The α-subunit of the trimeric GTPase Go2 regulates axonal growth

,1 ,1

*Center for Anatomy, Institute for Integrative Neuroanatomy, Functional Cell Biology, Charit�e-

Universit€atsmedizin Berlin, Germany

†Institute of Toxicology, Hannover Medical School (MHH), Germany

‡Laboratory of Neurobiology, Division of Intramural Research, National Institute of Environmental

Health Sciences, Research Triangle Park, North Carolina, USA

AbstractThe Goa splice variants Go1a and Go2a are subunits of themost abundant G-proteins in brain, Go1 and Go2. Only a fewinteracting partners binding to Go1a have been described sofar and splice variant-specific differences are not known. Usinga yeast two-hybrid screen with constitutively active Go2a asbait, we identified Rap1GTPase activating protein (Rap1GAP)and Girdin as interacting partners of Go2a, which wasconfirmed by co-immunoprecipitation. Comparison of subcel-lular fractions from brains of wild type and Go2a�/� micerevealed no differences in the overall expression level ofGirdin or Rap1GAP. However, we found higher amounts ofactive Rap1-GTP in brains of Go2a deficient mutants, indicat-

ing that Go2a may increase Rap1GAP activity, therebyeffecting the Rap1 activation/deactivation cycle. Rap1 hasbeen shown to be involved in neurite outgrowth and given aRap1GAP-Go2a interaction, we found that the loss of Go2aaffected axonal outgrowth. Axons of cultured cortical andhippocampal neurons prepared from embryonic Go2a�/�mice grew longer and developed more branches than thosefrom wild-type mice. Taken together, we provide evidence thatGo2a regulates axonal outgrowth and branching.Keywords: axonal growth, Girdin, Go2a, heterotrimericG-proteins, Rap1, Rap1GAP.J. Neurochem. (2013) 124, 782–794.

Heterotrimeric G-proteins, consisting of an a subunit and abc dimer, couple cell surface receptors to intracellular secondmessenger systems (Birnbaumer 2007). In addition, they arealso located on secretory granules and synaptic vesicles(Ahnert-Hilger et al. 1994; Pahner et al. 2003; Takamoriet al. 2006). There are four families of Ga-subunits, Gsa, Gi/oa, Gq/11a, and G12/13a (Simon et al. 1991). Goa belongsto the Gi/o subfamily and represents together with Gia up to1.5% of membrane protein in mammalian brains (Neer et al.1984; Sternweis and Robishaw 1984). Goa comprises twosplice variants Go1a and Go2a. Go1a has been shown to beselectively involved in the light response of ON-bipolar cells(Dhingra et al. 2002). Vesicle-associated Go2a inhibitsvesicular monoamine transporter 1 and 2 (Ahnert-Hilgeret al. 1998; Holtje et al. 2000) and affects the chloridedependence of vesicular glutamate transporters (Winter et al.2005). Furthermore, Go2a has been shown to inhibitadenylyl cyclase activity in reconstituted assay systems(Kobayashi et al. 1990), to reduce voltage-sensitive calcium

currents in neurons of the pond snail helisoma (Man-Son-Hing et al. 1992), and to regulate glucose-induced release ofinsulin (Wang et al. 2010). Besides these Go2a-specific

Received August 17, 2012; revised manuscript received November 23,2012; accepted December 9, 2012.Address correspondence and reprint requests to Irene Brunk or Gudrun

Ahnert-Hilger, Institute for Integrative Neuroanatomy, Philippstr. 12,10115 Berlin, Germany.E-mails: [email protected]; [email protected] authors contributed equally to the article.Abbreviations used: AD, GAL4 activating domain; AGS, activator of

G-protein signaling; DNA-BD, GAL4 DNA binding domain; GAP,GTPase-activating protein; GEF, guanine nucleotide exchange factor;GIV, Ga-interacting vesicle-associated protein; GPSM, G-proteinsignaling modulator; GRIN, G-Protein regulated inducer of neuriteoutgrowth; H, homogenate; LP1, lysed pellet 1, synaptic vesicle; LP2,lysed pellet 2, synaptic vesicle; MAP, microtubule associated protein;NFP, neurofilament protein; OD, optical density; P2, synaptosomes;RGS, regulator of G-protein signaling; syb, synaptobrevin; syp,synaptophysin; VEGF, vascular endothelial growth factor.

782 © 2012 International Society for Neurochemistry, J. Neurochem. (2013) 124, 782--794

JOURNAL OF NEUROCHEMISTRY | 2013 | 124 | 782–794 doi: 10.1111/jnc.12123

effects, most of the reported Goa interactions refer only toGo1a. Accordingly, Goa/Go1a has been shown to interactwith regulator of G-protein signaling (RGS)14, RGS17 andG-Protein-Regulated-Inducer-of Neurite-Outgrowth 1 and 2(GRIN 1 and 2) (Chen et al. 1999; Traver et al. 2000;Nakata and Kozasa 2005), Rap1GAP, IP6 and Purkinje cellprotein 2 (PCP2) (Jordan et al. 1999; Luo and Denker 1999),whereas proteins directly interacting with Go2a have notbeen identified at this time.There is growing evidence for a cross talk between

heterotrimeric and small GTPases. In this respect, it has beenshown that the activities of GTP exchanging factors (GEF)and GTPase activating proteins (GAPs) are modulated byheterotrimeric G proteins (Aittaleb et al. 2011). In neuro-blastoma cell lines activated Go1a has been shown tomodulate neurite outgrowth by interacting with Rap1 viaRap1GAP (He et al. 2006) or Rit (Kim et al. 2008);however, the role of both Goa splice variants was notdifferentially regarded.In this study, we performed a yeast two-hybrid screen

using constitutively active Go2a (Go2aQ205L) as well asGo1a (Go1aQ205L) and Gqa (GqaQ209L) for comparison.We found that Rap1GAP and the non-receptor GEF Girdin,also known as Ga-interacting vesicle-associated protein(GIV) (Garcia-Marcos et al. 2009), interact with bothGo1aQ205L and Go2aQ205L with a preference for theGo2a-subunit. We provide evidence that the lack of Go2ahas an impact on levels of Rap1GTP, which is regulated byRap1GAP, modulating axonal growth.

Materials and methods

Antibodies

Mouse monoclonal antibodies raised against both Goa splicevariants (clone 101.1) or specifically recognizing Go2a (clone101.4) were previously described (Winter et al. 2005). Antibodiesagainst Goa (mouse), Girdin (rabbit), and Rap1 (rabbit) werepurchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).The anti-Goa antibody from Santa Cruz has been previously shownto detect only Go1a (Brunk et al. 2008). Antibodies againstRap1GAP (rabbit monoclonal and polyclonal) were obtained fromAbcam (Cambridge, UK) and Calbiochem (Merck, Darmstadt,Germany), respectively.

Monoclonal antibodies against synaptophysin (Jahn et al. 1985)and synaptobrevin and a rabbit polyclonal antibody against actinwere obtained from Synaptic Systems (G€ottingen, Germany) orSigma-Aldrich (St. Louis, MO, USA), respectively.

Neuronal cultures were stained by a mouse monoclonal antibodyagainst neurofilament protein (NFP; 200 kDa) and a rabbitpolyclonal antiserum against microtubule associated protein 2(MAP2), both from Millipore (Billerica, MA, USA) to identifyaxons or dendrites, respectively (Ahnert-Hilger et al. 2004).

Secondary antibodies, horse anti-mouse and goat anti-rabbitconjugated to horseradish peroxidase, were purchased from Vec-tor Laboratories (Burlingame, CA, USA). Secondary antibodiesconjugated to fluorochromes (goat anti-mouse IgG and goat anti-

rabbit IgG, either Cy2 or Cy3) were obtained from Dianova(Hamburg, Germany).

Mice

Go1a and Go2a splice variant-specific deletion mutants were bredand genotyped as described (Jiang et al. 1998; Dhingra et al. 2002).For all experiments, except for hippocampal cultures, mutant andwild-type littermates were obtained by interbreeding of heterozy-gous parents. Institutional approval (approval number T0119/11)was obtained for killing the animals used in this study.

Molecular cloning and yeast two-hybrid experiments

A mouse brain cDNA-library cloned into the vector pACT2(Clontech-Takara Bio Europe, Saint-Germain-en-Laye, France)was amplified in E.coli BNN132 according to the manufacturer’sinstructions. Afterward, plasmid-DNA was isolated using theQiagen Plasmid Giga Kit (Qiagen GmbH, Hilden, Germany).

cDNAs of Go2aQ205L, Go1aQ205L as well as GqaQ209L werecloned into pAGA2 (Hsu et al. 1990; Wei et al. 1991) and thenwere amplified by PCR using the following primer pairs:

Go1a: 5′-ACCGAATTCATGGGATGTACTCTGAGC-3′5′-AATGTCGACTCAGTACAAGCCACAGC-3′

Go2a: 5′-ACCGAATTCATGGGATGTACTCTGAGC-3′5′-AATGTCGACTCAGTACAAGCCACAGCCCCGCAGA-3′

Gqa: 5′-AGCGAATTCATGACTCTGGAGTCC-3′5′- AGCCTCGAGTTAGACCAGATTGTACTCC-3′

Ga subunit amplicons were cloned into the multiple cloning site ofthe pGBKT7 plasmid using EcoRI/SalI and EcoRI/XhoI restrictionsites, and the absence of PCR-induced mutations was confirmed byDNA sequencing. Plasmid expression in yeast yields to bait fusionproteins in which the Gal4-DNA-binding domain (DNA-BD) isfused to the N-terminus of Ga subunits.

The yeast strain AH109 was sequentially transformed usingpGBKT7Go2Q205L and the mouse brain cDNA-library. Thetransformed cells were grown in quadruple-dropout-selection-medium (QDO) containing 1 mM 3-aminotriazole and lackingtryptophan, leucine, adenine, and histidine at 30°C for up to2 weeks. Colonies were re-grown three times using the samemedium supplemented with 40 lg/mL X-a-Gal (5-Bromo-4-chloro-3-indolyl-a-D-galactopyranoside) to detect a-galactosidase expres-sion. Plasmid-DNAs of a-galactosidase positive colonies wereisolated using the Zymoprep II Yeast Plasmid Minipreparation Kit(Zymo Research Corporation, Orange, CA, USA) and amplifiedafter transformation of E. coli DH5a cells. Finally, after E. coliplasmid preparation, library cDNAs cloned into pACT2-vector weresequenced and analyzed using the BLAST-tools of the EuropeanBioinformatics Institute (EBI).

To confirm the protein interaction and to analyze the interactionspecificity of the identified clones, yeast mating experiments wereperformed. To this end, the yeast AH109 strain mating type a wasretransformed with the purified plasmid-DNA encoding identifiedinteracting proteins (preys). The yeast Y187 strain mating type awas transformed using either control-vector-DNA or the pGBKT7vector carrying cDNAs encoding constitutive active Go1a, Go2a, orGqa cloned into EcoRI/SalI or EcoRI/XhoI restriction sites (baits).Yeast strains were then grown on SD (synthetic dropout) – mediumlacking leucine or tryptophan for selection of positive co-transfor-

© 2012 International Society for Neurochemistry, J. Neurochem. (2013) 124, 782--794

Go2a regulates axonal growth 783

mants. After mating in 200 lL YPD-medium over night at 30°C,diploid cells were selectively grown on SD-Leu-Trp-medium for3 days. Afterward, growth of yeast cells was analyzed on QDO-medium-plates as described above.

For the p-nitrophenyl-a-D-galactopyranoside (PNP-a-Gal) assay,samples were prepared by yeast mating as described above bycrossing cells of the Y187 strain mating type a bearing the baitplasmids pGBKT7-Go2aQ205L, -Go1aQ205L, -GqaQ209L, -Lamin C, -p53, or the empty vector pGBKT7 with cells of theAH109 strain mating type a, bearing the prey plasmids pACT2-Girdin, -Rap1Gap, Synembryn, pGADT7-T, or the empty pGADT7vector (Table 1). After mating and selection of diploid cells onminimal agar lacking Trp and Leu, 2 mL of the same medium orhigh stringency SD medium (SD –Trp –Leu –His –Ade +1 mM 3-AT) were inoculated with one large or up to three small coloniesexpressing the indicated pair of proteins, and incubated overnight at30°C while shaking at 250 rpm. The cell density of the suspensionwas adjusted to an OD600 of between 0.4 and 1.0. One milliliter ofyeast cells were then collected by centrifugation at 10 000 g for2 min. Three 16 lL aliquots of each supernatant as well as ofmedium for blank values were mixed with 48 lL of assay buffercontaining 33.3 mM PNP-a-Gal and 0.33 M NaOAc, pH 4.5 in a 96well microtiterplate and incubated for 1 h at 30°C. A p-nitrophenolstandard was run afterward. The reaction was stopped by adding136 lL of stop solution containing 1 M Na2CO3 and colordevelopment was determined at 405 nm using a microtiterplateELISA reader (Anthos Labtec, Salzburg, WALS, Austria; HT-2).

Units of the a-galactosidase were calculated by the followingmethod:

mU=ðmL� cellÞ ¼ OD405 � Vf � 1000=½ðe� bÞ � t � Vi

� ðOD600=DFÞ�

OD405, optical density of p-nitrophenol after enzymatic reaction;t, elapsed time of incubation (min); Vf, final volume of assay(200 lL); Vi, volume of culture medium supernatant added (16 lL);OD600, optical density of overnight culture; DF, dilution factor ofOD600; e 9 b, p-nitrophenol molar absorption at 405 nm multipliedby the light path (cm) and the proportionality constant of a linearstandard regression line.

Significance was tested from five (Girdin interaction) or four(Rap1GAP interaction) independent experiments using the Kruskal–Wallis test on the basis of a significance level of p < 0.05.

Immunoblot analysis

Subcellular fractions were prepared from mouse brains as describedpreviously (Huttner et al. 1983; Becher et al. 1999). Samples wereanalyzed by sodium dodecyl sulfate–polyacrylamide gel electro-phoresis (SDS-PAGE) and western blot techniques using the ECLdetection system (GE Healthcare, Munich, Germany). For quanti-fication, ECL-processed films were scanned and protein bands weredensitometrically measured using the Labimage 1D program(KAPELAN, Halle, Germany) ensuring that signals were in thelinear range of the ECL detection system. Comparative quantifica-tion of protein samples from wild-type mice and deletion mutantswas performed in the same gel using actin or synaptophysin asinternal controls.

Immunoprecipitation

Synaptic vesicle preparations (LP2; 1 mg) were extracted with 1 mLextraction buffer containing 140 mM KCl, 2 mM EDTA, 20 mMHEPES-KOH (pH 7.4), and 1% (v/v) Triton X-100; and thenincubated for 1 h at 4°C under rotation, followed by centrifugation at1300 g for 5 min. Two hundred microliter of each extract were thenimmunoprecipitated using the following antibodies: rabbit anti-Rap1GAP, rabbit anti-Girdin, mouse anti-Go2a, normal mouse IgG,normal rabbit IgG. All antibodies were diluted to give a finalimmunoglobulin concentration of 2–4 lg per sample depending onthe antibody charge. Immunoprecipitation was performed usingDynabeadse (Invitrogen, Darmstadt, Germany). Following thebinding of antibodies to 50 lL beads (incubation for 1 h at 20°Cwhile rotating) and washing, 200 lL of brain extract was added andsamples were incubated over night at 4°C. The supernatants werekept for further analysis, whereas the beads were washed three timesin extraction buffer. Finally, the bead pellets were resuspended inloading buffer and boiled for 10 min. Supernatants and pellets wereanalyzed by SDS-PAGE and western blotting.

Rap1 pull-down assay

The Rap1-binding domain of RalGDS (RalGDS-RBD) wasexpressed as a GST fusion protein in E. coli and purified by affinitychromatography using glutathione-sepharose. Frozen mouse brains(males) were homogenized on ice in a Potter-Elvehjem homoge-nizer, in freshly prepared ice-cold buffer (150 mM NaCl, 50 mMTris pH 7.2, 5 mM MgCl2, 1 mM phenylmethanesulfonylfluoride,

Table 1 Mating pairs of transformed yeast cells

Sample Y187/DNA-BD/bait AH109/AD/prey

1 pGBKT7-Go2aQ205L pGADT72 pGBKT7-Go1aQ205L pGADT7

3 pGBKT7-GqaQ209L pGADT74 pGBKT7-LaminC pGADT75 pGBKT7 pGADT76 pGBKT7-Go2aQ205L pACT2-Girdin

7 pGBKT7-Go1aQ205L pACT2-Girdin8 pGBKT7-GqaQ209L pACT2-Girdin9 pGBKT7-LaminC pACT2-Girdin

10 pGBKT7 pACT2-Girdin11 pGBKT7-Go2aQ205L pACT2-Rap1Gap12 pGBKT7-Go1aQ205L pACT2-Rap1Gap

13 pGBKT7-GqaQ209L pACT2-Rap1Gap14 pGBKT7-LaminC pACT2-Rap1Gap15 pGBKT7 pCT2-Rap1Gap

16 pGBKT7-Go2aQ205L pACT2-Synembryn17 pGBKT7-Go1aQ205L pACT2-Synembryn18 pGBKT7-GqaQ209L pACT2-Synembryn19 pGBKT7-LaminC pACT2-Synembryn

20 pGBKT7 pACT2-Synembryn21 pGBKT7-p53 pGADT7-large T antigen

Indicated are the plasmids with which yeast cells of the respectivestrain were transformed. Transformed Y187 yeast cells (mating type a

were mated with transformed AH109 yeast cells (mating type a) as

shown.


784 J. Baron et al.

5 mM dithiothreitol, 1% NP-40 and protease inhibitor cocktail), byhand using 8 to 10 up and down strokes of the Teflon pestle andthen sonicated. The solubilized tissue was spun at 13 000 g for10 min at 4°C, DNAse digestion was then performed at 20°C for15 min and lysates were clarified by centrifugation at maximalspeed for 10 min at 4°C. After 15 min, the GTP-bound state ofRap1 was stabilized with 60 mM MgCl2. Glutathione S-transferase(GST)-tagged Ral-GDS beads were added to the supernatants andincubated for 1 h (4°C). The beads were washed three times, andbound proteins were solubilized by incubation with loading bufferat 95°C for 10 min. Samples were subjected to SDS-PAGE andwestern blot analysis using a polyclonal antibody raised againstRap1 (Cell Signalling, Danvers, MA, USA). Finally, all signalswere analyzed densitometrically using the KODAK 1D softwareImage Analysis Software (Eastman Kodak Company, Stuttgart,Germany) and normalized to b-actin signals. Pull-down resultswere statistically tested with five animals per genotype using a two-sample paired t-test on the basis of a significance level of p < 0.05.

Cell culture

Neurons were prepared from fetal brains of wild type, heterozygous,and Go2a�/� mice or from wild type and Go1a�/� mice atembryonic day 16 (E16) as described previously (Ahnert-Hilgeret al. 2004). Pieces of the hippocampus or neocortex were rinsedwith phosphate-buffered saline (PBS), then with dissociationmedium [modified Eagle medium (MEM) supplemented with 10%fetal calf serum, 100 IU/L insulin, 0.5 mM glutamine, 100 U/mLpenicillin/streptomycin, 44 mM glucose, and 10 mM HEPESbuffer] and then dissociated mechanically. Following centrifugation,cells were resuspended in starter medium (serum-free neurobasalmedium supplemented with B27, 0.5 mM glutamine, 100 U/mLpenicillin/streptomycin, and 25 lM glutamate) and plated at adensity of 2 9 104 cells/well on poly-L-lysine/collagen pre-coatedglass coverslips. All ingredients were obtained from Gibco/BRLLife Technologies (Eggenstein, Germany).

For hippocampal cultures, wild type and Go2a�/� strains werebred separately and hippocampi of each genotype were collected.For littermate neocortical cultures, wild type (129/Sv x C57BL/6)and Go2a splice variant-specific mutants or wild type and Go1asplice variant-specific mutants were cross-bred, and the resultingheterozygous offsprings were used to generate littermates with thesame genetic background. Neocortical neurons of each individualembryo were cultured separately and the genotype of the individualcultures was determined.

Immunocytochemistry and morphometric analysis of neuronal

cultures

After 5 days in vitro (DIV) neurons were fixed with 4% formalin for15 min and subsequently permeabilized for 30 min at 20°C using0.3% Triton X-100 dissolved in PBS. For confocal laser scanningmicroscopy, neurons were stained with the indicated primaryantibodies overnight at 4°C. For morphometric analysis, antibodiesagainst NFP200 and MAP2 were used to mark axons and dendrites,respectively. After washing in PBS, secondary antibodies wereapplied for 1 h at 20°C. After three rinses with PBS and one rinsewith water, coverslips were mounted on glass slides for microscopicanalysis of fluorescent labeling.

For confocal laser scanning microscopy we used a Leica DMREmicroscope (Leica Microsystems GmbH, Heidelberg, Germany),

with an 963 oil immersion objective lens, with excitation frequen-cies of 488 nm (argon laser) and 543 nm (helium-neon laser). Imageacquisition and analysis was performed using the built-in LeicaConfocal Software.

Image acquisition preceding morphometric analysis was per-formed by a Leica DMLB microscope using an 940 objective lens.Images were captured at a resolution of 1024 9 1024 pixels. Totallength and overall number of branching nodes of axons anddendrites were analyzed morphometrically using the Neurolucidasoftware (MicroBrightField, Williston, VT, USA). The parameter‘axon length’ represents the integral total length of all visible partsof an axon, including its higher order branches (Ahnert-Hilger et al.2004). Experiments were carried out twice for hippocampal andtwice for neocortical neurons from cultures prepared on the sameday. Typically, five coverslips were prepared per genotype and sixneurons were evaluated on each coverslip. Data from five coverslips(30 individual neurons) were pooled and given as means � SD. Thedata displayed in the graphs of Fig. 5 refer to a single, representativeexperiment, if not mentioned otherwise. Genotype related differ-ences were statistically analyzed using the Kruskal–Wallis andMann–Whitney-U-test, on the basis of a significance level ofp < 0.05.

Results

Interaction partners of constitutively active Go2aTo identify interaction partners of Go2a, a yeast two-hybridscreen was performed using a mouse brain cDNA-library asprey and constitutively active Go2aQ205L as bait. Screeningof 3.5 9 106 clones initially yielded 20 different interactingproteins, which were reduced to 11 after performing yeastmating controls (Table 2). Proteins already identified asimportant regulators of Ga- signaling were found to interact

Table 2 Interaction partners of Go2aQ205L

Clone Go2aQ205L Go1aQ205L GqaQ209L

Armadillo repeat

containing x-linked 1

+ + �

Girdin + + �G-protein signaling

modulator 1

+ + �

G-protein signalingmodulator 2

+ + �

Rap1Gap (KIAA0474) + + �RGS14 + + �RGS17 + + �RGS19 + + �RGS20 + + �Synembryn + + +

TNFAIP8 + + �

Indicated are interaction partners of the constitutively active mutant ofGo2a identified by a yeast two-hybrid screen. Interaction with Go1a

and Gqa was investigated to determine selectivity of interaction.+, interaction; �, no interaction.



with Go2a. These include regulators of G-protein signaling(RGS-proteins) 14, 17, 19, and 20, as well as G-proteinsignaling modulator 1 (Gpsm1 also called AGS3), andGpsm2 also called LGN, which have several GoLoco motifs,and potentially positively or negatively regulate G-proteinsignaling (Kerov et al. 2005). Known binding partners ofGo1a, like Rap1GAP (Jordan et al. 1999), or synembryn(Tall et al. 2003), were also identified as was Girdin, whichhas been described to bind to Goa, Gsa, and Gi1-3a before,however, the Goa splice variant involved was not elucidated(Le-Niculescu et al. 2005). Moreover, we identified proteinsfor which no interaction with a-subunits of heterotrimeric G-proteins has been reported so far, such as the Armadillorepeat containing x-linked 1 (ALEX1) (Kurochkin et al.2001), which may function as a tumor suppressor.Specificity of these interactions was controlled by yeast

mating experiments using the AH109 and Y187 strains,carrying the identified interacting partners (prey) and theconstitutively active Ga-subunits (baits) fused to GAL4domains, respectively. To determine isoform specificity ofthe identified interactions, Go1aQ205L was chosen for adirect comparison with Go2aQ205L. In addition, an inter-action with GqaQ209L belonging to the Gq/11-family ofGa-subunits, was analyzed to determine G-protein familyspecificity (Table 2). We found, that diploid yeast colonies,expressing Rap1GAP or Girdin in combination withGo2aQ205L, grew faster compared with diploid yeastcolonies bearing Rap1GAP or Girdin in combination withGo1aQ205L. For Girdin, two partial cDNAs were recoveredin the screen (coding for amino acids G1318-S1873 andL1351-S1873), whereas for Rap1GAP three full lengthclones were recovered. Synembryn also interacted with bothGoa splice variants, however, also with Gqa.To further analyze this effect, we used a quantitative

a-galactosidase assay (Fig. 1). Unspecific binding of theGal4-DNA-activating domain to bait fusion proteins andunspecific interactions of the Gal4-DNA-binding domain toprey fusion proteins were excluded (Fig. 1a). In addition, theabsence of a-Gal activation during mating, when using theLaminC-binding domain fusion protein in combination withthe respective prey-activating domain fusion protein, servedas negative control. As can be seen in Fig. 1b, theGo2aQ205L/Girdin interaction was 2.5-fold larger thanthe Girdin/Go1aQ205L interaction. On the same lines, theinteraction between Rap1GAP and Go2aQ205L (Fig. 1c)was 1.9-fold stronger than with Go1aQ205L. There was nointeraction of either protein with the activated form of Gqa(Fig. 1).The interaction of Go1a and Go2a with Rap1GAP and

Girdin was confirmed by immunoprecipitation (Fig. 2). Inagreement with the results obtained from the yeast two-hybrid screen, immunoprecipitation performed with anti-Go2a (Fig. 2a) led to co-precipitation of Rap1GAP andGirdin, whereas precipitation with normal mouse IgG did not

yield any specific precipitate. Immunoprecipitation usinganti-Girdin (Fig. 2b) and anti-Rap1GAP (Fig. 2c) led to co-precipitation of Go1a and Go2a.

Expression of Girdin and Rap1GAP in wild type and

Go2a�/� miceAs expected, synaptosomes (P2) from Go2a knock out brainsare devoid of Go2a as identified by the subtype-specificantibody (Fig. 3a). Following subcellular fractionation ofwhole adult brains, Girdin was predominantly found in theLP2 fraction (Fig. 3b). Girdin binds to actin filaments and isfound on ER-Golgi transport vesicles (Le-Niculescu et al.2005), as well as associating with the plasma membrane,depending on its phosphorylation state (Enomoto et al.2005). Comparison of wild type and Go2a�/� mice did notreveal significant differences in expression levels of Girdin indifferent fractions derived from whole brains (Fig. 3b).Similar amounts of Rap1GAP were found in brain

homogenates, synaptosomes, and synaptic vesicles of wildtype and Go2a�/� mice (Fig. 3c). Only in the LP1 fractionRap1GAP levels were decreased in comparison to wild-typemice (p < 0.04). The LP1 fraction is known to consist ofmaterial from plasma membranes. Go2a may attachRap1GAP to membrane compartments, so lack of Go2amay cause a shift in sorting. Rap1GAP is known to beenriched in the striatum (McAvoy et al. 2009). Therefore, weanalyzed striatal synaptosomes of wild type and Go2a�/�mice; however, no difference of Rap1GAP expression levelswas observed (data not shown). Expression patterns ofRap1GAP in the cerebellum, brainstem, striatum, andmesencephalon did not differ between wild type andGo2a�/� mice (data not shown).

Increased amounts of activated Rap1 in Go2a�/� mice

We have shown that constitutively active Go2a interacts withRap1GAP, the activation of which stimulates the GTPaseactivity of the Ras-like small G-protein Rap1 to render Rap1inactive (Rubinfeld et al. 1991). Therefore, we analyzed theexpression of Rap1 in different brain fractions and the levelsof active GTP-bound Rap1 in brains of wild type andGo2a�/� mice.Rap1 was comparably expressed in brain fractions of both

genotypes (Fig. 4a). Pull-down assays of activated Rap1-GTP from total brains, however, revealed a significantlyincreased ratio of activated to total Rap1 in Go2a deletionmutants compared with wild-type animals (Fig. 4b). Thus,loss of Go2a results in an increased level of activated Rap1,most likely by decreasing the Rap1GAP activity.

Morphometric analysis of neuronal cultures from

wild type and Go2a�/� mice

Rap1 is involved in regulation of neurite outgrowth anddendritic development (Jordan et al. 2005; Xie et al. 2005;McAvoy et al. 2009). As the amount of Rap1-GTP is


786 J. Baron et al.

increased in brains of Go2a deletion mutants, compared withwild types, we therefore analyzed axon and dendrite lengthand branching in neuronal cultures from these genotypes.In Go2a deletion mutants axons grew longer and had morebranching nodes in cortical (Fig. 5a left panel, b) andhippocampal (Fig. 5a right panel, c) primary neuronalcultures in comparison to cultures from wild-type mice.Axon lengths in neocortical cultures, from heterozygous

mice, were comparable to those from Go2a�/� mice;however, the number of branching nodes was significantlydecreased compared with the deletion mutants. Still, therewas an increase in branching points in the heterozygouscompared with the wild-type neurons (Fig. 5b). These dataindicate a gene dose effect of Go2a differentially effectinggrowth and branching of axons. Dendritic length andbranching did not differ between the genotypes (Fig. 5b,

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Rap

1GAP

-AD

Go2QL-

BD +

Rap

1GAP

-AD

LaminC

-BD +

Rap

1GAP

-AD

BD +

Rap

1GAP

-AD

GqQL-

BD +

Rap

1GAP

-AD

0

60708090

100

5040302010

*

(c)

Fig. 1 Relative strength of Go2a/Girdin and Go2a/Rap1Gap interac-tion determined by quantitative a-galactosidase assay. Mating-derived

diploid cells were probed for a-Galactosidase (a-Gal) expression andsecretion that resulted from interacting Gal4 fusion proteins asindicated. Lamin C acted as a negative control. BD, binding domain;AD, activating domain. (a) The displayed representative experiment

illustrates the considerably stronger interaction of Girdin and Rap1Gapwith Go2aQ205L compared with Go1aQ205L and the lack of aninteraction between GqaQ209L with both preys. In addition, the

following negative controls are depicted: Empty prey plasmids werecombined with each bait: Go2QL-BD+AD, Go1QL-BD+AD, GqQL-BD+AD. Empty bait plasmids were combined with each prey: BD+Girdin-

AD, BD+Rap1GAP-AD. The LaminC containing bait plasmid wascombined with each prey, because LaminC is known to interact with

almost no other protein: LaminC-BD+AD, LaminC-BD+Girdin-AD,LaminC-BD+Rap1GAP-AD. Empty prey and bait plasmids were

combined: BD+AD. (b) Summary of five independent experiments:The Go2aQ205L/Girdin interaction exceeds Girdin´s interaction withGo1aQ205L 2.5-fold; significance was confirmed by the Kruskal–Wallis test (p < 0.02); star denotes significance. There is no interaction

of Girdin with GqaQ209L since the level of a-Gal activity is comparableto the negative controls. (c) Summary of four independent experi-ments: The interaction between Rap1GAP and Go2aQ205L is 1.9-fold

stronger than with Go1aQ205L; significance was confirmed by theKruskal–Wallis test (p < 0.03); star denotes significance. There is nointeraction of Rap1Gap with GqaQ209L since the level of a-Gal activity

is comparable to the negative controls.



c). Taken together, Go2a appears to specifically regulateaxonal outgrowth and branching, which is perhaps explainedby the interaction of Go2a with Rap1GAP leading toincreased Rap1GTP in Go2a deletion mutants suggestingthat an enhancement of Rap1 activity might be involved.

Expression of Girdin, Rap1GAP and Rap1 in embryonic

neurons of wild type, Go1a�/� mice and Go2a�/� mice

Girdin, Rap1GAP and Rap1, which interact with Go2a andaffect axonal branching, were analyzed in post-nuclear

Immunoprecipitation with anti-Girdin

Immunoprecipitation with anti-Rap1Gap

Rap1Gap

Go1

Go2

Syp

Girdin

Go1

Go2

SypInput

SN IP

InputSN IP

Rap1Gap

Go2

Girdin

InputSN - anti-Go2

IP- anti-

SN - ms IgG

IP- ms IgG

Non-ExtractGo2

Immunoprecipitation with anti-Go2(a)

(b)

(c)

Fig. 2 Co-immunoprecipitation of Girdin and Rap1GAP with Go2a.SN, supernatant; IP, immunoprecipitate. (a) Immunoprecipitates frommouse LP1 derived from total brain using an antibody against Go2a

and normal mouse IgG as negative control. Confirming the results fromthe yeast two-hybrid screen both Girdin and Rap1GAP were co-precipitated with Go2a. Immunoprecipitation using mouse IgG yielded

no specific precipitate. (b) Immunoprecipitates from mouse LP2derived from total brain using a Girdin antibody contained besidesGirdin, Go1a and Go2a, confirming the results from the yeast two-

hybrid screen. Synaptophysin was not co-precipitated and served as anegative control. (c) In agreement with the results from the yeast two-hybrid screen, the Rap1GAP antibody precipitated Rap1GAP together

with Go2a and Go1a using a Triton X-100 extract from mouse synapticvesicles. Synaptophysin was not co-precipitated and served as anegative control.

Girdin

Syp

wt Go2

H P2 LP1LP2

wtGo2 –/–

0

0.16

0.12

0.08

0.04

H P2 LP1 LP2 H P2 LP1 LP2

P2 P2

Rap1Gap

Syp

wt

H P2 LP1LP2

wtGo2 –/–

Rel

ativ

e O

DR

elat

ive

OD

0

1.6

0.20.40.60.81.01.21.4

*


Syb

Go2

Go1+2

wt Go2 –/–

Girdin in subcellular fractions from wildtype and Go2 –/– brains

–/–

Rap1Gap in subcellular fractions from wildtype and Go2 –/– brains

Go2 –/–

(a)

(b)

(c)

Fig. 3 Expression of Girdin and Rap1GAP in brains of wild type and

Go2a�/� mice. (a) P2 fractions from wild type and Go2a�/� brainswere analyzed using antibodies against Go2a (clone 101.4) or againstboth Goa splice variants (clone 101.1) affirming absence of Go2a indeletion mutants. Synaptobrevin served as an internal control. (b)

Subcellular fractions prepared from wild type and Go2a�/� brainswere analyzed by western blotting using a Girdin antibody. Expressionof Girdin did not differ between the genotypes. The graphs show

quantifications of lanes loaded with 10 µg protein. (c) Subcellularfractions prepared from wild type and Go2a�/� brains were analyzedby western blotting using a Rap1GAP antibody. The amount of

Rap1GAP was lower in LP1 from Go2a�/� mice than in wild-type LP1.Significance was affirmed by the Kruskal–Wallis-test (p < 0.04); stardenotes significance. Regarding the other brain fractions, there wereno differences between the genotypes. The graphs show quantifica-

tions of lanes loaded with 10 µg protein. (b–c) Quantification is givenas relative optical density (OD) and was assessed using synaptophy-sin as an internal control. Values represent the mean of four animals

per genotype � SD. H, homogenate; P2, synaptosomes; LP1 (lysedpellet 1): LP2, synaptic vesicles.


788 J. Baron et al.

supernatants from embryonic brains of wild type andGo2a�/� mice. Expression levels of Girdin, Rap1GAPand Rap1 were comparable in post-nuclear supernatants ofE16 brains from both genotypes (Fig. 6).Possible co-localization of Rap1GAP (Fig. 7) with Go1a

and Go2a was investigated by confocal microscopy ofneuronal cultures from wild type and Go1a�/� mice. The

latter genotype has been used since the Goa antibodysuitable for immunofluorescence detection recognizes bothsplice variants. The expression pattern of Rap1GAP does notdiffer in wild type and Go1a�/� neurons (Fig. 7a).Rap1GAP could be detected in the cytosol of perikaryaand processes of wild type and Go1a�/� mice. Theexpression pattern of Rap1GAP also superimposes that ofGoa (Fig. 7a, b). Co-localization of Go2a and Rap1GAP isshown by merging immunosignals of anti-Rap1GAP andanti-Goa 1 + 2 in neuronal cell cultures of Go1a�/� mice(Fig. 7a). Determination of fluorescence intensity of bothsignals clearly confirms their overlay and thereby the co-localization of Go2a and Rap1GAP in processes of primaryneurons (Fig. 7b). The same results could be obtained forRap1 and Girdin (data not shown). Neurons from deletionmutants lacking both Goa splice variants were devoid ofimmunosignal after incubation with the Goa antibodyconfirming specificity. Specificity of the Rap1GAP immu-nolabeling was confirmed by blocking the antibody reactionwith the respective antigen used for immunization (Fig. 7c).

Discussion

To our knowledge, this is the first study aiming at theidentification of interaction partners specific for the splicevariant Go2a which in the brain represents one third of thehighly abundant Go heterotrimeric G-protein. Constitutivelyactive Goa variants were chosen as baits for the yeast two-hybrid screen to identify interacting partners and hence arepossible effectors of Go2a. We identified several proteinsinteracting with constitutively active Go2a, which alsointeracted with constitutively active Go1a. For two of them,Girdin and Rap1GAP, the interaction with Go2a was shownto be stronger compared with Go1a. Physiological relevanceof the Rap1GAP-Go2a interaction was confirmed whendeletion of Go2a led to an increase of cerebral Rap1GTPand positively affected axonal growth of embryonicneurons.

Interaction of Girdin and Go2aQ205LGirdin, otherwise known as GIV, is highly expressed in thebrain and testis and located on intracellular vesicles,especially Golgi transport vesicles, having been shown tobind to wild-type Gi1-3a, Gsa, Gza, and Goa subunits(Le-Niculescu et al. 2005). According to our yeast two-hybrid screen, Girdin interacts with both Go2aQ205L andGo1aQ205L, respectively, with a preference forGo2aQ205L. Girdin did not interact with Gqa.Girdin is believed to be a non-receptor GTP-exchange

factor (GEF). In this respect, Girdin may promote activationof vesicular Go2a, which has been shown to regulatevesicular monoamine accumulation (Ahnert-Hilger et al.1998), although a direct association of Girdin with synapticvesicles has, so far, not been shown.

Rap1

Rap1A/B input

GTP-Rap1

-actin

Syp

wt

wt

H P2 LP1LP2

wtGo2 –/–

Rel

ativ

e O

D

0

0.6

0.1

0.2

0.3

0.4

wt Go2

)nitca/1paR(/)nitc a/1pa

R-P T

G(

0

0.01

0.02

0.03

0.04

0.5


*

Rap1 in subcellular fractions from wildtypeand Go2 –/– brains

Go2 –/–

Go2 –/–

–/–

(a)

(b)

Fig. 4 Amounts of entire and activated Rap1 in brains of wild type andGo2a�/� mice. (a) Subcellular fractions prepared from wild type and

Go2a�/� brains were analyzed by western blotting using a Rap1antibody. The expression of Rap1 did not differ between thegenotypes. The graphs show quantifications of lanes loaded with

10 µg protein. Quantification is given as relative optical density (OD)and was assessed using synaptophysin as an internal control. Valuesrepresent the mean of four animals per genotype � SD. H, homog-

enate; P2, synaptosomes; LP1 (lysed pellet 1): LP2, synaptic vesicles.(b) Increased amount of GTP-bound Rap1 in brains of Go2a�/� micecompared with wild-type littermates as seen in a pull-down assay ofactivated GTP-bound Rap1. GTP-bound Rap1 was quantified from five

male animals per genotype. Values represent the mean (� SEM), stardenotes significance (paired t-test, p < 0.02).



Neocortical neurons Hippocampal neurons

wt

Go2 –/–Go2 /–

noruen/mµ

noruen/rebmun

0 0

250

200

150

100

50

5

2

1

3

4

Dendrite length Dendrite nodes

wt

Go2 –/–Go2 /–

noruen/mµ

noruen/rebmun

0 0

700600500400300200100

8

2

4

6

(b)Axon length

Neocortical neuronsAxon nodes

*

* ***

noruen/mµ

noruen/rebmun

0 0

500

400

300

200

100

5

2

1

3

4

Dendrite length Dendrite nodes

noruen/mµ

num

ber/n

euro

n

0 0

700800

600500400300200100

12

2

4

6

8

10

(c)Axon length

Hippocampal neuronsAxon nodes

**

(a)

Fig. 5 Morphometric analysis of neuronal cultures from wild type and

Go2a�/� mice. Total length and overall number of branching nodesfrom axons and dendrites were analyzed morphometrically in neocor-tical and hippocampal neuronal cultures from wild type, heterozygous

(only for neocortical cultures) and Go2a�/� mice after 5 DIV. TheFigure illustrates the results from one representative experiment (seedetails in material and methods). For each genotype data from 30

neurons were used for calculation of significance. The experiment wasperformed twice with hippocampal and twice with neocortical neurons.(a) Examples of neocortical (left) and hippocampal (right) neurons from

wild type and Go2a�/� mice immunostained for NFP200 (green) andMAP2 (red) to visualize axons and dendrites, respectively. Barrepresents 20 µm for upper panels and 10 µm for lower panels. (b)Neocortical neuronal cultures prepared from wild type, Go2a+/� and

Go2a�/� littermates were analyzed. Axonal length and branching

(upper panels) were significantly increased in Go2a deletion mutants

compared with wild-type mice (p < 0.005). Axon length of neuronsfrom heterozygous animals did not differ from axon length in deletionmutants, but from axon length in wild-type animals (p = 0.002). The

number of axonal branching nodes per neuron fell between those fromwild type and Go2a�/� animals (p < 0.005). Dendrite length andbranching (lower panels) did not differ between the genotypes.

Significance was tested by the Kruskal–Wallis test; stars denotesignificance. (c) Hippocampal neuronal cultures prepared from wildtype and Go2a�/� mice were analyzed: Axonal length and branching

(upper panels) were significantly increased in Go2a deletion mutantscompared with wild-type mice (p < 0.005 for both). Dendrite length andbranching (lower panels) did not differ between wild type and Go2a�/�mice. Significance was tested by the Kruskal–Wallis test; stars denote

significance.


790 J. Baron et al.

Rap1Gap

Rap1

Actin

Girdin

5 10 15 5 10 15 µg protein

5 10 15 5 10 15 µg protein

Go1+2

Syb

Go

Syp

wt wtGo2 –/– Go2 –/–(a) (b)2

Fig. 6 Expression of Rap1GAP, Rap1, and Girdin in embryonic brainsfrom wild type and Go2a�/� mice. (a) Post-nuclear supernatantsprepared from embryonic wild type and Go2a�/� brains at E16 wereanalyzed by western blotting using anti-Girdin, -Rap1GAP, and -Rap1

antibodies. Similar amounts of the proteins were expressed in bothgenotypes. Detection of actin and synaptophysin served as control.Lanes were loaded with 5 µg, 10 µg, and 15 µg of protein, respec-tively. The lack of Go2a was analyzed in parallel (b).

Rap1Gap Merge

wt

(a)

1 2 3 4 5 6 7Distance [µm]

40

I F I F

80

120

160

200

240

8 9 1 2 3 4 5 6 7

40

80

120

160

200

240

Distance [µm]

anti-Go1+2(c)

Go1+2 –/–wt

anti-Rap1GAP

Control + peptide

Go 1+2

Go1 –/–

–/–Go1

(b)

Fig. 7 Expression of Rap1GAP in primaryneuronal cell cultures from embryonic (E16)

brains of wild type and Go1a�/� mice.Expression patterns and co-localizationof Rap1GAP and Goa-subunits were

investigated by confocal laser scanningmicroscopy using an antibody againstRap1GAP and an antibody recognizingboth Go1a and Go2a. Therefore, co-

localization with Go2a can be seen incultures from Go1a�/� brains. (a) Theexpression pattern of Rap1GAP does not

differ between wild type and Go1a�/� mice.Co-localization of Rap1GAP with Go1a/Go2ain wild-type mice and with Go2a in Go1a�/�mice is demonstrated by the detailedphotos on the right. Bars represent 20 µmand 4 µm (right pictures), respectively. (b)

Quantification of Rap1GAP- and Goa-immunolabeling in neuronal processes ofGo1a deletion mutants confirms the co-localization with Go2a. (c) Specificity of

antibody labeling Upper panels: Theantibody against Goa (Go2a + Go1a)yields specific immunolabeling in wild-type

neurons; no signal can be detected inneurons from Go1a + Go2a�/� mice.Lower panels: Pre-incubation of Rap1GAP

antibody with the respective peptide used forimmunization blocks immunostaining inneuronal cultures confirming specificity. Bar

represents 50 µm.



Girdin is phosphorylated by Akt, an important regulatorof cell survival, growth, and migration; and is associatedwith actin filaments (Anai et al. 2005; Enomoto et al. 2005).Phosphorylation of Girdin by Akt has been suggested toplay an important role in cell motility, progression ofmalignant neoplasms, and VEGF-mediated angiogenesis(Jiang et al. 2008; Kitamura et al. 2008). In mammaliancells, the role of Girdin in cell migration appears to beregulated by Gi3a dependent on the state of bound GTP.Girdin has been described to be a GEF for Gia (Ghosh et al.2008; Garcia-Marcos et al. 2009) and to a lesser extent forGo1a (Garcia-Marcos et al. 2010); whether Girdin alsofunctions as a GEF for Go2a is not clear. Given aninteraction between activated Go2a and Girdin, Go2a maymodulate the GEF function of Girdin for Gia or Go1a.Girdin also interacts with Disrupted-In-Schizophrenia 1(DISC1) and both play a critical role in post-natal neuro-genesis of the dentate gyrus (Enomoto et al. 2009). Thus,Go2a may have an effect on neuronal development via itsinteraction with Girdin similar to effects reported hereregarding axonal growth of embryonic hippocampal andneocortical neurons. Since adult Go2a�/� mice did notshow severe morphological abnormalities, the impact ofGo2a deletion on the assembly of neuronal network appearsto be subtle becoming only evident during examination ofspecific behavioral tasks. For example, behavioral sensitiza-tion was absent in Go2a deletion mutants (Brunk et al.2008; Brunk et al. 2010).

Interaction of Rap1GAP and Go2aQ205LLike Girdin, Rap1GAP interacts with both Go2aQ205L andGo1aQ205L. Previous analyses described an interaction ofRap1GAP with wild-type Go1a, but not with the constitu-tively active Q205L mutant (Jordan et al. 1999) as we haveshown. Still, this finding supports the observation from ouryeast two-hybrid screen. Furthermore, the yeast two-hybrida-Gal-assay proves a preferential binding of Rap1GAP toGo2aQ205L over Go1aQ205L. Rap1GAP has also beenreported to bind to constitutively active Gza (Meng et al.1999).Rap1GAP is a GTPase activating protein for the small

GTPase Rap1 and is most abundant in fetal tissues, but is alsofound in adult brain (Rubinfeld et al. 1991). There are twohuman isoforms of Rap1GAP, one of which possesses an N-terminal extension, which reveals a GoLoco domain known tobind Ga subunits. The mouse strain we used, expresses onlyone isoform containing a GoLoco domain. The expressionlevel of Rap1GAP in different brain fractions was similar inboth genotypes except for the LP1 fraction, where Rap1GAPlevels were decreased in Go2a�/� compared with wild-typemice possibly because of changes in sorting.The Rap1GAP effector Rap1 is involved in a variety of

cellular functions, including cell proliferation and differen-tiation via regulation of ERK signaling as well as cell–cell

adhesion and migration via integrin mediated pathways(Hattori and Minato 2003). Within the nervous system,regulation of Rap1 activity by Rap1GAP contributes toneurite outgrowth, dendritic development, and dendriticspine plasticity (Chen et al. 2005; Jordan et al. 2005; Xieet al. 2005; McAvoy et al. 2009). Cannabinoid receptor-induced, Rap1-mediated neurite outgrowth has been linkedto Go1a (Jordan et al. 2005) with the wild-type Go1ainteracting predominantly with human Rap1GAP containingthe GoLoco motif. Go1a seems to target Rap1GAP fordegradation by the ubiquitin-proteasome system, therebyincreasing Rap1 activity (Jordan et al. 2005). Another wayby which Rap1GAP and as a consequence Rap1 can beregulated is phosphorylation (McAvoy et al. 2009) leadingto inhibition of GAP activity and, thus, to an increased Rap1activity. In brains of Go2a�/� mice the ratio of active Rap1-GTP to total Rap1 was increased when compared with wild-type littermates. Given that active Go2a binds Rap1GAP, itmay be speculated (contrary to the interaction of wild-typeGo1a and Rap1GAP) that activated Go2a increasesRap1GAP activity. Whether this interaction with activeGo2a leads to a decrease of phosphorylation or interfereswith the Go1a mediated targeting to degradation remainsunknown; however, both mechanisms lead to a decrease ofRap1 activity. Consequently, increased levels of active GTP-bound Rap1 in Go2a deletion mutants may be caused by animpairment of Rap1GAP activity. Thus, depending on theirstate of activity Go1a and Go2a may differentially controlRap1 activity and thereby influence Rap1 actions in neuronaldevelopment. Indeed axonal growth and branching ispromoted in Go2a deletion mutants potentially through anincrease of activated Rap1. An increase in Go1a levels inGo2a deletion mutants as explanation for higher amounts ofRap1GTP could be excluded, as previous quantification ofGo1a expression in these mice showed no difference fromwild type (Brunk et al. 2008).Taken together, our data show that Go2a specifically

regulates axonal outgrowth and branching. Interaction withRap1GAP and a higher ratio of Rap1GTP/Rap1 in the Go2adeletion mutants indicate that Go2a may decrease Rap1activity. Considering that Go1a, in its resting state, has beenshown to activate Rap1 by promoting degradation ofRap1GAP the two splice variants of Goa may differentiallyregulate Rap1 activity and neurite outgrowth.

Acknowledgement

The authors are indebted to Birgit Metze, Marion M€obes, and AntjeDr€ager for skillfull technical assistance, to Sam Booker for linguisticcorrections and to the Deutsche Forschungsgemeinschaft for finan-cial support (DFG Ah67/3-3). Part of this research was supported bythe Intramural Research Program of the NIH (Project Z01-ES-101643 to LB). None of the authors has to declare any conflict ofinterest.


792 J. Baron et al.

References

Ahnert-Hilger G., Schafer T., Spicher K., Grund C., Schultz G. andWiedenmann B. (1994) Detection of G-protein heterotrimers onlarge dense core and small synaptic vesicles of neuroendocrine andneuronal cells. Eur. J. Cell Biol. 65, 26–38.

Ahnert-Hilger G., Nurnberg B., Exner T., Schafer T. and Jahn R. (1998)The heterotrimeric G-protein Go2 regulates catecholamine uptakeby secretory vesicles. EMBO J. 17, 406–413.

Ahnert-Hilger G., Holtje M., Grosse G., Pickert G., Mucke C., Nixdorf-Bergweiler B., Boquet P., Hofmann F. and Just I. (2004)Differential effects of Rho GTPases on axonal and dendriticdevelopment in hippocampal neurones. J. Neurochem. 90, 9–18.

Aittaleb M., Boguth C. A. and Tesmer J. J. (2011) Structure and functionof heterotrimeric G protein-regulated Rho guanine nucleotideexchange factors. Mol. Pharmacol. 77, 111–125.

Anai M., Shojima N., Katagiri H. et al. (2005) A novel protein kinase B(PKB)/AKT-binding protein enhances PKB kinase activity andregulates DNA synthesis. J. Biol. Chem. 280, 18525–18535.

Becher A., Drenckhahn A., Pahner I. and Ahnert-Hilger G. (1999) Thesynaptophysin-synaptobrevin complex is developmentallyupregulated in cultivated neurons but is absent in neuroendocrinecells. Eur. J. Cell Biol. 78, 650–656.

Birnbaumer L. (2007) Expansion of signal transduction by G proteins.The second 15 years or so: from 3 to 16 alpha subunits plusbetagamma dimers. Biochim. Biophys. Acta 1768, 772–793.

Brunk I., Blex C., Sanchis-Segura C. et al. (2008) Deletion of Go2alphaabolishes cocaine-induced behavioral sensitization by disturbingthe striatal dopamine system. FASEB J. 22, 3736–3746.

Brunk I., Sanchis-Segura C., Blex C., Perreau-Lenz S., Bilbao A.,Spanagel R. and Ahnert-Hilger G. (2010) Amphetamine regulatesNR2B expression in Go2a knockout mice and thereby sustainsbehavioral sensitization. J. Neurochem. 115, 234–246.

Chen L. T., Gilman A. G. and Kozasa T. (1999) A candidate target for Gprotein action in brain. J. Biol. Chem. 274, 26931–26938.

Chen Y., Wang P. Y. and Ghosh A. (2005) Regulation of corticaldendrite development by Rap1 signaling. Mol. Cell. Neurosci. 28,215–228.

Dhingra A., Jiang M., Wang T. L., Lyubarsky A., Savchenko A., Bar-Yehuda T., Sterling P., Birnbaumer L. and Vardi N. (2002) Lightresponse of retinal ON bipolar cells requires a specific splicevariant of Galpha(o). J. Neurosci. 22, 4878–4884.

Enomoto A., Murakami H., Asai N., Morone N., Watanabe T., KawaiK., Murakumo Y., Usukura J., Kaibuchi K. and Takahashi M.(2005) Akt/PKB regulates actin organization and cell motility viaGirdin/APE. Dev. Cell 9, 389–402.

Enomoto A., Asai N., Namba T. et al. (2009) Roles of disrupted-in-schizophrenia 1-interacting protein girdin in postnatal developmentof the dentate gyrus. Neuron 63, 774–787.

Garcia-Marcos M., Ghosh P. and Farquhar M. G. (2009) GIV is anonreceptor GEF for G alpha i with a unique motif that regulatesAkt signaling. Proc. Natl Acad. Sci. USA 106, 3178–3183.

Garcia-Marcos M., Ghosh P., Ear J. and Farquhar M. G. (2010) Astructural determinant that renders G{alpha}i sensitive to activationby GIV/Girdin is required to promote cell migration. J. Biol. Chem.285, 12765–12777.

Ghosh P., Garcia-Marcos M., Bornheimer S. J. and Farquhar M. G.(2008) Activation of Galphai3 triggers cell migration via regulationof GIV. J. Cell Biol. 182, 381–393.

Hattori M. and Minato N. (2003) Rap1 GTPase: functions, regulation,and malignancy. J. Biochem. 134, 479–484.

He J. C., Neves S. R., Jordan J. D. and Iyengar R. (2006) Role of theGo/i signaling network in the regulation of neurite outgrowth. Can.J. Physiol. Pharmacol. 84, 687–694.

Holtje M., von Jagow B., Pahner I., Lautenschlager M., Hortnagl H.,Nurnberg B., Jahn R. and Ahnert-Hilger G. (2000) The neuronalmonoamine transporter VMAT2 is regulated by the trimericGTPase Go(2). J. Neurosci. 20, 2131–2141.

Hsu W. H., Rudolph U., Sanford J., Bertrand P., Olate J., Nelson C.,Moss L. G., Boyd A. E., Codina J. and Birnbaumer L. (1990)Molecular cloning of a novel splice variant of the alpha subunit ofthe mammalian Go protein. J. Biol. Chem. 265, 11220–11226.

Huttner W. B., Schiebler W., Greengard P. and De Camilli P. (1983)Synapsin I (protein I), a nerve terminal-specificphosphoprotein. III. Its association with synaptic vesiclesstudied in a highly purified synaptic vesicle preparation.J. Cell Biol. 96, 1374–1388.

Jahn R., Schiebler W., Ouimet C. and Greengard P. (1985) A 38,000-dalton membrane protein (p38) present in synaptic vesicles. Proc.Natl Acad. Sci. USA 82, 4137–4141.

Jiang M., Gold M. S., Boulay G., Spicher K., Peyton M., Brabet P.,Srinivasan Y., Rudolph U., Ellison G. and Birnbaumer L. (1998)Multiple neurological abnormalities in mice deficient in the Gprotein Go. Proc. Natl Acad. Sci. USA 95, 3269–3274.

Jiang P., Enomoto A., Jijiwa M., Kato T., Hasegawa T., Ishida M., SatoT., Asai N., Murakumo Y. and Takahashi M. (2008) An actin-binding protein Girdin regulates the motility of breast cancer cells.Cancer Res. 68, 1310–1318.

Jordan J. D., Carey K. D., Stork P. J. and Iyengar R. (1999) Modulationof rap activity by direct interaction of Galpha(o) with Rap1GTPase-activating protein. J. Biol. Chem. 274, 21507–21510.

Jordan J. D., He J. C., Eungdamrong N. J., Gomes I., Ali W., Nguyen T.,Bivona T. G., Philips M. R., Devi L. A. and Iyengar R. (2005)Cannabinoid receptor-induced neurite outgrowth is mediated byRap1 activation through G(alpha)o/i-triggered proteasomaldegradation of Rap1GAPII. J. Biol. Chem. 280, 11413–11421.

Kerov V. S., Natochin M. and Artemyev N. O. (2005) Interaction oftransducin-alpha with LGN, a G-protein modulator expressed inphotoreceptor cells. Mol. Cell. Neurosci. 28, 485–495.

Kim S. H., Kim S. and Ghil S. H. (2008) Rit contributes to neuriteoutgrowth triggered by the alpha subunit of Go. NeuroReport 19,521–525.

Kitamura T., Asai N., Enomoto A. et al. (2008) Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin. Nat. CellBiol. 10, 329–337.

Kobayashi I., Shibasaki H., Takahashi K., Tohyama K., Kurachi Y., ItoH., Ui M. and Katada T. (1990) Purification and characterization offive different alpha subunits of guanine-nucleotide-binding proteinsin bovine brain membranes. Their physiological propertiesconcerning the activities of adenylate cyclase and atrialmuscarinic K+ channels. Eur. J. Biochem. 191, 499–506.

Kurochkin I. V., Yonemitsu N., Funahashi S. I. and Nomura H. (2001)ALEX1, a novel human armadillo repeat protein that is expresseddifferentially in normal tissues and carcinomas. Biochem. Biophys.Res. Commun. 280, 340–347.

Le-Niculescu H., Niesman I., Fischer T., DeVries L. and Farquhar M. G.(2005) Identification and characterization of GIV, a novel Galphai/s-interacting protein found on COPI, endoplasmic reticulum-Golgi transport vesicles. J. Biol. Chem. 280, 22012–22020.

Luo Y. and Denker B. M. (1999) Interaction of heterotrimeric G proteinGalphao with Purkinje cell protein-2. Evidence for a novelnucleotide exchange factor. J. Biol. Chem. 274, 10685–10688.

Man-Son-Hing H. J., Codina J., Abramowitz J. and Haydon P. G. (1992)Microinjection of the alpha-subunit of the G protein Go2, but notGo1, reduces a voltage-sensitive calcium current. Cell. Signal. 4,429–441.

McAvoy T., Zhou M. M., Greengard P. and Nairn A. C. (2009)Phosphorylation of Rap1GAP, a striatally enriched protein, by



protein kinase A controls Rap1 activity and dendritic spinemorphology. Proc. Natl Acad. Sci. USA 106, 3531–3536.

Meng J., Glick J. L., Polakis P. and Casey P. J. (1999) Functionalinteraction between Galpha(z) and Rap1GAP suggests a novelform of cellular cross-talk. J. Biol. Chem. 274, 36663–36669.

Nakata H. and Kozasa T. (2005) Functional characterization of Galphaosignaling through G protein-regulated inducer of neurite outgrowth1. Mol. Pharmacol. 67, 695–702.

Neer E. J., Lok J. M. and Wolf L. G. (1984) Purification and propertiesof the inhibitory guanine nucleotide regulatory unit of brainadenylate cyclase. J. Biol. Chem. 259, 14222–14229.

Pahner I., Holtje M., Winter S., Takamori S., Bellocchio E. E.,Spicher K., Laake P., Nurnberg B., Ottersen O. P. and Ahnert-Hilger G. (2003) Functional G-protein heterotrimers areassociated with vesicles of putative glutamatergic terminals:implications for regulation of transmitter uptake. Mol. Cell.Neurosci. 23, 398–413.

Rubinfeld B., Munemitsu S., Clark R., Conroy L., Watt K., Crosier W.J., McCormick F. and Polakis P. (1991) Molecular cloning of aGTPase activating protein specific for the Krev-1 protein p21rap1.Cell 65, 1033–1042.

Simon M. I., Strathmann M. P. and Gautam N. (1991) Diversity of Gproteins in signal transduction. Science 252, 802–808.

Sternweis P. C. and Robishaw J. D. (1984) Isolation of two proteins withhigh affinity for guanine nucleotides from membranes of bovinebrain. J. Biol. Chem. 259, 13806–13813.

Takamori S., Holt M., Stenius K. et al. (2006) Molecular anatomy of atrafficking organelle. Cell 127, 831–846.

Tall G. G., Krumins A. M. and Gilman A. G. (2003) Mammalian Ric-8A(synembryn) is a heterotrimeric Galpha protein guanine nucleotideexchange factor. J. Biol. Chem. 278, 8356–8362.

Traver S., Bidot C., Spassky N., Baltauss T., De Tand M. F., Thomas J.L., Zalc B., Janoueix-Lerosey I. and Gunzburg J. D. (2000) RGS14is a novel Rap effector that preferentially regulates the GTPaseactivity of galphao. Biochem. J. 350(Pt 1), 19–29.

Wang Y., Park S., Bajpayee N. S., Nagaoka Y., Boulay G., BirnbaumerL. and Jiang M. (2010) Augmented glucose-induced insulin releasein mice lacking G(o2), but not G(o1) or G(i) proteins. Proc. NatlAcad. Sci. USA 108, 1693–1698.

Wei X. Y., Perez-Reyes E., Lacerda A. E., Schuster G., Brown A. M.and Birnbaumer L. (1991) Heterologous regulation of the cardiacCa2 + channel alpha 1 subunit by skeletal muscle beta and gammasubunits. Implications for the structure of cardiac L-type Ca2+channels. J. Biol. Chem. 266, 21943–21947.

Winter S., Brunk I., Walther D. J., Holtje M., Jiang M., Peter J. U.,Takamori S., Jahn R., Birnbaumer L. and Ahnert-Hilger G.(2005) Galphao2 regulates vesicular glutamate transporteractivity by changing its chloride dependence. J. Neurosci. 25,4672–4680.

Xie Z., Huganir R. L. and Penzes P. (2005) Activity-dependent dendriticspine structural plasticity is regulated by small GTPase Rap1 andits target AF-6. Neuron 48, 605–618.


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The α-subunit of the trimeric GTPase Go2 regulates axonal growth

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