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Dominant-negative Ga subunits are a mechanismof dysregulated heterotrimeric G protein signalingin human diseaseArthur Marivin,1* Anthony Leyme,1* Kshitij Parag-Sharma,1 Vincent DiGiacomo,1

Anthony Y. Cheung,1 Lien T. Nguyen,1 Isabel Dominguez,2 Mikel Garcia-Marcos1†

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Auriculo-condylar syndrome (ACS), a rare condition that impairs craniofacial development, is causedby mutations in a G protein–coupled receptor (GPCR) signaling pathway. In mice, disruption ofsignaling by the endothelin type A receptor (ETAR), which is mediated by the G protein (heterotrimericguanine nucleotide–binding protein) subunit Gaq/11 and subsequently phospholipase C (PLC), impairsneural crest cell differentiation that is required for normal craniofacial development. Some ACSpatients have mutations in GNAI3, which encodes Gai3, but it is unknown whether this G proteinhas a role within the ETAR pathway. We used a Xenopus model of vertebrate development, in vitrobiochemistry, and biosensors of G protein activity in mammalian cells to systematically characterize thephenotype and function of all known ACS-associated Gai3 mutants. We found that ACS-associatedmutations in GNAI3 produce dominant-negative Gai3 mutant proteins that couple to ETAR but cannotbind and hydrolyze guanosine triphosphate, resulting in the prevention of endothelin-mediated activationof Gaq/11 and PLC. Thus, ACS is caused by functionally dominant-negative mutations in a heterotrimericG protein subunit.

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INTRODUCTION

Heterotrimeric guanine nucleotide–binding proteins (G proteins) are gate-keepers of signal transduction that play critical roles in physiology. Theycycle between inactive [guanosine diphosphate (GDP)–bound] and active[guanosine triphosphate (GTP)–bound] states to control the flow of in-formation from extracellular cues to intracellular effectors (1–4). Restingheterotrimeric G proteins are composed of a GDP-bound Ga subunit incomplex with Gbg. G protein activation is predominantly carried out byG protein–coupled receptors (GPCRs), which promote the exchange ofGDP for GTP on Ga (1, 3). This leads to the dissociation of the hetero-trimer into Ga-GTP and free Gbg, both of which act on downstream ef-fector molecules. Ga subunits are classified into four major families,Gas, Gai/o, Gaq/11, and Ga12/13, which are characterized by their abilityto modulate different effector molecules and second messengers. Forexample, Gai subunits dampen the production of cyclic adenosine mono-phosphate by inhibiting adenylyl cyclase, whereas Gaq subunits increaseintracellular Ca2+ by activating phospholipase C (PLC). The evidence thatthe dysregulation of heterotrimeric G protein signaling causes various dis-eases is mounting rapidly, but the underlying molecular mechanisms inmany of these diseases are elusive. Most G protein–associated mutationshave been identified in genes encoding Ga subunits (5, 6) and are broadlyclassified as loss-of-function or gain-of-function mutations. Loss-of-function mutations in various Ga proteins are associated with the congen-ital diseases Albright’s hereditary osteodystrophy (Gas), Nougaret nightblindness (Gat), and two types of dystonia (Gaolf) (5, 7–10). On the otherhand, gain-of-function mutations are somatic and exert a dominant effectby rendering the Ga subunit constitutively active. The most frequent al-

1Department of Biochemistry, Boston University School of Medicine, Boston,MA 02118, USA. 2Department of Medicine, Boston University School of Med-icine, Boston, MA 02118, USA.*These authors contributed equally to this work.†Corresponding author. Email: mgm1@bu.edu

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teration associated with gain-of-function mutations is guanosine tripho-sphatase (GTPase) deficiency, as seen in aberrant Gas, Gaq, and Ga11in up to 80% of patients with some types of cancer (6).

Auriculo-condylar syndrome (ACS) (11–13) is a rare condition that im-pairs craniofacial development. Evidence from genetic studies in humansand animal models indicates that ACS is caused by disruption of an en-dothelin typeA receptor (ETAR)/Ga/PLCpathway that induces the expres-sion of genes encoding the distal-less homeobox (DLX) transcriptionfactors DLX5 andDLX6 required for specification and patterning of neuralcrest cells during craniofacial development (14). In humans, this hypothesisis supported by the identification of mutations in the genes encoding for thenatural ETAR ligand endothelin-1 (ET-1) (15) and the Gaq/11 effectorPLCb4 (11, 12). This is in agreement with evidence in mice showing thatknockout animals lacking Gaq and its close homolog Ga11 display cranio-facial defects that resemble ACS (16, 17). However, this model contrastswith the genetic evidence in humans; no Gaq or Ga11 mutations have beenfound inACSpatients to date. Instead, someACSpatients havemutations inanother Ga subunit, Gai3. Whether Gai3 functions between ETAR andPLCb4 and the mechanism of action by which Gai3 mutations affect thispathway are not yet known.

ACS is classified as type I, II, or III based on the presence of muta-tions in GNAI3 (encoding Gai3), PLCB4 (encoding PLCb4), or EDN1(encoding ET-1), respectively. Five autosomal dominant mutations inGai3 have been found in type I ACS (11–13). All five mutations affectconserved amino acid positions that cluster within the nucleotide bindingpocket (Fig. 1 and fig. S1). It has been speculated that ACS-associatedGai3 mutantsmay behave as dominant-negatives (meaning it interfereswiththe function of the normal gene product) or as constitutively active G pro-teins (11, 13, 14). However, none of these hypotheses or the possibility ofacting as loss-of-function mutants leading to haploinsufficiency has beenformally tested. Here, we investigated the molecular basis of type I ACSby systematically characterizing the functional consequences of the fiveGai3 mutations found inACS patients using invivo and invitro approaches.

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RESULTS

ACS mutations increase the frequency of developmentaldefects induced by the ectopic expression of Gai3 inXenopus laevis embryosAs a first approach to investigate the functional consequences of ACSmutations in Gai3, we performed experiments with Xenopus laevisembryos as a development-relevant model. For this, we took advantageof previous observations (18) showing that ectopic expression of Gai1induces developmental defects in X. laevis. During normal gastrulation,embryo cell movements cause the appearance of a new hollow cavity, thearchenteron, while simultaneously inducing the progressive removal ofanother, the blastocoel. In Gai1-injected embryos, this process is dis-rupted: the archenteron does not inflate and the blastocoel is not removed(18). The abnormal distribution of the internal cavities alters the flotationof the embryo and causes an inversion of its normal gravitational orien-tation (Fig. 2A). This phenotype can be easily scored at the neurula stagebecause the neural tube faces downward instead of upward. Although itis not known what specific signaling pathways are disrupted by the ec-topic expression of Gai, this system provides a suitable platform to testwhether G proteinmutants induce a gain or loss of function in the contextof embryonic development. First, we validated that ectopic expression ofGai3 caused the same defects as those previously observed for Gai1 (Fig.2A). We reasoned that if the ACS-associated mutations induce loss offunction in Gai3, their expression will not lead to a Gai3 overexpressionphenotype. We found that this is not the case because the ACS-associatedGai3 mutants exerted the opposite effect; expression of any of the fivemutants (G40R, G45V, S47R, T48N, and N269Y) increased the frequen-cy of gravitational inversion in embryos compared to those expressingwild-type Gai3; this was observed at two different injected doses ofmRNA (Fig. 2, B and C). Embryo bisections revealed that the gravita-tional inversion in ACS-associated Gai3 mutants (of which S47R isshown as a representative in Fig. 2B) was accompanied by defectivearchenteron inflation and incomplete blastocoel removal, indicatingthat the underlying cause of the phenotype is the same as in wild-typeGai3–injected embryos. We ruled out that the different abundance ofthe Gai3 mutant proteins compared to that of wild-type Gai3 caused

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the difference in phenotype frequency by confirming their equal quantitiesin immunoblots of embryo lysates (Fig. 2C). Together, these results dem-onstrate that ACS mutations increase the frequency of developmentaldefects caused by ectopic expression of Gai3, indicating that the mu-tations provide Gai3 with dominant properties rather than induce a lossof function.

ACS-associatedGai3mutantsdisplaydefectiveGTPbindingTo further characterize the functional consequences of the ACS muta-tions in Gai3, we investigated their biochemical properties in vitro. Weobtained very poor or no yields of soluble proteins when we attempted topurify Gai3 G40R, G45V, T48N, or N269Y from Escherichia coli, sug-gesting that the mutations compromise G protein stability. Only Gai3S47R was purified in sufficient quantities for biochemical studies.The ability of Gai3 S47R to bind nucleotides was investigated by usinga well-established assay that monitors G protein activation upon nucle-otide binding by measuring its susceptibility to limited trypsinolysis.Briefly, inactive, GDP-bound Ga is readily digested by trypsin, whereasactive Ga, generated by the binding of GTP mimetics guanosine 5′-O-(3′-thiotriphosphate) (GTPgS) or GDP-AlCl3/NaF (GDP-AlF4

−), adopts aconformation that is resistant to trypsin digestion outside of a short N-terminal sequence, which is cleaved off by trypsin (19). We found thatGai3 S47R protection from trypsinolysis after incubation with GTPgSor GDP-AlF4

− was markedly reduced compared to that of wild-typeGai3 (Fig. 3A), indicating that the mutant cannot bind nucleotides and/orit cannot change conformation efficiently upon GTP binding. Next, we in-vestigated whether the S47R mutation affects the steady-state GTPaseactivity of Gai3 and found that it was virtually abolished (Fig. 3B).The cause of this defect was failure to bind GTP, as determined by twoindependent approaches that measure binding of the nonhydrolyzableanalog GTPgS (Fig. 3, C and D). To elucidate whether the impaired ac-tivation of Gai3 S47R was due to a defect specific to GTP binding or tonucleotide binding in general, we analyzed its nucleotide content byhigh-performance liquid chromatography (HPLC). Gai3 proteins were ex-changed into nucleotide-free buffer and rapidly denatured to release anybound nucleotide. The HPLC analysis revealed identical chromatogramsfor wild-type Gai3 and Gai3 S47R corresponding to GDP peaks of iden-tical intensity (Fig. 3E). Together, these results demonstrate that Gai3S47R retains the capability to bind GDP but fails to bind GTP, renderingthe G protein unable to switch into an active conformation.

Next, we investigated whether the GTP-binding defect of Gai3 S47Ris a common feature among all the ACS mutants. Because Gai3 G40R,G45V, T48N, and N269Y mutants could not be purified from bacteria,we measured susceptibility to limited proteolysis after expression inmammalian cells. In initial experiments, we found that all the ACS mu-tant plasmids expressed lower protein quantities than wild-type Gai3 inhuman embryonic kidney 293T (HEK293T) cells. To overcome this lim-itation and be able to make comparisons, we equalized the amount ofprotein expression by transfecting larger quantities of the correspondingACSmutant plasmids (two to four times more than that of the wild type).We found that protection from trypsinolysis after GTPgS incubation wasreduced for all five ACS-associated Gai3 mutants (G40R, G45V, S47R,T48N, and N269Y) compared to that for wild-type Gai3 (Fig. 3F). Thesefindings indicate that defectiveGTP binding andG protein activation is acommon feature of ACS-associated Gai3 mutants.

ACS-associated Gai3 mutants cannot be activatedby a GPCROur in vitro data indicate that ACS mutations abolish the spontaneousexchange of GDP for GTP on Gai3. Therefore, we investigated whether

Fig. 1. ACSmutations affect re-sidues that cluster around thenucleotide binding pocket ofGai3. Top: Schematic diagramdepicting structural elementsofGai3 and the location of all fivemutations found to date in pa-tientswithACS.Switch regions I,II, and III involved in activation-inducedconformational changes

aredepicted ingray, and the fiveconservedGboxes involved in nucleotidebinding are shown inblue.Bottom: Three-dimensional representation of theGai nucleotide binding pocket [Protein Data Bank (PDB): 1GIA]. Aminoacidsmutated in ACS (beige) cluster around the nucleotide binding pocket(GTP in purple).

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they also affect GPCR-catalyzed Gai3 activation. GPCRs are guaninenucleotide exchange factors (GEFs) that accelerate the exchange ofGDP for GTP, which in turn dissociates Gabg trimers into Ga-GTP andGbg. Because association of Ga with Gbg is an obligatory requirementfor GPCR-catalyzed activation, we first examined whether ACS-associatedGai3 mutants bind to Gbg in cells. For this, we used a cell-based bio-luminescence resonance energy transfer (BRET) system that monitorsthe association of free Venus-Gbg (BRET acceptor) with the C-terminalfragment of its effector, G protein–coupled receptor kinase 3 (GRK3), fusedto a modified luciferase (masGRK3ct-Nluc, BRET donor) (20, 21). In theabsence of Ga, free Gbg robustly associates with the GRK3 probe, lead-ing to high BRET signals, whereas expression of Ga subunits favors theformation of Ga:Gbg complexes and diminishes the association of Gbgwith GRK3, thereby quenching the BRET signals (fig. S2A). As expected,we found that ectopic expression of wild-type Gai3 in HEK293T cells de-creased the Gbg:GRK3 BRET signals compared to cells not transfected

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with Gai3 (fig. S2B). When we trans-fected cells with plasmids expressing ACS-associated Gai3 mutants G40R, G45V,S47R, T48N, or N269Yat the appropriatequantities to obtain protein amounts simi-lar to wild-type Gai3 (fig. S2B, lower pan-el), we found that the BRET signal wasalso significantly reduced compared tocells expressing Gbg alone (fig. S2B, upperpanel). However, we also observed thatexpression of some of the mutants (G40R,G45V, T48N, and N269Y) did not quenchthe BRET signal as efficiently as did ex-pression of wild-type Gai3 (fig. S2B, upperpanel), which is indicative of reduced Gbgbinding. These results indicate that, al-though some of the ACS-associated Gai3mutants display mild to moderate Gbgbinding defects, all of them are capableof forming significant amounts of Gabgcomplexes that serve as GPCR substrates.

We used the same BRET-based experi-mental system described above to monitorthe kinetics of G protein activation uponstimulation of a prototypical Gi-coupledGPCR, the adenosine 1 receptor (A1R)(Fig. 4A and fig. S2C). In cells expressingwild-type Gai3, stimulation of A1R withadenosine resulted in a rapid increase ofthe BRET signal (Fig. 4B and fig. S2C),which is indicative of dissociation of Gbgfrom Gai3 upon G protein activation. Thisactivation was reversed upon GPCR inhi-bition because addition of the A1R antag-onist 8-cyclopentyl-1,3-dipropylxanthine(DPCPX) returned BRET signals to basalvalues (fig. S2C). When analogous experi-ments were performedwith cells expressingany of the five ACS-associated Gai3 mutants(G40R, G45V, S47R, T48N, or N269Y) inprotein amounts equivalent to wild-typeGai3 (as in fig. S2B), the BRET increaseupon adenosine stimulation was essentiallyabsent (Fig. 4B). Because, under these con-

ditions, some ACS-associated Gai3 mutants displayed increased basalBRET values that are indicative of impaired association with Gbg (fig.S2B), we performed additional experiments to rule out the possibility thatthe observed defect in GPCR-mediated activation was due to the impairedformation of Gabg heterotrimers. For this, we adjusted the transfectedamounts of wild-type Gai3 and mutant plasmids to obtain similar basalBRET values for all of them, which reflect equivalent Gabg heterotrimerformation.We found that, under these conditions, the BRETincrease uponadenosine stimulation was absent for all the Gai3 mutants. Because it isalso possible that impaired activation of Gai3 mutants by GPCRs is due toG protein mislocalization, we performed an additional control by investi-gating the subcellular localization of the Gai3 mutants when expressed inthe presence of Gbg. We found that the subcellular distribution of each ofthe five ACS-associated Gai3 mutants was similar to that of wild-typeGai3 (fig. S3), including localization at the plasma membrane as well asendomembranes, as previously reported (22, 23), indicating that defective

Fig. 2. ACS mutations increase the frequency of developmental defects induced by ectopic expressionof Gai3 in X. laevis embryos. (A) Validation of an assay to monitor Gai3-induced developmental defectsin X. laevis. Left: Schematic diagram illustrating the relationship between tissue remodeling defectsand inversion of the gravitational orientation of Gai-injected embryos. Top right: Representativeembryos injected with mRNA encoding wild-type Gai3 (Gai3 WT; 60 pg) compared with uninjectedcontrols at the late neurula stage. Arrowhead marks inverted gravitational orientation [neural tube(NT) facing downward]. Bottom right: Sagittal sections of a representative control and a Gai3-injectedembryo with impaired archenteron (arc) inflation and incomplete blastocoel (bc) removal. (B and C)Assessment of the percentage of embryos displaying a gravitational defect after injection with 30 or 60 pgof mRNA encoding Gai3 WT or ACS mutant. Representative images (B) are presented and marked as in(A). Quantification is shown in (C). Data are means ± SE of the phenotype proportion of the indicated totalnumber (n) of embryos from each test group. *P < 0.01 and **P < 0.001 for Gai3 mutants compared to WTusing Fisher’s exact test. Immunoblots (IB; C) confirm protein abundance in embryos injected with theindicated mRNAs.

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activation by GPCRs is not due to G protein mislocalization. Together,these results demonstrate that trimeric G proteins bearing Gai3 subunitswith ACS-associated mutations cannot be activated by GPCRs.

ACS-associated Gai3 mutants have increased bindingto GEFsThe results presented so far indicate that ACS-associated Gai3 mutantsexert a dominant effect in Xenopus bioassays, cannot bind GTP efficiently,and fail to be activated by the GEF activity of ligand-bound GPCRs. Theseresults suggest that these ACS-associated Gai3 mutants may work asdominant-negative proteins by forming nonproductive complexeswithGEFsbecause the dissociation that occurs upon GTP binding to the wild-type pro-tein (24–27) would not occur for the mutants. If this is the case, we reasonedthat ACS-associated Gai3 mutants would associate with GEFs better thanthe wild-type protein. We initially explored this idea with the nonrecep-tor GEF Ric-8A. Much like GPCR GEFs, Ric-8A binds with moderateaffinity to GDP-bound G proteins, forms a high-affinity complex with thenucleotide-free G protein, and dissociates once the G protein binds GTP(Fig. 5A) (25–27). To validate that Ric-8A can be used as a tool to probefor different Gai conformations in yeast two-hybrid experiments, we usedmutants that mimic different states of Gai3 along the activation pathway

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(Fig. 5A). As expected, binding of Ric-8A to Gai3 Q204L, a mutantconstitutively bound to GTP (19), was diminished compared to wild type,which is predominantly bound to GDP. Moreover, binding was enhancedbetween Ric-8A and Gai3 N269D (Fig. 5, B and C), a synthetic “GEF-trapping” mutant that mimics the nucleotide-free intermediate state andforms a nondissociable complex with GPCRs (28). We found that, similarto the Gai3 N269D mutant, all five ACS-associated Gai3 mutants showedincreased binding to Ric-8A compared to wild-type Gai3, suggesting thatthey assemble into more stable G protein–GEF complexes. Next, we per-formed coimmunoprecipitation experiments to directly test whether ACS-associated Gai3 mutants bind with higher affinity to ETAR, the GPCR thatgoverns the signaling pathway dysregulated in ACS (14). We found that allfive ACS-associated Gai3 mutants displayed increased binding (three- tofivefold) to myc-tagged ETAR compared to wild-type Gai3 (Fig. 5D). Col-lectively, these results indicate that ACS-associated Gai3 mutants bind toGEFs with high affinity.

ACS-associated Gai3 mutants exert a dominant-negativeaction on the ET-1/ETAR/Gaq pathwayACS is caused by disruption of the ET-1/ETAR signaling axis (14). To furtherexplore the idea thatACS-associatedGai3mutantswork as dominant-negative

Fig. 3. ACS-associatedGai3 mutants fail to bindGTP in vitro. (A) Coomassieblue–stained gel of purified proteins treated as indicated in a trypsin protec-tion assay to assess whether Gai3 S47R is activated by nucleotides. Arrowmarks full-length His-Gai3; * marks trypsin-resistant fragment of active His-Gai3. (B) Effect of S47R mutation on the steady-state GTPase activity ofGai3. Purified His-Gai3 proteins were incubated in the presence of radio-labeledGTP, and activity was determined bymeasuring the release of radio-active phosphate. (C and D) GTPgS binding by Gai3 mutant (S47R) and WTwas determined by intrinsic fluorescence measurements (F0, basal fluores-cence) (C) or radioligandbinding (D). (E) Assessment ofGDPoccupationon

Gai3 WT andS47R. Nucleotide content of equimolar amounts of His-Gai3 WT(black) or His-Gai3 S47R (gray) (top) was compared to GDP (black) or GTP(gray) standards (bottom) by HPLC (A280, nucleotide absorbance at 280 nm).(F andG) GTPgS binding as assessed by trypsin protection assays by Gai3ACS mutants G40R, G45V, S47R, T48N, and N269Y. Representative immu-noblots of Gai3 (arrow, full-length Gai3; *, trypsin-resistant fragment of activeGai3) inHEK293Tcell lysates treatedas indicated in (F) andquantified in (G).One experiment representative of three is shown in (A) to (D) and (F). Data in(G) are means ± SEM (n = 4 experiments). *P < 0.05 and **P < 0.01 com-pared to WT using Student’s t test. Endo, endogenous.

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proteins and that this underlies the molecular basis of the disease, we inves-tigated their impact on endothelin-dependent signaling. As a first step, weinvestigated the coupling efficiency of ETAR to Gq and Gi3 proteins usingthe BRET assay described above (fig. S4A). We found that both Gaq andGai3 associatewithGbgwith similar efficiency (fig. S4B), as determined byquenching of the BRET signal compared to cells expressing freeGbg alone.However,Gq, but notGi,was efficiently activated byET-1–stimulatedETAR(Fig. 6, A and B). In Gaq-expressing cells, ET-1 induced a robust and dose-dependent increase of BRET signals, whereas in Gai3-expressing cells,ET-1 induced only a modest increase at the highest concentrations tested(Fig. 6, A and B). These results indicate that, using an identical readout(free Gbg–mediated BRET), ETAR preferentially couples to Gq but showssome promiscuity toward Gi proteins. Because ACS-associated Gai3 mu-tants show increased binding toGEFs but cannot be activated,we reasonedthat they could work as dominant-negative proteins by precluding Gq cou-pling to ETAR (Fig. 6C). To investigate this, we coexpressed Gaq withsubstoichiometric amounts of wild-type Gai3 or ACS-associated mutants(8:1 ratio) and monitored Gaq activation upon stimulation with 0.3 mM

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ET-1. Under these conditions, the BRET changes reflect Gq activation be-cause Gi3 activation by 0.3 mM ET-1 was negligible (Fig. 6A). Moreover,the amount of ectopically expressed Gai3 proteins was low [the totalamount of Gai3 in transfected cells is indistinguishable from untransfectedcontrols (Fig. 6D)], and therefore, the vast majority of Gabg complexesthat serve as GPCR substrates are expected to contain Gaq and not Gai3.We found that expression of any of the five ACS-associated Gai3 mutantsdecreased Gq activation upon ET-1 stimulation by ~65 to 85% (Fig. 6D).On the other hand, expression of wild-type Gai3 did not change Gq acti-vation, which ruled out that the effect of the mutants was due to seques-tration of available Gbg to form the Gaq-Gbg complexes. We performedanalogous experiments to test the effect of substoichiometric amounts ofACS-associated Gai3 mutants on the modest activation of wild-type Gai3by ET-1/ETAR (Fig. 6D).We found that cells coexpressing wild-typeGai3and ACS-associated Gai3 mutants had similar BRET response compared tocells expressing wild-type Gai3 alone (Fig. 6D), which suggests that the inhi-bitory effect of ACS-associated Gai3 mutants is specific toward ET-1/ETAR/Gaq, but not ET-1/ETAR/Gai3, signaling. From these experiments, we con-clude that ACS-associated Gai3 mutants exert a dominant-negative effect onthe ET-1/ETAR/Gaq pathway by precluding the coupling of the GPCR to Gq.

DISCUSSION

The main finding of this work is the identification of a new mechanismbywhichmutations in trimeric G proteins cause human disease. Our dataindicate that ACS-associated Gai3 mutants are dominant-negative pro-teins that disrupt a G protein–dependent signaling pathway required forproper craniofacial development. This pathway consists of the activationof ETAR by ET-1, which in mice leads to activation of Gaq/11-dependentsignaling and subsequent transcriptional regulation of neural crest celldevelopment (14). Although ETAR displays some promiscuity in termsof G protein selectivity, previous work (29) and our work here haveshown that it preferentially activates Gaq over Gai. We propose that thedominant-negative action of ACS-associated Gai3 mutants is due to theirunproductive coupling to ETAR, which in turn precludes Gaq activation(Fig. 7). The unproductive coupling of ACS-associated Gai3 mutants toETAR is due to their inability to bind GTP (Fig. 3) and their increasedbinding to GEFs (Fig. 5), which lead to irreversible association with Gbgand favor sustained association with ETAR even after ligand stimulation.In this scenario, the availability of ETAR for Gaq activation is diminished,and endothelin-dependent signaling important for craniofacial developmentis impaired.

Previous genetic studies in humans and animal models indicate thatdisruption of an ET-1/ETAR/Ga/PLCb4 pathway that controls cranio-facial development leads to ACS (14). Two items support that Ga sub-units of the Gaq/11 family fulfill the role of mediators in this pathway.One is that PLCb4, contrary to the rest of PLCb isoforms, is activatedexclusively by Gq-related Ga subunits and not by Gbg subunits, whichcan originate from heterotrimers containing any type of Ga (30, 31). Theother one is that Gaq/Ga11 double knockout mice display craniofacialdevelopmental defects analogous to those found in ACS (16, 17). How-ever, results from mouse models should be interpreted with caution be-cause there is a precedent that the signaling mechanism that controlscraniofacial development in mice might have differences with humans.For example, knockoutmice lacking PLCb4 do not display a craniofacialphenotype (32), whereas there is at least one reported case of autosomalrecessive ACS caused by mutations in PLCb4 (33). Moreover, no muta-tions in Gaq and/or Ga11 have been found in ACS patients. Conversely,the presence ofmutations in Gai3 indicates that this G protein plays a rolein the signaling pathway disrupted in ACS, but the mechanism involved has

Fig. 4. ACS-associated Gai3 mutants are not activated by the GPCR A1R.(A) Schematic diagramdepicting the BRET assay used tomonitor the disso-ciation ofGai3:Gbg trimers uponGPCRstimulation. Under resting conditions,Venus-tagged Gbg (V-Gbg) associates with Gai3 and BRET signals are low.Upon stimulation of A1Rwith adenosine, Gi trimers dissociate, and free V-Gbgbinds to mas-GRK3ct-Nluc (GRK), leading to an increase of BRET signal.(B) Assessment of A1R-induced dissociation of Gi trimers containing Gai3WTorACSmutant subunits. HEK293Tcells were transfectedwith plasmidsencoding for Venus(155–239)-Gb1 (VC-Gb1), Venus(1–155)-Gg2 (VN-Gg2),mas-GRK3ct-Nluc, and A1R along with Gai3 WT or ACS mutants (G40R,G45V, S47R, T48N, or N269Y), and BRET was measured every second (asdescribed in Materials and Methods). Equal amounts of V-Gbg and thedifferent Gai3 proteins were verified by immunoblotting (fig. S2). After 30 sunder resting conditions, cells were stimulated with adenosine (1 mM). Theaverage of BRET signal during the first 30 s (basal BRET) was subtractedfrom each data point to present the data as increase of BRET (DBRET).One representative experiment of four is shown.

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remained unclear until now. Our resultssupport that ACS-associated Gai3 mutantsdisrupt signaling “horizontally” by blockingGPCR-mediated activation of another G pro-tein, Gaq. Although it is possible that ETAR-Gai3 signaling participates in craniofacialdevelopment, our results indicate that ACS-associated Gai3 mutants do not exert theirdominant-negative effect under conditionsin which ETAR-Gaq signaling is efficientlyinhibited (Fig. 6D).

The lack of dominant-negative inhibitionon Gai3 is somewhat puzzling because theunproductive coupling of ACS-associatedGai3 mutantswith ETARwould be expectedto interferewith the binding of anyGproteinto this GPCR. However, the dominance ofACS-associated Gai3 mutants could be dif-ferent depending on the nature of the Gq-ETARandGi-ETAR interactions. It is possiblethat the dominant effect is dampened forGai3 if Gi proteins have higher affinity forETAR than Gq and/or if Gi proteins, butnot Gq, “precouple” to ETAR before agoniststimulation. Although further investiga-tion is required to clarify the specificity ofthe dominant-negative function of ACS-associated Gai3 mutants, our results suggestthat the primary mechanism by which theycause ACS is “horizontal” interference withGaq-mediated signaling.

Another question that remains open iswhy ACS patients bearing mutations inGai3 do not display more pleiotropic phe-notypes than the observed effect restrictedto neural crest cell differentiation during de-velopment. It is possible that the dominant-negative function of ACS-associated Gai3mutants is dampened in other situations dueto differences in overallGai3 expression. Be-cause ACS-associated Gai3 mutant proteinsare intrinsically unstable, the dominant-negative function would become apparentonly when sufficient amounts accumulate,for example, by increased gene transcriptionand/or posttranslational stabilization. An-other possibility is that the dominant functionisdampened in thepresenceofhigher amountsof other Gai subunits and/or when multipleGPCRs are present and activated simulta-neously. Similarly, if the ACS phenotypeis specifically associatedwith the disruptionof Gaq-dependent signaling, the relativeamounts of Gaq and Gai3 are bound to becritical for thedevelopment of ACS. In sum-mary, obtaining additional information onthe expression patterns of Gai3, Gaq, andother signaling components in developinghumans will be important to interpret themechanistic results presented here.

Fig. 5. Gai3 ACS mutations increase binding to GEFs. (A) Schematic of Ric-8A binding to different

conformations of Gai3 during its activation cycle and description of constructs that mimic each one ofthese conformations. (B) Ric-8A binding by Gai3 WT or ACS mutant using yeast two-hybrid assays (sche-matic, top; AD, Gal4 activation domain; BD, Gal4 DNA binding domain). Data are means ± SEM (n = 3experiments: *P < 0.05 and **P < 0.01, Student’s t test). (C) Immunoblot of the strains used in (B). (D)Relative affinity of ACS-associated Gai3 mutants for the ETAR GPCR as determined by immunoprecipitation(IP) with myc or immunoglobulin G (IgG) antibody in lysates of HEK293T cells transfected with myc-ETAR and the indicated Gai3 constructs. Blots are representative of three experiments.

Fig. 6. ACS-associated Gai3 mutants prevent ETAR-mediated activation of Gaq. (A and B) BRET assay in

HEK293T cells transfected with Gaq (left) or Gai3 (right) along with V-Gbg, mas-GRK3-Nuc, and ETAR,stimulated (arrow) with the indicated concentrations of ET-1, analyzed as in Fig. 4B. One representativeexperiment is shown in (A). Data in (B) are means ± SEM (n = 3 experiments). (C) Putative mechanism ofdominant-negative action of ACS-associated Gai3 mutants on Gaq activation by ETAR as determined byBRET assays. In cells expressing Gai3 WT (top), ETAR couples predominantly to Gq. In cells express-ing Gai3 mutants (bottom), high-affinity binding of mutants to ETAR precludes Gq activation. (D) HEK293Tcells transfected with Gaq (left) or Gai3 WT (right) (1 mg) and substoichiometric amounts of Gai3 WT or anACS mutant (0.12 mg) along with V-Gbg, mas-GRK3-Nuc, and ETAR were stimulated (arrow) with ET-1 (0.3 mM Gaq/3 mM Gai3). One representative of three experiments, with respective immunoblots (below),is shown. HA, hemagglutinin.

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In the past, the characterization of synthetic Ga mutants with dominant-negative function has been instrumental in understanding the molecular basisof G protein regulation (28, 34–37). The present work is the first descriptionof naturally occurring Ga mutants with dominant-negative function, whichdemonstrates that this type of mutants is important not only as a research toolbut also as an underlying cause of human disease. ACS-associated Gai3mutants have similarities and differences with some of the previouslydescribed synthetic dominant-negatives. For example, both an N→Dmutant in Ga G-4 box originally identified in yeast and Gat S43N (lo-cated in the G-1 box/P-loop) bind to Gbg and have increased affinity forGEFs (28, 36, 38), much like Gai3 in ACS. These mutants adopt a con-formation that mimics an intermediate in the G protein activation cyclethat traps GPCRs in an unproductive complex (28, 36). The positionsmutated in Ga N→D and Gat S43N correspond to N269 and S47 inGai3, two of the positions mutated in ACS. However, they are mutatedto different residues, meaning N269Yand S47R, which may account forsome of their different properties. The main difference is that Ga N→DandGat S43N are capable of binding GTP spontaneously (28, 36), whereasGai3 N269Yand S47R (as well as the rest of ACS mutants) do not. Thisunique feature of ACS-associated Gai3 mutants is bound to contribute totheir dominant-negative effect by precluding Gai3 from adopting an activeconformation and blocking the progression of the G protein cycle.

Another feature shared with other dominant-negative proteins like GaN→D is decreased stability (28, 38), which is most readily explained bytheir mimicry of an unstable nucleotide-free intermediary in the activationcycle (25, 39). All or four of five ACS-associated Gai3 mutants expressedpoorly in mammalian cells and bacteria, respectively. On the other hand, theamount of ACS-associated Gai3 mutant proteins ectopically expressed inXenopus or yeast was similar to wild-type Gai3. This discrepancy is bestexplained by technical differences among experimental systems. G proteinswere expressed at lower temperatures in Xenopus and yeast (16° and 30°C,

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respectively) compared to mammalian cells (37°C). In addition, the mutantswere coexpressed with Ric-8A in yeast, which is a folding chaperone forGai (25). Regardless, these results suggest that ACS-associated Gai3 mutantsare potent dominant-negative proteins because they can efficiently disruptsignaling evenwhen present at quantities lower than endogenousGai3 inmam-malian cells.

As for other diseases caused by inherited G protein mutations like dys-tonias, Nougaret night blindness, or Albright’s hereditary osteodystrophy,the genetic pattern of type I ACS inheritance is dominant. However, ourresults indicate that themolecular basis for type IACSdominant inheritanceis different from other diseases caused by G protein mutants. In type I ACS,a single mutant allele is sufficient to cause the disease because of thedominant-negative action of Gai3 variants, whereas in other diseases, asingle G protein mutant allele causes haploinsufficiency. For example,primary torsion dystonia, craniocervical dystonia, or Albright’s heredi-tary osteodystrophy can be caused by nonsense or missense mutationsthat lead to absence of protein or severe protein structural defects (5, 9, 10).Nougaret night blindness is caused by a missense mutation in Gat, but ithas been unequivocally established that the disease arises from a loss offunction not accompanied by any dominant-negative function (7, 8). Con-versely, our results rule out haploinsufficiency as the cause of type I ACSbecause Gai3 mutants provoke an increase rather than a decrease of the pen-etrance of Gai3-dependent phenotypes in embryo development assays(Fig. 2). On the basis of this finding, we conclude that type I ACS is the firsthuman disease caused by G protein mutants that arises from a dominant-negative function.

MATERIALS AND METHODS

Reagents and antibodiesUnless otherwise indicated, all reagents were of analytical grade and ob-tained from Sigma or Fisher Scientific. The cell culture medium and theE. coli strain BL21(DE3) were purchased from Invitrogen. All restrictionendonucleases were from Thermo Scientific, and E. coli strain DH5awas purchased from New England Biolabs. PfuUltra DNA Polymerasewas purchased from Agilent. DPCPX, ET-1, and adenosine were fromSigma. [g-32P]GTP and [35S]GTPgS were from PerkinElmer Life Sciences.Goat anti-rabbit and goat anti-mouse Alexa Fluor 680 or IRDye 800 F(ab′)2were from LI-COR Biosciences. Rabbit antibodies raised against Gai3(C10) and Gb (M-14) and mouse monoclonal antibodies against green flu-orescent protein (GFP) (B-2) were from Santa Cruz Biotechnology. Rabbitpolyclonal antibodies for myc tag were from Sigma (C3956). Mousemonoclonal antibodies raised against tubulin (12G10) and myc tag(9E10) were from the Developmental Studies Hybridoma Bank (Universi-ty of Iowa), and the mouse monoclonal antibody for HA tag (12CA5) wasfrom Roche.

BioinformaticsThe protein sequences of all 16 Ga subunits in Homo sapiens wereretrieved from UniProt, aligned using ClustalW, and shaded with Box-Shade 3.21. Protein structure images were generated with PyMOL Mo-lecular Graphics System (Schrödinger, LLC) using the PDB: 1GIA.

Plasmids and in vitro mRNA synthesisCloning of rat Gai3 into pcDNA3 and pET28b has been described pre-viously (22, 24). pcDNA3-Gaq-HA [internally tagged; (40)] was fromP. Wedegaertner (Thomas Jefferson University). Rat Gai3 was clonedinto the Eco RI/Sal I sites of the pGBKT7 vector to generate the Gal4AD-Gai3 fusion protein used in yeast two-hybrid experiments. A fragment of

Fig. 7. Proposed model. Left: In healthy individuals, ETAR couples predomi-

nantly to Gaq/11, which promotes PLCb4 activation and the expression ofDLX5/6 transcription factors required for neural crest cell differentiationand normal craniofacial development. Right: In type I ACS patients,ACS-associated Gai3 mutants bind with high affinity to ETAR and form anunproductive complex. This reduces the availability of ETAR for Gaq acti-vation, which blocks the signaling pathway required for normal craniofacialdevelopment.

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Ric-8A corresponding to amino acids 12 to 491 was amplified from aplasmid (25) provided by S. Sprang (University of Montana) andcloned into the Nde I/Eco RI sites of the pGADT7 vector to generatethe Gal4BD–Ric-8A fusion protein used in yeast two-hybrid experiments.A rat Gai3 construct internally tagged with yellow fluorescent protein(YFP) was generated by introducing the fluorescent protein in the b/c loopof the all-helical domain, which is a strategy previously described for Gai1that preserves the native properties of the G protein (41). Briefly, YFP wasinserted between S113 and A114 (b/c loop) of Gai3 by introducing si-lent mutations in pcDNA3.1(−)-Gai3 to create an artificial Afe I site inthis location, followed by digestion and ligation of the sequence encodingfor the fluorescent protein. pcDNA3.1-A1R, pcDNA3.1-VN-Gg2,pcDNA3.1-VC-Gb1, pcDNA3.1-Gg2, pcDNA3.1-Gb1, and myc-ETAR-Rluc8 were provided by N. Lambert (Georgia Regents University)(20). pcDNA3.1-masGRK3ct-Nluc (21) and pcMin ETAR (42) weregifts from K. Martemyanov (Scripps Research Institute) and P. Polgar(Boston University), respectively.

Rat Gai3 was cloned into the Eco RI and Xho I sites of pCS2(+) togenerate the DNA template for in vitro mRNA transcription. Briefly,pCS2(+)-Gai3 was linearized by digestion with Not I, and DNA puri-fied by alkaline phenol/chloroform extraction followed by ethanol pre-cipitation. Purified DNA (1 mg) was used as template for each mRNAin vitro transcription reaction with the SP6 mMessage mMachine Kit(Ambion). In vitro mRNA transcription reactions were treated by de-oxyribonuclease I to eliminate the template and mRNA purified by al-kaline phenol/choloroform extraction, followed sequential precipitationsin isopropanol and ethanol. Purified mRNAs were quantified spectro-scopically and their quality checked in a 1% agarose/formaldehydegel. mRNAs were diluted to the desired final concentrations and storedat −80°C.

Gai3 mutants were generated using specific primers (sequences availa-ble upon request) following the manufacturer’s instructions (QuickChangeII, Agilent). All constructs were checked by DNA sequencing.

X. laevis embryo manipulationsFrog studies were carried out with wild-type animals (Nasco) according tothe Boston University Institutional Animal Care and Use Committee–approved protocol, in compliance with the Guide for the Care and Use ofLaboratory Animals. Egg laying was induced by dorsal lymph injection ofhuman chorionic gonadotropin (500U) (Intervet). Invitro fertilization andembryo culturewere carried out in 0.1×Marc’smodified Ringer’smediumas described (43). Stagingwas according toNieuwkoop and Faber. InvitrotranscribedmRNAs (30 or 60 pg) were injected equatorially in both dorsalblastomeres of four- or eight-cell stage embryos, whichwere subsequentlyincubated at 16°C. Embryos were fixed at late neurulation (stages 18 to19) in MEMFA [100 mM Mops (pH 7.4), 2 mM EGTA, 1 mM MgSO4,and 3.7% (v/v) formaldehyde], photographed, and subjected to phenotypicanalysis. Embryos were observed using a Leica MZ6 dissection microscopeand scored as “gravitational defect”when the neural tubewas facing down-ward (18). Sagittal bisections of MEMFA fixed embryos at stage 15 wereperformed with a razor blade and photographed in 1× phosphate-bufferedsaline (PBS).All pictureswere takenwith aCanonXSi camera connected tothe microscope.

For the analysis of Gai3 by immunoblotting, two embryoswere resus-pended in60ml of lysis buffer [20mMHepes (pH7.2), 5mMMg(CH3COO)2,125mMK(CH3COO), 0.4%TritonX-100, and 1mMdithiothreitol (DTT),supplementedwith a protease inhibitor cocktail (Sigma, catalog no. S8830)]and homogenized by pipetting.After centrifugation for 10min at 14,000g at4°C, supernatants were supplemented with Laemmli sample buffer andboiled for 5 min.

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Cell culture, transfections, and BRET assayHEK293T cells were grown at 37°C in Dulbecco’s modified Eagle’s me-dium supplemented with 10% fetal bovine serum, penicillin (100 U/ml),streptomycin (100 mg/ml), 1% L-glutamine, and 5% CO2. All DNA plas-mids were transfected using the calcium phosphate method in six-wellplates, and BRET measurements were done 16 to 24 hours after trans-fection. Equal amounts (0.2 mg) of masGRK3ct-Nluc, VC-Gb1, VN-Gg2,and GPCR (either A1R or ETAR) were transfected for BRETexperiments.Gai3 and Gaq were typically transfected at 1 mg of DNA per well. Thisamount of DNAwas adjusted (1 to 4 mg) in some experiments to producesimilar amounts of protein among different G proteins mutants and wildtype. For the experiments investigating the dominant-negative effect ofGai3on Gaq activation, the plasmids were transfected at 1:8 ratio (0.12 mg:1 mg,Gai3:Gaq). BRETexperiments were carried out and analyzed as describedpreviously (20, 21) with minor modifications. Briefly, 16 to 24 hours aftertransfection, the cells were gently scrapped in PBS, centrifuged, and re-suspended in Tyrode’s solution [140 mMNaCl, 5 mM KCl, 1 mMMgCl2,1mMCaCl2, 0.37mMNaH2PO4, 24mMNaHCO3, 10mMHepes (pH 7.4),and 0.1% glucose] at a density of 106 cell/ml. Cell suspensions (25 ml)were added to a white opaque 96-well plate (Opti-Plate, Perkin Elmer) andmixedwith an equal volume of theNanoLuc substrateNano-Glo (Promega,diluted 1:50). After 2 min of incubation, luminescence was measured atroom temperature in a Synergy H1 plate reader (BioTek) at 460 ± 20 nmand 528 ± 10 nm. BRET signals were calculated as the ratio of the emis-sion intensity at 528 ± 10 nm divided by the emission intensity at 460 ±20 nm. For the kinetic experiments, BRET measurements were done everysecond. Basal BRETwas measured for 30 s, after which adenosine or ET-1was added to thewell. With the exception of fig. S2D, kinetic BRET dataare presented as the increase in BRET (DBRET) for clarity. DBRETwascalculated by subtracting the average of BRET signal before agoniststimulation (t = 0 to 30 s) to every point of the time trace. An aliquot ofthe cell suspensionswas processed for immunoblot analysis. Briefly, cellswere pelleted, resuspended in lysis buffer [20 mM Hepes (pH 7.2), 5 mMMg(CH3COO)2, 125 mM K(CH3COO), 0.4% Triton X-100, 1 mM DTT,supplemented with a protease inhibitor cocktail (Sigma, catalog no. S8830)],and cleared by centrifugation at 14,000g at 4°C for 10 min. After centrifu-gation, supernatants were supplemented with Laemmli sample buffer andboiled for 5 min.

In vitro G protein biochemistry assaysSteady-stateGTPase assayswere performed using radiolabeled [g-32P]GTPand measuring the release of [g-32P]Pi at 30°C. GTPgS binding was deter-mined by measuring intrinsic tryptophan fluorescence (excitation, 284 nm;emission, 340 nm) in a Hitachi F-4500 fluorescence spectrophotometeror by directly measuring the binding of radiolabeled [g-35S]GTPgS at30°C. Limited proteolysis assays were carried out by incubating purifiedG proteins or lysates of HEK293T cells expressing different G proteinswith GDP, GTPgS, or GDP plus AlF4

− at 30°C before adding trypsin.HPLC analyses of G protein nucleotide content were performed afterexchanging the purifiedGproteins into nucleotide-free buffer and concentratingthem to50mM.Nucleotide standards at50mMwereprepared in the samebuffer.

ImmunoblottingProteins were separated by SDS–polyacrylamide gel electrophoresis(SDS-PAGE) and transferred onto polyvinylidene difluoride membranes,which were sequentially incubated with primary and secondary antibodies(goat anti-rabbit Alexa Fluor 680 and goat anti-mouse or IRDye 800,1:10,000). The primary antibodies were used at the following dilutions:Gai3, 1:250; pan-Gb, 1:250; a-tubulin, 1:2500; HA, 1:1000; and rabbitmyc, 1:1000. Infrared imaging of immunoblots was performed according

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to the manufacturer’s protocols using an Odyssey Infrared Imaging System(LI-COR Biosciences). Images were processed using ImageJ software[National Institutes of Health (NIH)] and assembled for presentation usingPhotoshop and Illustrator software (Adobe).

Protein purificationHis-tagged Gai3 proteins were expressed in E. coli strain BL21(DE3)(Invitrogen) and purified as described previously (44). Briefly, bacterialcultures were induced with 1 mM isopropyl-b-D-1-thiogalactopyranosideovernight at 23°C. Pelleted bacteria from 1 liter of culturewere resuspendedin25mlofHis lysis buffer [50mMNaH2PO4 (pH7.4), 300mMNaCl, 10mMimidazole, 1% (v/v) Triton X-100, 25 mM GDP, 1 mM leupeptin, 2.5 mMpepstatin, 0.2 mM aprotinin, and 1 mM phenylmethylsulfonyl fluoride].After sonication (four cycles, with pulses lasting 30 s per cycle, and with1-min interval between cycles to prevent heating), lysateswere centrifugedat 12,000 g for 20 min at 4°C. Solubilized proteins were affinity-purifiedon HisPur Cobalt Resin (Pierce), eluted with imidazole, dialyzed againstPBS, andbuffer-exchanged/concentrated in20mM tris-HCl (pH7.4), 20mMNaCl, 1mMMgCl2, 1mMDTT, 10 mMGDP, and 5% (v/v) glycerol beforestorage at −80°C.

Limited proteolysis assay with purified proteinsThis assay was carried out as described previously (44, 45) with minormodifications. Briefly, His-Gai3 (0.4mg/ml) was incubated for 90min at30 °C in buffer [20 mMNa-Hepes (pH 8), 100 mMNaCl, 1 mM EDTA,10mMMgCl2, 1mMDTT, and 0.05% (w/v) C12E10] supplementedwithGDP (30 mM), GTPgS (30 mM), or GDP-AlF4

− (30 mM GDP, 30 mMAlCl3, and 10 mM NaF). Then, trypsin was added to the tubes (final con-centration, 80 mg/ml), and samples were incubated for 10 min at 30 °C.Samples were rapidly transferred to ice, reactions were stopped bythe addition of Laemmli sample buffer, and samples were incubated at65 °C for 5 min. Proteins were separated by SDS-PAGE and stained withCoomassie blue.

Limited proteolysis assay with HEK293T cell lysatesThis assay was carried out as described previously (45) with minor mod-ifications. HEK293T cells were transfected with plasmids encoding forwild-type Gai3 or ACS mutants as described in the “Cell culture, trans-fections, and BRETassay” section. The amount of each plasmid requiredto achieve equal protein expression for wild-type and mutant Gai3 was de-termined empirically in preliminary experiments. A quarter of the HEK293Tcells from a well of a six-well plate was lysed in 48 ml of buffer [20 mMHepes (pH 7.2), 5 mMMg(CH3COO)2, 125mMK(CH3COO), 0.4% TritonX-100, and 1 mM DTT] supplemented with 125 mM GDP or 125 mMGTPgS. Samples were vortexed, incubated in ice for 10 min, and centri-fuged at 14,000g for 10min at 4°C. The supernatants (40 ml) were incubatedfor 2 hours and 30 min at 30°C to allow the loading of nucleotide on theG protein. Trypsin (12.5 mg/ml) was added to the tubes and incubated at30°C for 20min. Reactionswere stopped by the addition of 12 ml of Laemmlisample buffer and boiled for 5 min. The “% protection by GTPgS” wascalculated by quantifying the bands corresponding to “+trypsin/+GTPgS”condition divided by the band of “−trypsin” condition and multiplying theresult by 100. To determine the specific protection of each ectopically ex-pressed Gai3 protein, the quantification values of the corresponding bands(that is, GTPgS-protected or total) of endogenous Gai3 (that is, transfectedwith an empty vector)were subtracted before the calculation described above.

Steady-state GTPase assayThis assay was performed as described previously (44, 45). Briefly, re-actions were initiated at 30°C by mixing assay buffer [20 mMNa-Hepes

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(pH 8), 100mMNaCl, 1mMEDTA, 2mMMgCl2, 1mMDTT, and 0.05%(w/v) C12E10] containing [g-

32P]GTP (1 mM, ~100 cpm/fmol) with an equalvolume of His-Gai3 (100 nM) in the same buffer. Duplicate aliquots (50 ml)were removed at 0, 2, 4, 6, 10, and 15 min, and reactions stopped with950 ml of ice-cold 5% (w/v) activated charcoal in 20 mMH3PO4 (pH 3).Samples were centrifuged for 10min at 10,000g, and 500 ml of the resultantsupernatant was scintillation-counted to quantify the amount of [32P]Pireleased. Data are presented as raw radioactivity counts.

Measurement of GTPgS binding by intrinsictryptophan fluorescenceThis assay was performed as described previously (45). Purified His-Gai3(1 mM) was equilibrated at 30°C in a cuvette with 1 ml of buffer [20 mMNa-Hepes (pH 8), 100 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 1 mMDTT, and 0.05% (w/v) C12E10]. GTPgS (1.25 mM)was added to the cuvetteafter ~3 min, and the G protein activation rate was monitored by measuringthe change in intrinsic fluorescence (excitation at 284 nm and emission at340 nm) due to the structural rearrangement of the switch II tryptophanresidue W211. Data were collected using a Hitachi F-4500 fluorescencespectrophotometer, background-corrected (buffer fluorescence), andpresented as a ratio of the basal fluorescence (F/F0).

Measurement of radiolabeled GTPgS bindingThis assay was performed as described previously (44–46). Briefly, reac-tions were initiated at 30°C by mixing assay buffer [20 mM Na-Hepes(pH 8), 100 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 1 mM DTT, and0.05% (w/v) C12E10] containing [

35S]GTPgS (1 mM, ~50 cpm/fmol) withan equal volume of His-Gai3 (100 nM) in the same buffer. Duplicate aliquots(25 ml) were removed at the indicated time points, and binding of radio-active nucleotidewas stopped by the addition of 2 ml of ice-cold wash buffer[20mm tris-HCl (pH8.0), 100mmNaCl, and 25mmMgCl2]. The quenchedreactionswere rapidly passed throughBA85nitrocellulose filters (Whatman).Filterswere dried and subjected to scintillation counting.Data are presented asraw radioactivity counts.

Nucleotide HPLC analysisHPLC analyses of G protein nucleotide content were performed as previ-ously described (47). His-Gai3 wild type and His-Gai3 S47Rmutant wereexchanged into nucleotide-free buffer [20mM tris-HCl (pH 7.4), 200mMNaCl, 1 mMMgCl2, 1 mMDTT, and 5% (v/v) glycerol] and concentratedto 50mM.Nucleotide standards at 50mMwere prepared in the same buffer.Acetonitrile was added to each protein sample or standard tube to obtain afinal concentration of 7.5% (v/v); the mixture was boiled for 5 min andthen centrifuged for 10 min at 20,000g to precipitate the denatured pro-teins. The supernatant was collected and used for the HPLC analysis. AZorbax C-18 reversed-phase column (4.6 × 150 mm) filled with 3.5-mmsilica (Agilent) was equilibrated with 100 mMKH2PO4 (pH 6.5), 10 mMtetrabutylammonium bromide (phase-transfer catalyst), 0.2% (w/v) NaN3,and 7.5% (v/v) acetonitrile using anAgilent 1260 InfinityQuaternaryPumpflowing at 1 ml/min. Freshly prepared sample (15 ml) was loaded into 20 mlof HPLC loading loop. The samples were then injected onto the C-18 col-umn and isocratically eluted at 1 ml/min. Absorbance of 280-nm wave-length light was performed by an Agilent 1260 infinity ultraviolet detector.Control standards of GDP and GTP were eluted at distinct retention timesof 2.25 and 2.56 min, respectively.

CoimmunoprecipitationThis assay was performed as previously described (46). HEK293T cellswere transfected in 10-cm dishes with plasmids encoding for untaggedGb1 (1.2 mg), untagged Gg2 (1.2 mg), myc-ETAR (6 mg), and wild-type

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Gai3-YFP (internal b/c loop) or ACS mutants as described in the “Cellculture, transfections, and BRETassay” section. The amount of plasmid re-quired to achieve equal protein expression for wild-type and mutant Gai3was determinedempirically inpreliminaryexperiments.HEK293Tcells froma 10-cm platewere lysed in 1ml of ice-cold buffer [20mMHepes (pH 7.2),5 mMMg(CH3COO)2, 125 mM K(CH3COO), 0.4% Triton X-100, and1 mM DTT], vortexed, passed through a 30-gauge insulin syringe fivetimes, and incubated in ice for 10 min. After centrifugation at 14,000g for10min at 4°C, the supernatant was split into two tubes for each condition.Anti-myc (9E10) or control mouse IgG ((2.5 µg; Santa Cruz Biotechnol-ogy) was added to each tube and incubated for 4 hours at 4°C with con-stant rotation. Protein G agarose beads (Thermo Scientific) were blockedwith 5% bovine serum albumin (BSA) for 2 hours at room temperature,washed, added to each of the tubes containing lysates and antibodies,and incubated for 90 min at 4 °C with rotation. Beads were then washedthree times with wash buffer [4.3 mM Na2HPO4, 1.4 mM KH2PO4

(pH 7.4), 137 mM NaCl, 2.7 mM KCl, 0.1% (v/v) Tween 20, 10 mMMgCl2, 5 mm EDTA, and 1 mM DTT], and proteins were eluted by in-cubation in Laemmli sample buffer for 10 min at 37°C.

Immunofluorescence microscopyThis assay was performed as previously described (48). HEK293T cellswere transfected with plasmids encoding for untagged Gb1, untagged Gg2,and Gai3-YFP (b/c loop internal) wild type or ACS mutants as describedin the “Cell culture, transfections, andBRETassay” section except that theywere seeded on poly-L-lyisine–coated glass coverslips. The amount of plas-mid required to achieve equal protein expression for wild-type andmutantGai3 was determined empirically in preliminary experiments. One day aftertransfection, cells were fixed with 3% paraformaldehyde for 30 min, per-meabilized, blocked in PBS containing 10% normal goat serum and 0.1%Triton X-100 for 30 min, and then sequentially incubated with primaryand secondary antibodies for 1 hour at room temperature. Antibody dilu-tions were as follows: primary mouse anti-GFP (B-2, Santa Cruz Biotech-nology), 1:200; secondary goat anti-mouse Alexa 488 (Life Technologies),1:300. Images were acquired with a Zeiss Axiovert 200 LSM510 laserscanning confocal microscope using a 63× oil immersion objective. Allindividual images were assembled for presentation using Photoshop andIllustrator software (both Adobe).

Yeast two-hybrid assayThis assay was performed usingMatchmaker Gold (Promega) accordingto the manufacturer’s instructions. Briefly, pGADT7–Ric-8Awas trans-formed into the Saccharomyces cerevisiae haploid strain Y187, andpGBKT7-Gai3 (wild type or mutants) into the haploid strain AH109, usingthe lithium acetate method (49). Transformants were selected in syntheticdefined (SD)medium plates lacking leucine (SD-Leu) and tryptophan (SD-Trp), respectively, and mated by co-inoculation of single colonies in YPD(yeast extract, peptone, and dextrose) medium and overnight incubation at30°C. Mated diploid strains were selected by inoculation of 20 ml of theovernight cultures on SD-Leu-Trp medium. Individual colonies were in-oculated into 3 ml of SD-Leu-Trp and incubated overnight at 30°C. Thisstarting culture was used to inoculate 20 ml of SD-Leu-Trp at an opticaldensity at 600 nm (OD600) of 0.3. Exponentially growing cells (OD600,~0.7 to 0.8; 4 to 5 hours) were pelleted to prepare samples for subsequentassays (see next sections).

b-Galactosidase activity assayThis assay was performed as described previously (49) with minor mod-ifications. Pellets corresponding to an OD600 of 0.5 were washed oncewithPBS + 0.1% (w/v) BSA and resuspended in 200 ml of assay buffer [60 mM

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Na2PO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgCl2, 0.25% (v/v)b-mercaptoethanol, 0.01% (w/v) SDS, and 10% (v/v) chloroform] andvortexed. One hundred microliters was transferred to 96-well plates,and reactions were started by the addition of 50 ml of the fluorogenicsubstrate fluorescein di-b-D-galactopyranoside (final concentration,100 mM). Fluorescence (excitation, 485 ± 10 nm; emission, 528 ± 10 nm)was measured every 2 min for 90 min at 30°C in a Synergy H1 platereader (Biotek). Enzymatic activity was calculated from the slope of fluo-rescence (arbitrary units) versus time (minutes). At least three independentclones determined in duplicate were measured for each condition, andresults were normalized (%) to the activity in cells expressing Ric-8A andwild-type Gai3.

Preparation of yeast samples for immunoblottingThis procedure was performed as described previously (49, 50) with minormodifications. Briefly, pellets corresponding to an OD600 of 5 were washedoncewith PBS+0.1%BSAand resuspended in 150ml of lysis buffer [10mMtris-HCl (pH8.0), 10%(w/v) trichloroacetic acid, 25mMNH4OAc, and1mMEDTA].Glass beads (100 ml) were added to each tube, and the sampleswerevortexed in a cold room for 5min. Lysateswere transferred by poking a holein the bottom of the tubes followed by centrifugation onto a new set oftubes. The process was repeated after the addition of 50 ml of lysis bufferto wash the glass beads. Proteins were precipitated by centrifugation(20,000g for 10 min) and resuspended in 60 ml of solubilization buffer[0.1 M tris-HCl (pH 11.0) and 3% SDS]. Samples were boiled for 5 min andcentrifuged (20,000g for 1min), and 50 ml of the supernatant was transferred tonew tubes containing 12.5 ml of Laemmli sample buffer and boiled for 5 min.

Statistical analysesEach experiment was performed at least three times. Data are presentedas means ± SEM or as representative results of biological replicates. Theerror bars of the phenotype distributions shown in Fig. 2C were calculated

as follows: SE=ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiPð100 � PÞ

n

q, whereP is the proportion (%) of the phenotype

and n is the total number of embryos analyzed. Statistical significancebetween various conditions was assessed with Student’s t test or Fisher’s exacttest (for the Xenopus embryo assays). P < 0.05 was considered significant.

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/9/423/ra37/DC1Fig. S1. ACS mutations in Gai3 affect residues conserved across all Ga proteins inhumans.Fig. S2. ACS-associated Gai3 mutants bind to Gbg.Fig. S3. The subcellular localization of ACS-associated Gai3 mutants is similar to that ofwild-type Gai3.Fig. S4. Gaq and Gai3 associate similarly with Gbg subunits.

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Acknowledgments: We are indebted to N. Lambert (Georgia Regents University) forproviding critical reagents and extensive discussions for the BRET assay. We thankS. Sprang (University of Montana), K. Martemyanov (Scripps Research Institute), andP. Polgar (Boston University) for providing plasmids; E. Simmons (Boston University)for giving access to the spectrofluorimeter; and V. Trinkaus-Randall (Boston Universi-ty) for providing access to the confocal microscope. We also thank A. J. McDonald andD. A. Harris (Boston University) for providing access to equipment and technical helpfor the HPLC experiments, and K. Steiling (Boston University) for assessment of the sta-tistical analyses. Funding: This work was supported by NIH grants R01GM108733 andR01GM112631 and American Cancer Society grant RSG-13-362-01-TBE to M.G.-M.; NIHgrant R01GM098367 and American Heart Association grant 10GRNT3010038 to I.D.; anda postdoctoral fellowship from the Hartwell Foundation to V.D.G. Author contributions:A.M., A.L., and M.G.-M. designed the study; A.M., A.L., K.P.-S., V.D.G., A.Y.C., L.T.N., and

w

M.G.-M. performed the experiments; I.D. contributed new reagents and analytic tools;A.M., A.L., K.P.-S., I.D., and M.G.-M. analyzed the data; and A.M. and M.G.-M. wrote thepaper with input from all the authors. Competing interests: The authors declare that theyhave no competing interests.

Submitted 13 August 2015Accepted 24 March 2016Final Publication 12 April 201610.1126/scisignal.aad2429Citation: A.Marivin, A. Leyme, K. Parag-Sharma, V. DiGiacomo, A. Y. Cheung, L. T. Nguyen,I. Dominguez, M. Garcia-Marcos, Dominant-negative Ga subunits are a mechanism ofdysregulated heterotrimeric G protein signaling in human disease. Sci. Signal. 9, ra37 (2016).

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signaling in human disease subunits are a mechanism of dysregulated heterotrimeric G proteinαDominant-negative G

Dominguez and Mikel Garcia-MarcosArthur Marivin, Anthony Leyme, Kshitij Parag-Sharma, Vincent DiGiacomo, Anthony Y. Cheung, Lien T. Nguyen, Isabel

DOI: 10.1126/scisignal.aad2429 (423), ra37.9Sci. Signal. 

G protein can impair another and cause disease.preventing intracellular propagation of the endothelin signal. The findings show that dominant-negative mutations in one

mutants lacked enzymatic activity, therebyi3αR, GA. Although able to bind ETq/11αthe binding of another G protein, GR and blockA to bind inappropriately to the endothelin receptor ETi3α mutations associated with ACS enable Gi3αthe G

embryos and biochemical analysis in mammalian cells revealed thatXenopus. Developmental analysis of transfected i3αsyndrome (ACS), who have defects in craniofacial development, have mutations in the heterotrimeric G protein subunit Gphysiology, and mutations in GPCR signaling pathway components cause disease. Some patients with auriculo-condylar

coupled receptors (GPCRs) regulates various aspects of development and adult−Signaling by G proteinqα gets in the way of GiαG

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