha to control adipogenic conversion of the 3T3-L1 fibroblasts to adipocytes. Gsalpha has been shown to play a key role in the differentiation of 3T3-L1 cells, as evidenced by the following observations. Gsalpha expression declines dramatically within 48 h of induction of differentiation; constitutive expression of Gsalpha in 3T3-L1 cells blocks induction of cell differentiation by known inducers; suppression of Gsalpha expression by antisense oligodeoxynucleotides (mimicking the inducer-driven decline in Gsalpha ) accelerates the cell differentiation from a 10-day to a 3-day process in the presence of inducers; and treatment with oligodeoxynucleotides antisense to Gsalpha alone provokes adipogenesis in the absence of the classical inducers (1, 6, 13-15). Overexpression of the G protein that antagonizes many Gsalpha effects, Gialpha 2, provokes adipogenesis in either the absence or the presence of the inducers (16).

The central question remains how Gsalpha controls cell differentiation. The ability of Gsalpha 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 Gsalpha , much like expression of the constitutively active mutant form of Gsalpha (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 Gsalpha is substituted for the corresponding region of Gialpha 2 (Gialpha 2 1-122/Gsalpha 145-235/Gialpha 2 236-394) inhibited cell differentiation as effectively as wild-type Gsalpha (1). These data indicate that the sequence harboring residues 146-235 of Gsalpha , which is not the region interacting with adenylate cyclase (17-19), is critical in controlling cell differentiation. The region 146-235 of Gsalpha includes Switch I and Switch II (20), which are involved both in contact with beta gamma 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 Gsalpha that are responsible for the control of adipogenesis, the differentiation of 3T3-L1 embryonic fibroblasts to adipocytes. The repressor domain of Gsalpha was trisected into smaller sequences that were substituted with the corresponding domains of Gialpha 2 and the chimeras stably expressed in 3T3-L1 cells. Sequences 147-171 and 200-235, but not 172-199 of Gsalpha 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 Gsalpha to repress differentiation of 3T3-L1 cells.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Construction of Chimeras-- Chimeras were constructed from pCW1Gsalpha , pCW1GsQ227L, pCW1Gialpha 2Q205L, and pCW1Galpha i/sBam (1, 17). The small HindIII fragment of pCW1Gsalpha and pCW1Gialpha 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 pAlterGsalpha was transferred into the BgllI-ClaI site of pAlterGialpha 2Q205L to avoid two AflII sites at the 3' end of the Gsalpha 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 Gsalpha , where 690CAGCTG695 and 773GCTTCGC779 of DNA sequences were changed to 690CAATTG695 and 773 CTTAAGA779, respectively. The codons for both Gsalpha and Gialpha 2 were not changed. The plasmid is designated pAlterGsalpha MA. To construct chimera Galpha s1-146/i125-149/s (sisEM), pAlterGialpha 2Q205L was amplified as using T7 primers and Gialpha 2EcoRI sense oligonucleotide 5'-AAGAATTCTCGGGCGTCATCCGGAGG. The fragment was digested with EcoRI and inserted into the EcoRI sites of pAlterGsalpha 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 pAlterGsalpha MA. To generate chimera Galpha s1-171/i150-177/s (sisMA), pCW1-Gialpha 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 pAlterGsalpha MA. To generate chimera Galpha s1-199/i178-213/s (sisAB), pCW1Galpha 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 pAlterGsalpha MA.

Site-directed Mutagenesis-- Mutations were introduced into pAlterGsalpha 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, pCW1Gsalpha Q227L was partially digested with HindIII, filled in by Klenow fragment, and then ligated. Plasmids lacking the HindIII site at the 3' end of Gsalpha 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 Gsalpha and Gialpha 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 Gsalpha (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 beta -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 Gsalpha , 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
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Abstract
Introduction
Procedures
Results
Discussion
References

The strategy adopted to the analysis of the sequence of Gsalpha responsible for the repression of adipogenesis was based upon our previous analysis in which the sequence of 145-235 of Gsalpha was identified as critical (1). Gsalpha sequence 145-235 embedded into the corresponding region of Gialpha 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 Gsalpha repressing adipogenesis. To make the task manageable, the region(s) of Gsalpha 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 Gsalpha was trisected and regions 125-149, 150-177, and 178-213 of Gialpha 2 substituted individually for the corresponding region of Gsalpha . The chimeras constructed in this fashion are displayed in Fig. 1. The inability of a chimera with the Gialpha 2 region embedded in Gsalpha to repress adipogenesis would identify region(s) as candidate(s) for further analysis by alanine-scanning mutagenesis.


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Fig. 1.   Chimera with Gialpha 2 sequences substituted for corresponding regions of Gsalpha define the regions of sequence 145-235 that repress adipogenesis. To identify the region within Gsalpha 145-235 responsible for repression of adipogenesis, the region of interest was trisected and the corresponding region of Gialpha 2 was substituted to form a chimera. Clones of 3T3-L1 stably transfected were selected for expression of each of the chimera at a level at least 1-fold greater than endogenous levels of Gsalpha . Constitutive expression of Gsalpha at these levels is sufficient to block adipogenesis. Levels of endogenous Gsalpha decline precipitously in the first 3 days of adipogenesis (1, 6).

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 Gsalpha 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 Gsalpha (CM129). Expression of Gsalpha in clones harboring the expression vector for a chimera minimally was approximately 1-fold greater than the endogenous levels of Gsalpha expressed by 3T3-L1 clones stably transfected with the empty vector pCW1 alone. Constitutive expression of Gsalpha at a level 50% greater or more that endogenous Gsalpha is readily able to repress the ability of dexamethasone and MIX to induce adipogenesis (1). Chimeras of Gsalpha in which a smaller region of Gialpha 2 (125-149, 150-177, or 178-213) was substituted for the corresponding region in Gsalpha had little effect on the cAMP response of the clones to stimulation by isoproterenol, when compared with cells transfected with wild-type Gsalpha alone. Basal cAMP accumulation of these clones in comparison to that of the transfectants expressing wild-type Gsalpha alone was reduced slightly, perhaps reflecting the predominant Gialpha 2 nature of the expressed protein (Fig. 2B). Earlier studies have demonstrated that Gsalpha -Gialpha 2 chimera with these types of substitutions retain their capacity to bind GTP and transduce signaling (17, 24).


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Fig. 2.   Stable expression of Gsalpha and Gsalpha /Gialpha 2 chimeras in mouse 3T3-L1 cell clones: analysis by immunoblotting and cyclic AMP accumulation. Panel A, crude cell membranes (50 µg/lane) were prepared from 3T3-L1 clones stably transfected with the expression vector alone (pCW1) (lane 1), Gsalpha (lane 2), sisEM (lane 3), sisMA (lane 4), and sisAB (lane 5). The membranes were subjected to SDS-polyacrylamide gel electrophoresis and the resolved proteins transferred to nitrocellulose blots. The blots were stained with anti-Gsalpha antibody (CM129) raised against the peptide corresponding to C-terminal sequence Gsalpha -(384-394). Immune complexes were made visible by a second goat anti-rabbit IgG to which calf alkaline phosphatase was coupled. Panel B, for cyclic AMP accumulation, cells were incubated in the absence or presence of isoproterenol (10 µM) for 15 min at 37 °C in medium supplemented with Ro compound (0.1 mM). Data are the mean values ± S.E., representing four independent experiments. Labeling: EV, empty vector; Gs, Gsalpha ; chimera sisEM; chimera sisMA; and chimera sisAB.

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 Gsalpha , 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 Gsalpha (panel D). These data demonstrate that regions 147-171 (sisEM) and 200-235 (sisAB) of Gsalpha are critical in the control of adipogenesis, when substituted with the corresponding region of Gialpha 2 the chimera lose the ability to repress adipogenesis. Region 172-199 of Gsalpha , to the contrary, appears to be dispensable and can be replaced by the corresponding region of Gialpha 2 without altering the ability of chimera to repress adipogenesis.


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Fig. 3.   Expression of chimera sisEM and chimera sisAB lose the ability to repress induction of adipogenesis: analysis by oil red O staining. Stably transfected clones with empty vector (pCW1), as well as those transfectants stably expressing Gsalpha (alpha s), sisEM, sisMA, and sisAB chimeras, as well as the D229S mutation of Gsalpha (alpha sD229S), were plated on coverslips and propagated in 24-well culture plates. At confluence (day 0), one set of cells were treated with dexamethasone and methylisobutylxanthine (+D/M). Dexamethasone + MIX were removed after incubation for 2 days, and the cells were maintained in DMEM containing 10% fetal bovine serum for 10 days. A second replicate set of cells was maintained in DMEM containing 10% fetal bovine serum in the absence (-D/M) of dexamethasone + MIX. At day 10, cells were fixed by 3% paraformaldehyde for 5 min and stained with oil red O for 10 min. Hematoxylin (1%) was used to stained nuclei. Adipogenesis was examined under a Zeiss Axiophot microscope. The darkly stained bodies of the cytosol are oil droplets. Bar, 100 µm. Data represent six different clones and at least two independent series of transfection experiments.

Having identified two smaller regions of Gsalpha 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 (Gsalpha M3, Gsalpha M5, and Gsalpha M6) and multiple (Gsalpha M1, Gsalpha M2, Gsalpha M4, and Gsalpha M7-M10) alanine substitutions in protein sequences 147-171 and 200-235 of Gsalpha (Fig. 4, A and B), presuming that alanine substitution of a critical residue would abolish the ability of the mutant Gsalpha to repress adipogenesis. Clones stably transfected with pCW1 harboring the cDNA of Gsalpha with one or more mutations displayed increased immunoreactive Gsalpha , reflecting the expression of the mutant forms in excess of the endogenous level of Gsalpha . Clones were selected that expressed the mutant Gsalpha molecules at levels approximately 1-fold greater than the staining observed in clones harboring empty vector alone (Fig. 5A). All of the clones expressing Gsalpha 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).


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Fig. 4.   Alignment of sequences of Gsalpha and Gialpha 2 with regions of Gsalpha subjected to alanine-scanning mutagenesis to identify amino acid residues of Gsalpha critical to the repression of adipogenesis. Protein sequence is provided using the three-letter symbol and the number of each residues commencing from the N terminus is provided above the sequence for Gialpha 2 and below for the sequences for Gsalpha . Residues in boxes are conserved between Gsalpha and Gialpha 2 and were not subjected to mutation analysis, since Gsalpha represses adipogenesis whereas Gialpha 2 does not. Residues in bold are the alanine substitutions created in the Gsalpha mutants. Panel A, alignment of sisEM region is provided to describe alanine substitutions in mutants in the series Gsalpha M1-M5. Panel B, alanine substitutions of sisAB region highlighted in mutations of series Gsalpha M6-M10 as well as the single Gsalpha D229S mutant. Panel C, single alanine substitutions Gsalpha (200-235) shows highlight nine mutants used to confirm and extend the analysis of the alanine mutations performed as clusters.


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Fig. 5.   Stable expression of wild-type and alanine-scanning mutant forms of Gsalpha in 3T3-L1 cell clones: analysis by immunoblotting and cyclic AMP accumulation. Panel A, immunoblotting of the crude cell membranes of 3T3-L1 clones stably transfected with the expression vector alone (EV) (lane 1) and vector harboring Gsalpha M1 (lane 2), Gsalpha M2 (lane 3), Gsalpha M3 (lane 4), Gsalpha M4 (lane 5), Gsalpha M5 (lane 6), Gsalpha M6 (lane 7), Gsalpha M7 (lane 8), Gsalpha M8 (lane 9), Gsalpha M9 (lane 10), and Gsalpha M10 (lane 11). Panel B, for cyclic AMP accumulation, cells were incubated in the absence or presence of isoproterenol (Iso, 10 µM) for 15 min at 37 C in medium supplemented with Ro compound (0.1 mM). Data are the mean values ± S.E. representing four independent experiments. Labeling: EV, empty vector; Gs, Gsalpha ; M1, Gsalpha M1; M2, Gsalpha M2; M3, Gsalpha M3; M4, Gsalpha M4; M5, Gsalpha M5; M6, Gsalpha M6; M7, Gsalpha M7; M8, Gsalpha M8; M9, Gsalpha M9; and M10, and Gsalpha M10.

In the context of region 147-171 (sisEM domain), alanine-scanning mutations of Gsalpha M1 through Gsalpha M4 displayed no effect on the ability of the mutant form of Gsalpha to repress adipogenesis in response to the inducers dexamethasone + MIX (Fig. 6). Clones constitutively expressing Gsalpha M1, 2, 3, and 4 (panels C, D, E, and F, respectively) were refractory to induction of adipogenesis. In contrast, expression of Gsalpha M5, a single alanine substitution for asparagine at position 167 abolishes the ability of the mutant form of Gsalpha to repress adipogenesis (panel G). Clones expressing N167A Gsalpha 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).


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Fig. 6.   Expression of Gsalpha M1, Gsalpha M2, Gsalpha M3, Gsalpha M4 and Gsalpha M8, but not Gsalpha M5-7, Gsalpha M9, nor Gsalpha M10, blocks induction of adipogenesis: analysis by oil red O staining. Stably transfected clones with empty vector (pCW1), Gsalpha (alpha s), and 10 Gsalpha mutants were plated on coverslips and propagated in 24-well culture plates, respectively. At confluence (day 0), one set of cells were treated with dexamethasone and methylisobutylxanthine (+D/M). Dexamethasone + MIX were removed after incubation for 2 days, and the cells were maintained in DMEM containing 10% fetal bovine serum for 10 days. A second replicate set of cells was maintained in DMEM containing 10% fetal bovine serum in the absence of dexamethasone + MIX (not differentiated and not shown). At day 10, cells were fixed by 3% paraformaldehyde for 5 min and stained with oil red O for 10 min. Expression of Gsalpha M1, Gsalpha M2, Gsalpha M3, Gsalpha M4, and Gsalpha M8 but not Gsalpha M5, Gsalpha M6, Gsalpha M7, Gsalpha M9 nor Gsalpha M10, mutants blocks induction of adipogenesis. The darkly stained bodies of the cytosol are oil droplets. Bar, 100 µm. Data represent six different clones and at least two independent series of transfection experiments.

Analysis of the sisAB region (Gsalpha residues 200-235) initially focused upon aspartic acid residue 229 which was the only residue within the region of 221-235 of Gsalpha that varied from that of Gialpha 2 (Fig. 4B). The D229S mutant form of Gsalpha 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 Gsalpha , 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 (Gsalpha M6) and the others multiple substitutions (Gsalpha M7-M10), were constructed and shown to be expressed in stably transfected clones at >1-fold over endogenous Gsalpha (Fig. 5A). These clones displayed increased basal cAMP accumulation over that observed for clones expressing the endogenous Gsalpha (Fig. 5B) and were examined further for their ability to respond to induction with dexamethasone + MIX.

Expression of Gsalpha M6 (C200A) abolished the ability of Gsalpha to repress adipogenesis in the clones (Fig. 6, panel H). Clones expressing C200A Gsalpha now stain prominently for accumulated lipid following treatment with dexamethasone + MIX. Expression of Gsalpha M7 (L203A and S205A), likewise, abolished the ability of Gsalpha 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 Gsalpha in the clones expressing Gsalpha M8 had no discernible effect on the ability of Gsalpha 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 Gsalpha M9 (Fig. 6, panel K). In the presence of the inducers dexamethasone + MIX, clones expressing the Gsalpha M9 mutant form of Gsalpha 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 Gsalpha to repress adipogenesis in response to dexamethasone + MIX (Fig. 6, panel L). Thus, the mutations found in Gsalpha M6, 7, 9, and 10 render the Gsalpha unable to repress adipogenesis as does the wild-type Gsalpha (Fig. 6, panel B).

The alanine-scanning mutagenesis of single residues had revealed that N167A Gsalpha and C200A Gsalpha 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 Gsalpha would require finer detailed analysis. Single point alanine substitutions were created for each of nine residues implicated as critical to the repressor activity of Gsalpha (Fig. 4C). Mutant forms of Gsalpha were stably transfected at levels approximately 1-fold greater than that of endogenous Gsalpha (Fig. 7). Although Gsalpha M7 with L203A and S205A double mutation has lost the ability of Gsalpha 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 Gsalpha .


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Fig. 7.   Endogenous Gsalpha and stable expression of point mutations of Gsalpha in 3T3-L1 cell clones: analysis by immunoblotting. Immunoblotting of the crude cell membranes of 3T3-L1 clones stably transfected with the expression vector alone (EV, lane 1) and vector harboring Gsalpha L203A (lane 2), Gsalpha S205A (lane 3), Gsalpha Q213A (lane 4), Gsalpha V214A (lane 5), Gsalpha D215A (lane 6), Gsalpha K216A (lane 7), Gsalpha V217A (lane 8), Gsalpha N218A (lane 9), and Gsalpha H220A (lane 10).


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Fig. 8.   Expression of GsL203A, GsS205A, GsQ213A, GsD215A, GsV217A, GsN218A, and GsH220A, but not GsV214A nor GsK216A, block induction of adipogenesis: analysis by oil red O staining. Stably transfected clones expressing empty vector (pCW1), Gsalpha (alpha s), and nine Gsalpha single alanine substitution mutants were plated on coverslips and propagated in 24-well culture plates. At confluence (day 0), one set of cells were treated with dexamethasone and methylisobutylxanthine (+D/M). Dexamethasone + MIX were removed after incubation for 2 days, and the cells were maintained in DMEM containing 10% fetal bovine serum for 10 days. A second replicate set of cells was maintained in DMEM containing 10% fetal bovine serum in the absence of dexamethasone + MIX (not differentiated and not shown). At day 10, cells were fixed by 3% paraformaldehyde for 5 min and stained with oil red O for 10 min. The darkly stained bodies of the cytosol are oil droplets. Bar, 100 µm. Data represent six different clones and at least two independent series of transfection experiments.

The expression of Gsalpha M9, a mutant form of Gsalpha 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 Gsalpha to repress adipogenesis. Similarly, mutation of D215A resulted in a mutant form of Gsalpha that fully repressed the ability of dexamethasone + MIX to induce differentiation, just as observed with constitutive expression of wild-type Gsalpha . In sharp contrast to the G213A and D215A mutations, alanine substitution of Val 214 abolished the ability of Gsalpha to repress adipogenesis of the clones in response to dexamethasone + MIX. These data reveal the basis of the Gsalpha M9 cassette of mutations to abolish the repressor activity of Gsalpha resides solely in the V214A mutation.

The Gsalpha M10 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 Gsalpha (Fig. 4, panel C). Although expression of the Gsalpha M10 cassette eliminates the ability of Gsalpha 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 Gsalpha 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 Gsalpha M10 cassette on loss of repressor activity by Gsalpha .

Single mutations of N167A, C200A, V214A, and K216A as well as the double mutation L203A,S205A abolished the ability of expressed Gsalpha to repress adipogenesis in 3T3-L1 cells. Mutant forms of Gsalpha were expressed at levels approximately 1-fold greater than that of endogenous Gsalpha (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).


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Fig. 9.   Stable expression of those point mutations of Gsalpha in 3T3-L1 cell clones that control adipogenesis: analysis by immunoblotting and cyclic AMP accumulation. Panel A, immunoblotting of the crude cell membranes of 3T3-L1 clones stably transfected with the expression vector alone (EV) (lane 1) and vector harboring Gsalpha (lane 2), Gsalpha N167A (lane 3), Gsalpha C200A (lane 4), Gsalpha L203A + S205A (lane 5), Gsalpha V214A (lane 6), and Gsalpha K216A (lane 7). Panel B, for cyclic AMP accumulation, cells were incubated in the absence or presence of isoproterenol (10 µM) for 15 min at 37 °C in medium supplemented with Ro compound (0.1 mM). Data are the mean values ± S.E. representing four independent experiments. Labeling: EV, empty vector; N167A, Gsalpha N167A; C200A, Gsalpha C200A; L203A + S205A Gsalpha L203A + S205A; V124A, Gsalpha V214A; and K216A, Gsalpha K216A.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Comparisons of the sequences of the domains in Gsalpha (Tyr147-Leu171 and Cys200-Ile235) that repress adipogenesis in 3T3-L1 cells with the corresponding sequences of other G protein alpha  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 beta gamma complex by Gtalpha are conserved in Gsalpha also (21). The protein sequence Met221-Ile235 of Gsalpha is highly conserved in virtually all of the heterotrimeric G protein alpha  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 Gsalpha and Gialpha 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 alpha  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 Gsalpha found in the sequence from 221 to 235, and D229S mutant form of Gsalpha 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 alpha  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 Gsalpha , exploiting our knowledge of the repressor domain by alanine-scanning mutagenesis. Repressor activity of protein sequence 146-235 of Gsalpha was identified (6) through the construction and then expression of various chimeras between Gsalpha (repressor) and corresponding regions of Gialpha 2 (activator). The ability of these Gsalpha /Gialpha 2 chimeras to block adipogenesis in 3T3-L1 clones challenged with well-known inducers of differentiation was evaluated (1). Since Gialpha 2 and Gialpha 1 display more than 95% identify in their primary sequence (22), the crystal structure of Gialpha 1-GTPgamma S was adapted for a first-approximation description of the regions of Gialpha 2 analyzed in the present work (Fig. 10). The structure displayed is that of Gialpha 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 Gsalpha residues upon the corresponding structure of Gialpha 2 would place Leu203 and Ser205 at positions of Gialpha 1 residues Lys180 and Thr182, respectively (Fig. 10, panel C). In Gialpha 1, Lys180 and Thr182 appear as exposed residues, available for protein-protein contact. It is of interest that Lys180 and Thr182 of Gialpha 1 have been shown to participate in the binding of the GTPase activator for Gialpha 1, RGS4 (24). Both Leu203 and Ser205 are essential residues to the repressor activity of Gsalpha , substitution of both to alanine abolishes the ability of the mutant Gsalpha to block adipogenesis. The results predict that this domain (Fig. 10, panel D) is an important contact site for Gsalpha with the effector controlling differentiation in these cells or that these residues, when altered via mutagenesis, interrupt some extended conformation of Gsalpha critical to the repressor activity of the molecule.


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Fig. 10.   Residues of Gsalpha critical for repressor activity of adipogenesis projected onto the corresponding regions of the crystal structure of GTPgamma S-liganded Gialpha 1. The structure displayed is that of Gialpha 1-GTPgamma S 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. The GTP molecule is displayed in white. Please see "Discussion" for further details.

The Cys200 residue is a unique feature of Gsalpha , not shared by any other heterotrimeric G protein alpha  subunit (Fig. 4). Cys200 is a critical residue for Gsalpha function with respect to repression of adipogenesis. Alanine substitution of Cys200 effectively abolished the ability of Gsalpha to exert its repressor activity on adipogenesis. Projection of this information on the structure of Gialpha 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 Gsalpha is embedded in a sequence highly conserved among G protein alpha  subunits Leu Arg Cys Arg Val Xaa Thr, including Gialpha 1, Gialpha 2, and Goalpha . 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 Gsalpha 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 Gialpha 1 structure (Fig. 10, panels C and D). Asn167, located at the flex region between alpha  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 alpha  subunits including Gsalpha , Gialpha 1, Gialpha 2, and Goalpha . 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 Gsalpha 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 alpha  subunits, including Gialpha 1, Gialpha 2, and Goalpha . The protein sequence 213-218 of Gsalpha 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 Gsalpha to block adipogenesis.

The recent elucidation of the crystal structure of Gsalpha at 2.5 A in a complex with GTPgamma S (25) affords the opportunity to relate the mutagenesis data to the structure of Gsalpha (Fig. 11). To facilitate the discussion, the domain of Gsalpha 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 Gsalpha , 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 Gsalpha -GTPgamma S structure than in the Gialpha 1-GTPgamma S structure (Fig. 11, panel A). A stick model of Gsalpha , in which the GTP molecule has been purposely deleted from the structure, illuminates all of the residues essential for the repressor activity of Gsalpha (Fig. 11, panel C). Inspection of the ribbon and coil diagram of Gsalpha 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).


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Fig. 11.   Residues of Gsalpha critical for repressor activity of adipogenesis displayed on the crystal structure of GTPgamma S-liganded Gsalpha . The crystal structure of Gsalpha at 2.5 Å in a complex with GTPgamma S (25) is displayed in panels A-D, as space-filling (front, panel A; back, panel B), stick (panel C), and ribbon (panel D). The mutagenesis data are project onto the structure of Gsalpha . To facilitate the discussion, the domain of Gsalpha 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 (panel A). The GTP molecule has been eliminated from the image to allow greater clarity of the switch regions. Please see "Discussion" for further details.

The current study adds to our expanding understanding of the structure-activity relationships in the Gsalpha molecule. Constitutive expression of Gsalpha blocks adipogenesis and induction of adipogenesis occurs through a rapid loss of Gsalpha in 3T3-L1 adipocytes. Using construction of chimeric alpha  subunits as well as alanine-scanning mutagenesis we provide an insight into the regions of Gsalpha 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 Gsalpha 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 Gsalpha .

    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 GTPgamma S-bound form of Gsalpha .

    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; GTPgamma S, adenosine-5'-O(thiotriphosphate).

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Abstract
Introduction
Procedures
Results
Discussion
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