RESEARCH ARTICLE

GABAB receptor promotes its own surface expression by recruitinga Rap1-dependent signaling cascadeZongyong Zhang1,*, Wenhua Zhang1,*, Siluo Huang1,*,‡, Qian Sun1, YunyunWang1, Yongjian Hu1, Ninghua Sun1,Yilei Zhang1, Zhihua Jiang1, Nagahiro Minato2, Jean-Philippe Pin3, Li Su1,‡ and Jianfeng Liu1,‡

ABSTRACTG-protein-coupled receptors (GPCRs) are key players in cellsignaling, and their cell surface expression is tightly regulated. Formany GPCRs such as β2-AR (β2-adrenergic receptor), receptoractivation leads to downregulation of receptor surface expression, aphenomenon that has been extensively characterized. By contrast,some other GPCRs, such as GABAB receptor, remain relativelystable at the cell surface even after prolonged agonist treatment;however, the underlying mechanisms are unclear. Here, we identifythe small GTPase Rap1 as a key regulator for promoting GABAB

receptor surface expression. Agonist stimulation of GABAB receptorsignals through Gαi/o to inhibit Rap1GAPII (also known asRap1GAP1b, an isoform of Rap1GAP1), thereby activating Rap1(which has two isoforms, Rap1a and Rap1b) in cultured cerebellargranule neurons (CGNs). The active form of Rap1 is then recruited toGABAB receptor through physical interactions in CGNs. This Rap1-dependent signaling cascade promotes GABAB receptor surfaceexpression by stimulating receptor recycling. Our results uncover anew mechanism regulating GPCR surface expression and alsoprovide a potential explanation for the slow, long-lasting inhibitoryaction of GABA neurotransmitter.

KEY WORDS: GABAB receptor, Rap1, Protein–protein interaction,Internalization, Recycling

INTRODUCTIONFor many GPCRs, signal transduction is controlled by theiragonist-induced desensitization and subsequent internalization toprotect cells against receptor over-stimulation (Moore et al.,2007). The best characterized such example is β2-adrenergicreceptor (β2-AR). Upon agonist stimulation, GPCRs first undergophosphorylation catalyzed by G-protein receptor kinases (GRKs)or other kinases. Subsequently, β-arrestin is recruited to thereceptor to preclude receptor–G-protein interaction, leading tointernalization of the receptor from the plasma membrane(Gainetdinov et al., 2004; Luttrell and Lefkowitz, 2002). Onceinternalized, receptors are dephosphorylated and recycled back tothe plasma membrane or are targeted to lysosomes for degradation(Marchese et al., 2008).

By contrast, some other GPCRs such as GABAB receptormaintain a rather stable expression level at the cell surface even afterprolonged agonist stimulation (Couve et al., 2002; Fairfax et al., 2004;Kuramoto et al., 2007).Other examples includeMrgX1, somatostatin-4,β3-AR, andκ-opioid receptors (Chu et al., 1997;Nantel et al., 1993;Rosenfeld et al., 2002; Schreff et al., 2000). GABAB receptor belongsto the class C type of GPCRs and is the metabotropic receptor for γ-aminobutyric acid (GABA), the primary inhibitory neurotransmitterin the mammalian nervous system (Bettler et al., 1998). GABAB

receptor functions as a heterodimer composed of two subunits GB1and GB2 (also known as GABABR1 and GABABR2) (Jones et al.,1998; Kaupmann et al., 1998; White et al., 1998). Each subunitcontains an extracellular domain, a seven transmembrane domain, andan intracellular C terminal. Only the GB1 extracellular domain bindsagonists, whereas the seven transmembrane domain of GB2 isresponsible for the activation of Gi/o proteins (Galvez et al., 2001;Kniazeff et al., 2002). It is a common therapeutic target for a widerange of neurological disorders, including epilepsy, schizophrenia,addiction, depression, anxiety and chronic pain (Bettler et al., 2004;Gassmann et al., 2004; Thuault et al., 2004). Unlike GABAA andGABAC receptors, which are fast-acting ion channels, as a GPCRGABAB receptor is best known to mediate the slow, prolongedinhibitory effect of GABAneurotransmitter (Couve et al., 2000). Thisfeature demands persistent expression of functional GABAB receptorat the cell surface. Thus, the unique cell surface stability of GABAB

receptor is well suited for its role in mediating long-term synapticinhibition in the central nervous system (CNS).

However, the mechanism underlying the surface stability ofGABAB receptor upon prolonged agonist stimulation is unknownand somewhat controversial. Although GABAB receptor is knownto undergo basal endocytosis, it remains controversial whetheragonist stimulation facilitates receptor internalization (Fairfax et al.,2004; Grampp et al., 2008, 2007). Thus, some studies attribute thecell surface stability of GABAB receptor to a lack of agonist-inducedinternalization (Fairfax et al., 2004; Perroy et al., 2003), whereasothers suggest that this might be due to re-insertion of the receptorinto the plasma membrane by an unknown mechanism (Gramppet al., 2008, 2007; Wilkins et al., 2008).

Here, we sought to investigate the mechanism by which GABAB

receptor maintains its surface expression level under prolongedagonist stimulation. Through biochemical purification, weidentified the small GTPase Rap1 (which has two isoforms,Rap1a and Rap1b) as a new GABAB-receptor-binding partner.Activation of GABAB receptor initiates a signaling cascade,including Gi/o, Rap1GAPII (also known as Rap1GAP1b, anisoform of Rap1GAP1) and Rap1 that binds to the receptor topromote its surface expression by facilitating receptor recycling.Our results identify a newmechanism that promotes GPCR surfaceexpression, providing insights into the slow and long-lasting actionof the neurotransmitter GABA.Received 11 December 2014; Accepted 5 May 2015

1Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministryof Education, School of Life Science and Technology and the CollaborativeInnovation Center for Brain Science, Huazhong University of Science andTechnology, Wuhan 430074, China. 2Department of Immunology and Cell Biology,Kyoto University, Kyoto 606-8501, Japan. 3Institut de Genomique Fonctionnelle,CNRS, UMR 5203, Universite Montpellier 1 et 2, Montpellier cedex 5 34094, France.*These authors contributed equally to this work

‡Authors for correspondence (slhuang@mail.hust.edu.cn, lisu@mail.hust.edu.cn,jfliu@mail.hust.edu.cn)

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RESULTSIdentification of Rap1 as an interacting partner of GABABreceptorThe C-terminal region of GPCRs usually has many binding sites forsoluble and scaffold proteins that are involved in various regulatoryprocesses such as desensitization, internalization and recycling(Bockaert et al., 2004). As no proteins known to bind to theC-terminus of the GB1 or GB2 subunits appear to regulate the cellsurface stability of GABAB receptor (Benzing et al., 2000; Brocket al., 2005; Guetg et al., 2010; Kuramoto et al., 2007; Nehringet al., 2000; Schwenk et al., 2010; Tan et al., 2004), we set out toidentify new GABAB-receptor-interacting proteins. To this end, wechose to work with cerebellar granule neurons (CGNs) that expressendogenous GABAB receptor, as the function of GABAB receptorhas been well characterized by us and many others in these neurons(Lin et al., 2012; Tu et al., 2007, 2010). We stimulated CGNs withthe prototypical GABAB receptor agonist baclofen, and then usedglutathione-S-transferase (GST) fusion proteins containing theC-terminal part of GB1 or GB2 (GST–GB1-C or GST–GB2-C)(Couve et al., 2004; Fairfax et al., 2004; Kuramoto et al., 2007;Nehring et al., 2000; Perroy et al., 2003) as a bait to pull downpotential interacting proteins from the cell lysate (Fig. 1A). On theSDS-PAGE gel visualized by silver staining, several protein bandsappeared to be pulled down by GST–GB1-C, which werecharacterized further by trypsin digestion followed by massspectrometry (Fig. 1B; supplementary material Table S1). Using

this strategy, we identified the small GTPase Rap1 as a bindingpartner of GB1.

To verify this interaction, we probed the pulldown fraction withan anti-Rap1 antibody and found that it indeed interacted with theGB1 C-terminus but not with GST alone (Fig. 1C). By contrast, thesame region in GB2 failed to pull down Rap1, suggesting that Rap1specifically interacts with GB1 (Fig. 1D). A similar phenomenonwas observed in HEK293 cells transfected with GABAB receptorupon treatment with baclofen, a specific agonist for GABAB

receptor (Fig. 1E). Interestingly, no such interaction was detected inthe absence of baclofen treatment, indicating that Rap1 wasrecruited to GABAB receptor upon receptor activation. Theseresults demonstrate that Rap1 was recruited to GABAB receptorupon receptor activation by binding to the C-terminal end of theGB1 but not the GB2 subunit of the receptor.

GABAB receptor binds to Rap1-GTP, the active form of Rap1As a molecular switch, Rap1 exists in at least two different forms:the active form Rap1-GTP and the inactive form Rap1-GDP. Toascertain whether GABAB receptor binds to Rap1-GTP or Rap1-GDP, we tested the constitutive active mutant of Rap1 (Rap1V12)which is GTP-bound, as well as its dominant negative mutantof Rap1 (Rap1N17) which is GDP-bound. We found that GB1C-terminus can interact with Rap1V12 but not Rap1N17 (Fig. 1F),suggesting that GABAB receptor interacts with Rap1-GTP but notRap1-GDP.

Fig. 1. Identification of Rap1 as an interacting partner of GABAB receptor. (A) Primary cultured mouse CGNs were treated with or without baclofen (100 µM,10 min) and lysed in immunoprecipitation buffer. Cell lysates were incubated for 60 min with glutathione-bead-boundGST–GB1-C (amino acids 854–961) protein.Interacting proteins were captured and detected by SDS-PAGE and visualized by silver staining. (B) Protein bands at 20–24 kDa were excised from SDS-PAGEgel and digested with trypsin, and peptides were separated and analyzed by MDLC-dependent tandem mass spectrometry. (C) GST–GB1-C or GST alone wasincubated with baclofen-treated (100 µM, 10 min) or untreated CGN lysates. Protein was captured, eluted and detected by immunoblotting with anti-Rap1antibodies. (D) GST–GB1-C, GST–GB2-C, GST or GST–RBD was incubated with baclofen-treated (100 µM, 10 min) mouse CGN cell lysates. (E) HEK293 cellswere transiently transfected with GB1 and GB2 subunits or empty vector. After baclofen treatment (100 µM, 10 min), cell lysates were incubated for 60 min withglutathione-bead-bound GST–GB1-C or GST only. Bound protein was detected by western blotting with anti-Rap1 antibodies. (F) HEK293 cells was transientlytransfected with a constitutively active form of Rap1A (pSRa-SK-Rap1V12) or dominant-negative form of Rap1A (pSRa-SK-Rap1N17). Captured proteins wereseparated by SDS-PAGE and detected with anti-Rap1 antibodies by western blotting. All data are representative of at least three independent experiments.

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Agonist stimulation of GABAB receptor induces persistentactivation of Rap1The observations that GB1 interacts with Rap1 in a baclofen-dependent manner and that it only interacts with the active form ofRap1 suggest that agonist stimulation of GABAB receptor activatesRap1. To test this, we performed an RBD assay to detect the activeform of Rap1 (Rap1-GTP). Briefly, the GST–RalGDS-RBD fusionprotein is purified and used to bind the activated GTP-boundRap1, but not inactivated GDP-bound, Rap1 (Tsukamoto et al.,1999), which can then be pulled down with glutathione resin.Rap1 activation levels are then determined by western blottingusing an anti-Rap1 antibody. Indeed, baclofen induced a persistentactivation of Rap1 in a time-dependent manner in both CGNs(Fig. 2A; supplementary material Fig. S1B) and HEK293 cellstransfected with GABAB receptor (Fig. 2B; supplementary materialFig. S1C). A similar phenomenon was observed upon treatment

with CGP7930, a specific positive allosteric modulator of GABAB

receptor, which activates GABAB receptor by binding to the HDdomain of GB2 subunit (Binet et al., 2004) (supplementary materialFig. S1A,B). Furthermore, CGP54626, a specific antagonist ofGABAB receptor, could block baclofen-induced Rap1 activation(Fig. 2C). Taken together, these results demonstrate that stimulationof GABAB receptor induces persistent activation of Rap1.

GABAB receptor signals to Rap1 through Gαi/o and RapGAP2How does GABAB receptor transmit signals to Rap1? GABAB

receptor is known to activate its downstream effectors through theGi/o type of heterotrimeric G proteins (Mannoury la Cour et al.,2008). Activation of GABAB receptor signals through Gαi/o andGβγ subunits. We first tested whether Gi/o-type G proteins wereinvolved in GABAB-receptor-induced Rap1 activation by usingpertussis toxin (PTX). We found that PTX inhibited baclofen-

Fig. 2. GABAB receptor activates Rap1 through Gαi/o and Rap1GAPII. (A) Effect of baclofen on Rap1 activation in CGNs. CGNs were treated with baclofen(100 µM) for different times as indicated. The activated form of GTP-boundRap1was detected with an RBD pulldown assay (top panel). The total amount of Rap1in cell lysate was probed by Rap1 antibodies (bottom panel). (B) Baclofen stimulation of GABAB receptor activates Rap1 in heterologous cells. HEK293 cellstransfected with GABAB receptor were treated with baclofen (100 µM) for different times as indicated. Rap1GTP was detected with an RBD pulldown assay (toppanel). The total amount of Rap1 in the cell lysate was probed with Rap1 antibodies (bottom panel). (C) Effect of CGP54626 on baclofen-induced (100 µM,10 min) Rap1 activation in CGNs. CGNs were pretreated with CGP54626 (10 µM, 20 min). (D) Effect of PTX on baclofen-induced (100 µM, 10 min) Rap1activation in CGNs. CGNs were pretreated with PTX (200 ng/ml, 16 h). (E) Role of Gβγ subunits on baclofen-induced (100 µM, 10 min) Rap1 activation. HEK293cells were co-transfected with GABAB receptor and empty vector (pRK6) or CD8–βARK. ERK1/2 serves as a positive control, as it is known to be stimulated byGABAB receptor signaling, an effect mediated by Gβγ subunits. (F) Effect of baclofen (100 µM) on the phosphorylation of Rap1GAPII. HEK293 cells were co-transfected with GABAB receptor and GST–Rap1GAPII, then treated with baclofen for different times as indicated. Cell lysates were precipitated with glutathionebeads. Serine phosphorylation of precipitated GST–Rap1GAPII was detected by an antibody against phosphorylated serine (pSer). The levels of totalRap1GAPII, phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2 were also probed by western blotting. (G) Effect of Rap1GAPII on baclofen-induced (100 µM,10 min) Rap1 activation. HEK293 cells were co-transfected with both GABAB receptor and wild-type Rap1GAPII or control (an inactive form of Rap1GAPII). Alldata are representative of at least three independent experiments.

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induced Rap1-GTP formation in CGNs (Fig. 2D). Furthermore, byusing the previously characterized Gβγ-scavenger, consisting of theC-terminal region of GRK2 (βARK) fused to the extracellular andtransmembrane domains of CD8, which then provides a membraneanchor for C-terminal tail of βARK (CD8–βARK), which is knownto block Gβγ-mediated signaling such as GABAB-receptor-inducedphosphorylation of ERK1/2 (also known as MAPK3 and MAPK1,respectively) (Tu et al., 2007), failed to inhibit Rap1 activationinduced by baclofen, whereas it did inhibit baclofen-induced ERK1/2 phosphorylation in HEK293 cells transfected with GABAB

receptor (Fig. 2E). These results indicate that GABAB receptorinduces the formation of Rap1-GTP by signaling through the Gαi/osubunit.We then asked how GABAB receptor induces Rap1 activation

through Gαi/o. Rap proteins are active when bound to GTP andinactive when bound to GDP. Rap activation is mediated by specificguanine nucleotide exchange factors (GEFs),whereas inactivation ofRap1-GTP can be achieved with the aid of specific GTPase-activating proteins (GAPs). Notably, the Gαi/o subunit is known toinhibit the activity of Rap1GAPII, a GAP for Rap1 (Jordan et al.,1999; Mochizuki et al., 1999). Phosphorylation of Rap1GAPIIcontrols Rap1 activity (McAvoy et al., 2009).We therefore tested theeffect of baclofen activation of GABAB-receptor-mediated Gαi/osignaling on Rap1GAPII phosphorylation. We found that baclofentreatment rapidly and persistently potentiated Rap1GAPIIphosphorylation, indicating that activation of GABAB-receptor–Gαi/o signaling inhibits Rap1GAPII in HEK293 cells transfectedwith GABAB receptor and GST–Rap1GAPII (Fig. 2F).To provide additional evidence, we reasoned that if inhibition

of Rap1GAPII sustains Rap1 activity, then overexpression ofRap1GAPII should reduce Rap1 activation induced by stimulationof GABAB receptor. Consistent with this prediction, overexpressionof wild-type Rap1GAPII in GABAB-receptor-expressing HEK293cells blocked the formation of Rap1–GFP induced by baclofen. As acontrol, we showed that an inactive form of Rap1GAPII failed toelicit such an effect (Fig. 2G). As Rap1GAPII is a GAP for Rap1,these results suggest a model in which GABAB receptor activatesRap1 by signaling through the Gαi/o subunit and Rap1GAPII.

Mapping of specific sites required for mediating theinteraction between Rap1-GTP and GABAB receptorTo gain a better insight into how Rap1 interacts with GB1, wesought to map the sites that are required for the interaction. As a firststep, we examined Rap1. Previous reports show that severalnegatively charged residues [aspartate (D) and glutamate (E)] inRap1 mediate its interaction with the RBD domain of RalGDS, aknown Rap1-binding protein (Fig. 3A) (Liu et al., 2011). As such,we mutated these negatively charged residues in Rap1 to alanine(i.e. D33A, E37A and D38A) to test whether they are important formediating the interaction between Rap1 and GB1 C-terminus(supplementary material Table S2). D33A and E37A pointmutations in Rap1 disrupted its interaction with the C-terminalend of GB1. A D38A point mutation in Rap1 also reduced thisinteraction. As a control, Rap1N17, a GDP-bound form of Rap1,failed to bind to GB1 (Fig. 3B). The identification of D33, E37 andD38 in Rap1 as three crucial residues required for Rap1 and GB1interaction provides further evidence that the observed interaction isspecific.We then sought to map the sites in GB1 C-terminus that are

required for mediating its interaction with Rap1. To do so, we firstaligned the sequences between GB-1 C-terminus and the RBDregion of RalGDS, a domain known to interact with Rap1-GTP.

We noted that a small region spanning from R947 to K960 in GB1C-terminus is analogous to the key residues in the GalGDS-RBDdomain that have been found to be important in mediating theinteraction with Rap1-GTP in RalGDS (Nassar et al., 1995). Wethen aligned the sequences of this region in GB1 and GB2 fromseveral organisms, and found that the residues 953–960(SRVHLLYK) in GB1 are conserved in most organisms rangingfrom X. tropicalis to Homo sapiens but show no similarity withGB2-C terminus in this regions[residues 934–940 (FRVMVSGL)](Fig. 3C). To test whether this short motif in GB1 is crucial formediating the interaction, we made a series of deletions and pointmutations in this motif and examined their effects (supplementarymaterial Table S2; Fig. S1D). Deletion of S953 or S959 abolishedthe ability of GB1 C-terminus to interact with Rap1-GTP in CGNs(Fig. 3D). Two point mutations R954A and K960A also disruptedthe interaction (Fig. 3D). Thus, the residues SRVHLLYK in GB1appear to play an important role in mediating the interactionbetween GABAB receptor and Rap1.

To provide further evidence, we designed and synthesized peptides(supplementary material Table S3), aiming to directly impair theinteraction between Rap1-GTP and the GB1 C-terminus (Fig. 3E).These peptides are membrane permeable, making it possible to usethem to disrupt Rap1-GB1 interaction in situ (supplementary materialFig. S2A). As expected, Pep (DGSRVHLLYK) and Pep-S7(RVHLLYK) inhibited the interaction between GB1 C-terminus andRap1-GTPboth inCGNs (Fig. 3F) andHEK293 cells (supplementarymaterial Fig. S2B). As negative controls, two unrelated peptides Pep-GB2 (FRVMVSGL) and PepN (PPDRLSCDGS) were unable toblock the interaction (Fig. 3F; supplementary material Fig. S2B).Furthermore, two other peptides Pep-K960A (DGSRVHLLYA) andPep-R954A (DGSAVHLLYK), which encompass point mutations,also failed to do so. This provides additional controls supporting thespecificity of Pep andPep-S7 in blocking the interaction betweenGB1C-terminus and Rap1-GTP (Fig. 3F; supplementary materialFig. S2B). These analyses identify specific sites in Rap1 and GB1C-terminus that are important for mediating the interaction betweenRap1-GTP and GABAB receptor, offering further evidence that theobserved interaction is specific.

Taken together, our data suggest a model in which agoniststimulation of GABAB receptor activates Rap1 by signaling throughGαi/o and Rap1GAPII, leading to recruitment of the active form ofRap1 to the receptor complex through a direct interaction betweenRap1 and the GB1 subunit of the receptor.

Rap1–GB1 interaction is important for the cell surfaceexpression of GABAB receptorHaving demonstrated that agonist stimulation of GABAB receptoractivates and recruits Rap1 to the receptor through a direct protein–protein interaction, we then asked what functions such an interactionmediates. We therefore decided to disrupt the interaction betweenRap1-GTP and the GB1 subunit to examine whether and how itaffects the function ofGABAB receptor.ActivityofGABAB receptorwas quantified by a commonly used functional assay to measureinositol trisphosphate (IP3) production elicited by baclofen inHEK293 cells co-expressing GABAB receptor and the chimericG-protein Gαqi9 (the last nine C-terminal residues of Gαq proteinwere replaced by those from Gαi2), which facilitates the coupling ofGi-coupled receptors to the phospholipase C signalling pathway.Disruption of the Rap1–GB1 interaction by Pep treatmentgreatly suppressed the activity of GABAB receptor in transfectedHEK293 cells (Fig. 4A; supplementary material Fig. S3A).Interestingly, the EC50 value of baclofen was not affected by Pep

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treatment (Fig. 4A). This implies that disruption of Rap1–GB1interaction might not affect the functional properties of GABAB

receptor, but instead might compromise the overall amount offunctional receptors at the cell surface.We thus performed a series of assays to explore whether the

interaction between Rap1 and GB1 regulates the cell surfaceexpression of GABAB receptor. As a first step, we used an enzyme-linked immunosorbent assay (ELISA) to measure the amount ofGABAB receptor expressed at the cell surface. We took advantageof the peptides that can disrupt the Rap1–GB1 interaction (Fig. 3E,F; supplementarymaterial Fig. S2B). Treatment with Pep, but not itscontrol PepN, reduced the amount of baclofen-induced GABAB

receptor at the cell surface in a time- and concentration-dependentmanner (Fig. 4B; supplementary material Fig. S3B). To gatheradditional evidence, we performed a flow cytometry assay toquantify the surface expression level of GABAB receptor intransfected HEK293 cells, and observed a similar phenomenon(supplementary material Fig. S3C). In addition, disrupting theinteraction between GB1 and Rap1 (i.e. GB1ΔS953) greatlyreduced GABAB receptor surface expression in this assay

(supplementary material Fig. S3D). The results from both ELISAand flow cytometry assays demonstrate that agonist-inducedrecruitment of Rap1-GTP to the GABAB receptor complex isimportant for the cell surface expression of the receptor.

Agonist stimulation of GABAB receptor promotes receptorinternalizationTo take a closer view at how GABAB receptor behaves at the cellsurface, we performed a biotinylation assay to follow the fate of thereceptor expressed at the cell surface upon agonist stimulation. If theinteraction between Rap1 and GB1 is important for the surfaceexpression of GABAB receptor, as suggested by the ELISA and flowcytometry analyses, then disruption of such interaction might lead toan accumulation of GABAB receptor inside the cell. Indeed, Pepdisruption of the Rap1–GB1 interaction gave rise to a dramaticincrease in the amount of internalized GABAB receptor uponbaclofen stimulation (Fig. 4C).Another interesting observation is thatbaclofen stimulation alone in untreated CGNs did not increase theamount of internalized receptors. These two observations togetherpointed to an interesting model in which agonist stimulation of

Fig. 3. Direct protein–protein interactions betweenRap1 andGB1C-terminus. (A) Predication of the key residues in Rap1 for Rap1–GB1 interactions. Shownis a structure of Rap1A deduced from the complex of Rap1A and RafRBD (PDB accession number 3KUC). A secondary structure prediction and domainassignment in the SWISS-MODEL workspace predicts that the negative charged amino acids in Rap1 might interact with GB1 C-terminus. (B) Effect of mutationsof key residues in the predicted interaction region on Rap1–GB1 interaction. Single point mutations to alanine residues for D33, E37 and D38 were introduced intoRap1V12, a dominant-active form of Rap1. Plasmids were transfected into HEK293 cells. Rap1–GB1 interaction was evaluated by pulling downGST–GB-1-C fromcell lysates (top panel). The total amount of Rap1 protein was probedwith anti-Rap1 by western blotting (bottom panel). (C) Sequence alignment of amino acids inthe GB1 C-terminus and GB2 C-terminus (GB1-C and GB2-C, respectively) from various organisms. (D) Effect of deletions and point mutations in the GB1 C-terminus on the Rap1–GB1 interaction. GST fusions of GB1 C-terminus harboring deletions or point mutations were used to pulldown Rap1 from baclofen-treated(100 µM, 10 min) cell lysates of CGNs. Deletion mutants include GB1-C-ΔS953 and GB1-C-ΔY959, and point mutations include GB1-C-R954A, GB1-C-H956Aand GB1-C-K960A. (E) Peptides tested for impairment of the Rap1–GB1 interaction. PepN (scrambled) serves as a negative control. (F) Effect of peptides(100 µg/ml; 60 min) on Rap1–GB1 interaction. The assay was performed as described in D. All data are representative of at least three independent experiments.

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GABAB receptor potentiates receptor internalization; however, asRap1–GB1 interaction probably also promotes the recycling ofinternalized receptors, the net amount of the receptors at the cellsurface then stays unchanged (Fig. 4C). In other words, both theinternalization and the recycling of the receptor might be potentiatedin response to agonist stimulation. Thismodel explains the seeminglyconfounding observations that baclofen stimulation of GABAB

receptor did not affect the net amount of internalized receptors, yetblocking Rap1–GB1 interaction led to an accumulation ofinternalized receptors inside the cell.To provide further evidence supporting the above model, we

directly visualized the fate of GABAB receptor present at the cellsurface before and after agonist stimulation. To do so, wespecifically labeled the GABAB receptor present at the cell

surface with primary antibodies and then followed its fate byimmunofluorescence staining. Consistent with the results from ourbiotinylation assay, baclofen stimulation of GABAB receptor didnot cause a notable change in the net amount of the receptor at thecell surface (Fig. 4D). By contrast, disruption of Rap1–GB1interaction by deleting the residues 953–960 in GB1 (GB1ΔS953)led to a dramatic decrease in the amount of GABAB receptor at thecell surface (Fig. 4D). Notably, this effect was strictly baclofen-dependent, as in the quiescent state (i.e. no baclofen treatment),disruption of the Rap1–GB1 interaction did not cause a notableeffect on the amount of the receptor found at the cell surface(Fig. 4D). Thus, the interaction between Rap1 and GB1 appears toregulate GABAB receptor surface expression in an activity-dependent manner.

Fig. 4. Agonist-induced Rap1-GB1 interaction controls GABAB receptor surface expression. (A) Effect of peptides on GABAB receptor activation. HEK293cells were transiently transfected with GABAB receptor and Gαqi9 chimeric protein. Cells were pretreated with Pep or its control PepN (100 µg/ml; 60 min) prior tobaclofen stimulation (100 µM, 30 min). Baclofen-induced IP3 accumulation was quantified. The inset shows the normalization of IP3 production in Pep-treated andPepN-treated cells – the two curves overlap with a similar EC50. Data are mean±s.e.m. and representative of three independent experiments. (B) Effect ofpeptides on GABAB receptor cell surface expression. HEK293 cells overexpressing GABAB receptor were pretreated with Pep or its control Pep-N (100 µg/ml;60 min) prior to baclofen stimulation (100 µM, 10 min). GB1 cell surface expression levels were measured by ELISA. Data are mean±s.e.m. and arerepresentative of three independent experiments. (C) Disruption of the GB1–Rap1 interaction with Pep increases agonist-induced GABAB receptor internalizationin CGNs. After biotinylation of surface proteins at 4°C, CGNs were incubated with baclofen (100 µM, 120 min) at 37°C. After removal of cell surface biotin withglutathione, biotinylated internalized proteins were purified with streptavidin–Sepharose beads and the abundance of GABAB receptor was determined bywestern blotting with GB1 antibodies. The total amount of surface receptors was determined by incubating CGNs on ice to prevent internalization, and these cellswere not subjected to cleavage with glutathione. *P<0.05 (ANOVA with Dunnett test). Data are mean±s.e.m. and are representative of three independentexperiments. (D) A GB1 mutation that impairs the GB1–Rap1 interaction can promote agonist-induced GABAB receptor internalization. HEK293 cells were co-transfected with either a HA-tagged wild-type GB1 or a GB1 deletion mutant (GB1ΔS953) plus GB2. Cell surface receptors were first labeled with primaryantibodies against GB1 (anti-HA) at 4°C. Cells were then stimulated with baclofen (100 µM, 120 min) at 4°C (control) or at 37°C to induce internalization. Cellsurface receptors were visualized by secondary antibodies coupled to a green fluorescence fluorophore at 4°C (green). After permeabilization of the cells,internalized receptors were detected by secondary antibodies carrying a red fluorescence fluorophore (red). Data aremean±s.e.m. and are representative of threeindependent experiments. **P<0.005 (ANOVA with Bonferroni’s multiple comparison test).

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Another interesting observation is that although baclofentreatment did not notably affect the net amount of internalizedGABAB receptor, once the Rap1–GB1 interaction was disrupted bythe GB1ΔS953 deletion, then the same baclofen treatment greatlyincreased the amount of the receptor found inside the cell (Fig. 4D).This data strongly suggests that baclofen stimulation of GABAB

receptor promotes receptor internalization. These results, togetherwith those from ELISA and biotinylation assays, support a model inwhich agonist stimulation of GABAB receptor promotes receptorinternalization and also recruits Rap1 to the receptor, which in turnmight potentiate receptor recycling. A combination of such twoactions would then lead to a relatively stable expression of thereceptor at the cell surface. Our data also help clarify a controversyover whether agonist stimulation of GABAB receptor facilitatesreceptor internalization.

Agonist stimulation ofGABAB receptor promotes recycling ofthe receptor to the cell surfaceThe above model suggests that agonist stimulation of GABAB

receptor promotes recycling of internalized receptors back to the cellsurface. If this is the case, then disrupting receptor recycling shouldreduce the surface expression of the receptor. To test this, we treatedthe cells with monensin, which blocks vesicle recycling, and foundthat it greatly reduced the surface expression level of GABAB

receptor upon agonist stimulation. As a control, we treated the cells

with brefeldin A (BFA), which inhibits de novo delivery of GABAB

receptor by impairing ER–Golgi trafficking of newly synthesizedmembrane receptors. BFA had no notable effect on the surfaceexpression of GABAB receptor (Fig. 5A). These results suggest thatagonist stimulation of GABAB receptor promotes receptorrecycling.

It might be argued that Rap1–GB1 interaction promotes GABAB

receptor surface expression by blocking agonist-induced receptorinternalization rather than by promoting receptor recycling. If this isthe case, then simultaneous inhibition of the Rap1–GB1 interactionand receptor recycling should have an additive effect. In otherwords, impairing the Rap1–GB1 interaction would reduce thesurface expression of GABAB receptor when receptor recycling isabsent. However, this is not the case, as disruption of the Rap1–GB1interaction with Pep treatment cannot further reduce the surfaceexpression of GABAB receptor after receptor recycling was blockedby monensin (Fig. 5A). Thus, it appears that the Rap1–GB1interaction and receptor recycling act in the same pathway,providing additional evidence that agonist stimulation of GABAB

receptor promotes receptor recycling. As a positive control, wefound that disruption of Rap1–GB1 interaction with Pep treatmentcan still suppress the surface expression of GABAB receptor inBFA-treated cells, further suggesting that recycling, rather than denovo delivery, of GABAB receptor contributes to its stable surfaceexpression upon agonist stimulation (Fig. 5A).

Fig. 5. Agonist-induced Rap1–GB1 interaction promotes GABAB receptor recycling. (A) Effects of monensin and BFA on GABAB receptor surfaceexpression. HEK293 cells were transfected with GABAB receptor. Cells were pretreated either with BFA (1 µM, 24 h) or monensin (50 µM, 30 min). Afterincubation with Pep (100 µg/ml, 60 min) or HBS buffer, cells were then treated with baclofen (100 µM, 10 min). Cell surface GB1 was measured by ELISA.***P<0.0001 (ANOVAwith Bonferroni’s multiple comparison test). (B,C) Effects of monensin on colocalization of GABAB receptor with either EEA1 (B) or LAMP1(C). The assay was performed on HEK293 cells co-transfected with GB1 (HA-tagged) and GB2. Cell surface GB1 was first labeled with HA-tag antibody at 4°C inthe presence or absence of monensin (50 µM, 30 min) followed by treatment of baclofen (100 µM, 120 min at 37°C). Cells were then fixed, permeabilized andstained for EEA1 or LAMP1. GB1, red; EEA1 or LAMP1, green. Scale bars: 10 µm. Quantification of colocalization is shown in D. Results are mean±s.e.m.**P<0.005 (Student’s t-test). All data are representative of at least three independent experiments.

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What is the fate of internalized GABAB receptor after its recyclingwas blocked? To address this question, we first examined to whichsubcellular compartments internalized GABAB receptor is localized.Internalized receptors are primarily directed to one of two distinctdestinations – either to lysosomes for degradation or they are recycledback to the cell surface. We found that ∼60% of GABAB receptorfound inside the cell colocalized with EEA1, an early endosomemarker, whereas merely 15% of the receptor colocalized withLAMP1, which marks late endosomes and lysosomes (Fig. 5B–D).By using monensin, which inhibits the fusion of intracellular vesicleswith the plasma membrane, then blocks recycling pathway (Gramppet al., 2008),we analyzedwhether recycling or degradation is themainpathway of endocytosed GABAB receptors. We found that monensintreatment drastically reduced the amount of GABAB receptorlocalized to early endosomes while greatly increased its presence inlate endosomes or lysosomes (Fig. 5B–D). This data suggests thatblockade of GABAB receptor recycling re-routes the receptor to lateendosomes and lysosomes for degradation.We also found that disruption of Rap1–GB1 interaction with Pep

treatment substantially reduced the amount of intracellular GABAB

receptor localized to early endosomewhile it increased its presence inlate endosomes and lysosomes (Fig. 6A,B). A similar observationwasmade with the GB1ΔS953 mutation, which disrupts the interactionbetween Rap1 and GB1 (supplementary material Fig. S4A,B).Furthermore, our results also show that disruption of the Rap1–GB1interaction with Pep treatment substantially reduced the amount ofintracellular GABAB receptor localized to recycling endosome(Fig. 7A,B). These findings are very similar to those obtained withmonensin, consistent with our model that the interaction betweenRap1–GB1 is important for GABAB receptor recycling.

DISCUSSIONOne prominent feature of many types of membrane receptors,particularly GPCRs, is the downregulation of their surfaceexpression following prolonged agonist treatment, a mechanismadopted by cells to protect them from receptor over-stimulation

(Ferguson, 2001). How the surface expression of these receptors isdownregulated in an activity-dependent manner has been very wellcharacterized (Calebiro et al., 2010; Premont and Gainetdinov,2007; Shenoy and Lefkowitz, 2011). However, some other GPCRssuch as GABAB receptor remain relatively stable at the cell surfaceeven after prolonged agonist treatment (Benke, 2010; Chu et al.,1997; Nantel et al., 1993; Schreff et al., 2000; Solinski et al., 2010).GABA is the primary inhibitory neurotransmitter in the mammalianCNS. The relative stable surface expression of GABAB receptor iswell suited for its role in mediating the slow, long-lasting inhibitoryfunction of GABA (Benke, 2010). Despite extensive studies, themechanisms underlying the stable surface expression of GABAB

receptor is unclear.Here, we identified a mechanism by which GABAB receptor

promotes its own surface expression under prolonged agoniststimulation (Fig. 7C). Our data show that it does so by recruitinga signaling cascade including Gi/o, Rap1GAPII and Rap1,with the latter protein recruited to the GB1 subunit of thereceptor through direct protein–protein interactions in anactivity-dependent manner. Specifically, in response to agoniststimulation, GABAB receptor activates the Gi/o protein, which inturn triggers phosphorylation of Rap1GAPII, a GAP for Rap1, toinhibit Rap1GAPII activity. This leads to an increase in half-lifeof the active form of Rap1 (Rap1-GTP). Rap1-GTP then binds tothe C-terminal end of GB1 subunit of GABAB receptor topromote its recycling to the plasma membrane. This effectcounteracts agonist-induced internalization of GABAB receptor,leading to a relatively stable surface expression of the receptor.Blocking this signaling cascade by inhibiting Rap1 activity,disrupting the interaction between Rap1 and the receptor, orrepressing vesicle recycling greatly compromised the surfaceexpression of GABAB receptor. To the best of our knowledge,this represents a new mechanism by which membrane receptorsregulate their own surface expression.

Our results show that the relatively stable surface expression ofGABAB receptor under chronic agonist stimulation is not a static

Fig. 6. Disruption of Rap1–GB1 interactionpromotes localization of GABAB receptor to lateendosomes or lysosomes. (A,B) Effects ofpeptide blockade of the GB1–Rap1 interaction oncolocalization of GABAB receptor with EEA1 (A) orwith LAMP1 (B). Experiments were performed asdescribed in Fig. 5, except that cells were treatedwith Pep (100 µg/ml, 60 min) or PepN (100 µg/ml,60 min) instead of monensin. Scale bars: 10 µm.Quantification of colocalization is shown in the right-hand panels. Results are mean±s.e.m. **P<0.005,***P<0.0001 (ANOVA with Bonferroni’s multiplecomparison test). All data are representative of atleast three independent experiments.

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but rather a dynamic process. Agonist stimulation of GABAB

receptor potentiates its internalization, which is supported bymultiple lines of evidence, including ELISA analysis, flowcytometry, biotinylation assay and immunofluorescence staining.In the meantime, agonist stimulation also facilitates recycling ofintracellular GABAB receptors. A combination of these two actionsleads to a relatively stable surface expression of the receptor. Theseresults also help clarify a long-standing controversy over whetherGABAB receptor potentiates internalization in response to agoniststimulation.It is not clear why agonist stimulation of GABAB receptor

facilitates both internalization and recycling (Benke, 2010; Gramppet al., 2008). On the one hand, this seems to constitute a futile cycle,which is energy inefficient. On the other hand, agonist-inducedinternalization of membrane receptors is considered a generalphenomenon and represents a default pathway for most, if not all,GPCRs (Marchese et al., 2003). In this case, to maintain a relativelystable surface expression of the receptor, it is logical for the cell topromote recycling of internalized receptor and/or increase de novodelivery of newly synthesized receptor to the cell surface (Tsao andvon Zastrow, 2000). Our results suggest that GABAB receptor

mainly adopts the former mechanism under our conditions (a 2-hwindow). Nevertheless, we do not exclude the possibility that thelatter mechanism also plays a role under other conditions, forexample, in a longer time frame.

We identified Rap1 as a new GABAB-receptor-binding partnerthrough a classic biochemical purification approach. Rap1 is a smallGTPase and regulates GABAB receptor activity by promoting itssurface expression. Conversely, GABAB receptor regulates Rap1activity by promoting the formation of its active form Rap1-GTP.This constitutes an interesting positive-feedback loop that regulatesGPCR function through small GTPases, a phenomenon that has notbeen reported. In addition, although several types of GPCR-interacting proteins have been identified, no small GTPases havebeen shown to directly bind to GPCRs. Rap1 represents the firstsuch small GTPases associated with GPCRs. Small GTPases, suchas Rab proteins, are known to regulate vesicle trafficking by actingas a sorting signal (Bhattacharya et al., 2004; Rosenfeld et al.,2002). It is possible that Rap1 might function through a similarmechanism. Nevertheless, future studies are needed to uncoverexactly how Rap1 promotes recycling of GABAB receptor. As Rap1and GABAB receptor are evolutionarily conserved from worms to

Fig. 7. Agonist-induced Rap1-GB1 interaction promotes GABAB receptor recycling. (A) Effects of peptide blockade of GB1–Rap1 interaction andmonensinon colocalization of GABAB receptor with Rab11. The assay was performed on HEK293 cells co-transfected with GB1 (HA-tagged) and GB2. Cell surface GB1was first labeled with HA-tag antibody at 4°C in the presence or absence of Pep (100 µg/ml, 60 min), PepN (100 µg/ml, 60 min) or monensin (50 µM, 30 min)followed by treatment with baclofen (120 min at 37°C). Cells were then fixed, permeabilized and stained for Rab11. GB1, red; Rab11, green. Scale bars: 10 µm.(B) Quantification of colocalization of GABAB receptor with Rab11 shown in A. Results are mean±s.e.m. ***P<0.0001 (ANOVA with Bonferroni’s multiplecomparison test). Data are representative of three independent experiments. (C) A schematic model. Stimulation of GABAB receptor with agonist (baclofen) or apositive allosteric modulator (CGP7930) triggers a series of signaling events involving activation of Gαi/o, inhibition of Rap1GAPII phosphorylation, activation ofRap1 (formation of Rap1-GTP), and recruitment of Rap1-GTP to GB1 through direct protein–protein interactions.

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humans, our findings raise the possibility that a similar phenomenonmight occur in other organisms.

MATERIALS AND METHODSPrimary neuron culture and cell linesAll experiments were approved by the Animal Experimentation EthicsCommittee of School of Life Science and Technology in HuazhongUniversity of Science and Technology and were specifically designed tominimize the number of animals used. Culturing of primary cerebellargranule neurons (CGNs) was as described previously (Tu et al., 2010).Briefly, the cerebella were dissected from 1-week-old KunMing mice ofeither sex from Hubei Provincial Center for Disease Control and Prevention,and neuronal cells were maintained in a 1:1 mixture of Dulbecco’s modifiedEagle’s medium (DMEM) and F-12 nutrients (Invitrogen), supplementedwith 30 mM glucose, 2 mM glutamine, 3 mM sodium bicarbonate, 5 mMHEPES buffer, 30 mM KCl and 10% fetal calf serum to improve neuronalsurvival. Pharmacological treatments were performed in Ca2+-free HEPES-buffered solution (HBS). HEK293 cells were cultured in DMEM mediumsupplemented with 10% fetal bovine serum (FBS).

Plasmids, antibodies and synthetic peptidesThe plasmids pRK5-HA-GB1 and pRK5-Flag-GB2 encoding the wild-typeGB1 and GB2 subunits were described previously (Rondard et al., 2008).pSRα-SK-T7-Rap1V12 and pSRα-SK-T7-Rap1N17, pEBG-Rap1GAPII(wild-type) and its negative control plasmid were gifts from N. Minato(Department of Immunology and Cell Biology, Kyoto University, Japan).The GST fusions of GB1-C (encoding amino acids 854–960 of GB1aC-terminus) and GB2-C (encoding amino acids 744-941 of GB2C-terminus) were cloned between BamHI and XhoI of pGEX-6p1. Theplasmids pRK5-HA-GB1, pSRα-SK-T7-Rap1 and pGEX-6p1-GB1-C-terminus were used for site-directed mutagenesis (stratagene). Antibodieswere as follows: anti-Rap1 (Sc-65) and anti-Rap1GAPII (G-17) were fromSanta Cruz Biotechnology; anti-Src, anti-HA-tag (C29F4) and anti-β-actinwere from Cell Signaling Technology, and anti-GB1 (ab55051) was fromAbcam. Peptides utilized in this study were synthesized and purified by theProteintech Group Inc. (CA, USA). The quality of peptides was assessed byhigh-performance liquid chromatography analysis, and the expectedmolecular mass was observed using matrix-assisted laser desorption(MDLC) mass spectrometry. Peptides were dissolved in DMSO anddiluted in phosphate-buffered saline (PBS, pH 7.4) to a concentration of5 mg/ml and stored at −80°C.

Mass spectrometry and data analysisMass spectrometry analysis was carried out as previously described (Leeet al., 2010; Perkins et al., 1999). Briefly, 60 µg GST–GB-C protein wasused to pulldown cell lysates from four 100-mm dishes of cultured CGNs.Protein bands at 20–24 kDa were excised from SDS-PAGE gel and digestedwith trypsin, and peptides were separated and analyzed by MDLC-dependent tandem mass spectrometry (MS/MS). We used the EttanTMMDLC system (GE Healthcare), and desalted tryptic peptide mixtures wereloaded onto the columns for separation. A FinniganTM LTQTM linear iontrap MS (Thermo Electron) equipped with an electrospray interface wasconnected to the liquid chromatography setup for detection of elutedpeptides. Data were obtained simultaneously based on MS/MS spectraagainst the non-redundant International Protein Index (IPI) human proteindatabase (version 3.26, 67,687 entries) using BioworksBrowser rev. 3.1.

GST and RBD pulldown assays for Rap1-GTP detectionThe GST pulldown assay for Rap1-GTP detection was performed aspreviously described (Guetg et al., 2010; Kuramoto et al., 2007; Whiteet al., 2000). Bacterial GST fusion proteins were immobilized on glutathione–Sepharose beads. After incubation with cell lysate, the beads were recoveredand washed four times with RBD buffer (150 nm NaCl, 50 nM Tris-HCl pH7.6, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mMNa3VO4, 10 mM NaF, 2 μg/ml leupeptin, 2 μg/ml aprotinin). Protein boundto the beads was eluted with SDS sample buffer and analyzed by westernblotting. The RBD pulldown assay for detecting intracellular Rap1-GTP was

performed as described previously (Raaijmakers and Bos, 2009). In brief, celllysate in RBD buffer was incubated with glutathione–Sepharose beadsconjugated to GST–RalGDS protein for 60 min at 4°C. After four washes,Rap1-GTP was analyzed by western blotting using a Rap1 antibody.

Immunoblotting analysisImmunoblotting analysis was performed as previously described(Baloucoune et al., 2012). Briefly, after determination of proteinconcentrations using Bradford reagent (Bio-Rad Laboratories), equalamounts of protein were resolved by SDS-PAGE and transferred ontonitrocellulose membrane. The blots were incubated with primary antibodies,then with horseradish peroxidase (HRP)-linked secondary antibodies,detected by enhanced chemiluminescence reagents (Pierce) and visualizedby using X-ray film.

Inositol phosphate measurementInositol phosphate (IP) accumulation in HEK293 cells co-transfected withGABAB receptor andGαqi9 chimeric proteinswasmeasured after stimulationwith baclofen for 30 min in 96-well microplates as previously described(Couve et al., 2000).After incubation in the presenceofLiCl (10 mM, 30 min)and termination of the reaction with 0.1 M formic acid, the supernatant wasrecovered andpurified by ion exchange chromatographyusingDOWEXresin.Radioactivitywasmeasured using aWallac 1450MicroBetamicroplate liquidscintillation counter (Perkin Elmer, Waltham, MA, USA).

ELISA and flow cytometryCell surfaceGB1 expressionwas detected byELISAand flowcytometry.HA-tagged GB1 and Flag-tagged GB2 were co-transfected into HEK293 cells for24 h, and then the cells were seeded into 96-well microplates. Cell surfaceGB1 expression was detected with a monoclonal rat anti-HA antibody (3F10,Roche) and a goat anti-rat second antibody coupled to HRP (JacksonImmunoresearch, West Grove, PA) as previously described (Rondard et al.,2008). Bound antibody was detected by chemoluminescence usingSuperSignal substrate (Pierce) and a 2103 EnVision™ Multilabel PlateReader (Perkin Elmer, Waltham, MA, USA). Flow cytometry analysis wasperformed on FACSAria (Beckman). HEK293 cells co-transfected with GB1and GB2 were incubated with a mouse monoclonal anti-GB1 antibodyab55051 (Abcam) and FITC-conjugated IgG. The surface mean greenfluorescence intensity (MFI) was acquired with CXP software (BeckmanCoulter, CA, USA).

Biotinylation assayCGNs grown on poly-L-lysine-coated dishes were incubated for 1 h inculture medium with Pep (100 µg/ml) or PepN (100 µg/ml) in presence ofleupeptin (100 µg/ml). Dishes were placed on ice and washed twice withice-cold buffer. Sulfo-NHS-SS-Biotin (Pierce) was freshly dissolved at aconcentration of 0.5 mg/ml in ice-cold buffer A (25 mM HEPES, pH 7.4,119 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 30 mM glucose),and cells were incubated with biotin for 15 min on ice. After three washeswith buffer A, cells were incubated with baclofen (100 µM; 120 min; 37°C)in the presence of Pep (100 µg/ml; 120 min; 37°C) or PepN (100 µg/ml;120 min; 37°C) followed by incubation on ice. Cell surface biotin wascleaved off by incubation with glutathione solution (75 mM glutathione,75 mM NaCl, 10 mM EDTA, 1% BSA) twice for 15 min each on ice.Finally, cells were washed twice in ice cold PBS, lysed in 300 µl of ice coldRIPA buffer (50 mM Tris-HCl pH 7.4, 5 mM EGTA, 5 mM EDTA, 50 mMNaF, 1% Nonidet P-40, 0.1% SDS, 2.5 mM sodium pyrophosphate, 1 mMsodium orthovanadate, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/mlpepstatin) followed by rotating for 30 min at 4°C. Cell lysates werecollected and centrifuged at 13,400 g for 15 min at 4°C to remove nuclearand cellular debris. The supernatant containing equal amounts of proteinwas precipitated with 50 µl of strepavidin–Sepharose (GE Healthcare)overnight at 4°C. Sepharose beads were then washed twice in RIPA buffercontaining 500 mM NaCl and once in RIPA buffer containing 150 mMNaCl. Beads were resuspended in 40 µl of SDS sample buffer and boiled for5 min at 95°C followed by SDS-PAGE and western blotting using a GB1antibody.

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Immunofluorescence-based internalization assayHEK293 cells transiently transfected with HA–GB1 or HA–GB1ΔS953 andFlag–GB2 were incubated with an HA antibody in buffer A (25 mMHEPES,pH 7.4, 119 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 30 mMglucose) containing 10% normal goat serum and 100 µg/ml leupeptin for40 min at 4°C. After extensive washes with ice-cold buffer A, cells wereincubated for 120 min at 37°C in the presence or absence of baclofen asindicated. Control cultures were left at 4°C, to prevent receptor internalization.For the immunofluorescence-based internalization assay, after washing thecells with ice-cold buffer A, we incubated cells with secondary antibodiescoupled to Alexa Fluor 488 at 4°C. Then cells were fixed with 4%paraformaldehyde, 4% sucrose for 10 min and permeabilized for 5 min with0.1% Triton X-100 in PBS. Cells were incubated with secondary antibodiescoupled to Cy3 (1:500; Invitrogen) for 1 h at room temperature. After threewashes with PBS, cells were analyzed by a Spinning-disk confocal imagingsystem (CSU-X1 Nipkow Yokogawa, Japan) with Andor IQ 1.8 software(Andor Technology plc, Springvale Business Park, UK). Images wereprocessed and fluorescence was measured by Image J (NIH).

Immunofluorescence staining for colocalizationFor colocalization, cells were fixed with 4% paraformaldehyde for 10 minand permeabilized for 5 min with 0.1% Triton X-100 in PBS. Then cellswere incubated either with EEA1 antibody or LAMP1 antibody for 1 h atroom temperature. After washing with PBS, we incubated cells withsecondary antibodies coupled either to Alexa Fluor 488 or Cy3 (1:500;Invitrogen) for 1 h at room temperature. After three washes with PBS, cellswere analyzed by a spinning-disk confocal imaging system (CSU-X1Nipkow Yokogawa, Japan) with Andor IQ 1.8 software. Images wereprocessed and fluorescence was quantified using Image J.

Statistical analysisData expressed as mean±s.e.m. were analyzed by GraphPad Software Prism6.0 and comparisons were made by one-way ANOVA analysis of variancewith Bonferroni’s multiple comparison tests or Student’s t-test. Values ofP<0.05 were taken as being indicative of statistical significance.

AcknowledgementsWe thank Philippe Rondard and X.Z. Shawn Xu for critically reading an early versionof the manuscript.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsJ.L. and L.S. conceived the project; J.L., L.S. and S.H. designed experiments; Z.Z.,W.Z., S.H., Q.S., Y.W., Y.H., N.S., Y.Z. and Z.J. performed the experiments; J.L.,L.S., S.H., Z.Z. and W.Z. analyzed the results. J.L. wrote the manuscript withcontributions from L.S. and S.H.

FundingThis work was supported by the National Natural Science Foundation of China(NSFC) [grant numbers 31130028, 30970661, 31225011, 31420103909,31100548]; the Ministry of Science and Technology [grant number2012CB518000, 2009DFA31940]; the Program of Introducing Talents ofDiscipline to the Universities of the Ministry of Education [grant number B08029]and Program for Changjiang Scholars and Innovative Research Team in University(PCSIRT) [grant number IRT13016]; Natural Science Foundation of HuBeiProvince [grant number 2014CFA010]; and the Merieux Research Grants Programof Institut-Merieux (to J.L.).

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.167056/-/DC1

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