Preso1 dynamically regulates group I metabotropic glutamate receptors

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1 ynmilly regultes group I metotropi glutmte reeptors Ji-Hu Hu 1, Linlin Yng 1, Pul J Kmmermeier 2, Chester G Moore 1, Pul R Brkemn 1,7, Jinheng Tu 1, Shouyng Yu 3, Ronl S Petrli 4, Zhe Li 1, Ping-Wu Zhng 1, Joo Min Prk 1, Xinzhong Dong 1,5, Bo Xio 1,3 & Pul F Worley 1,6 212 Nture Ameri, In. All rights reserve. Group I metotropi glutmte reeptors (mglurs), inluing mglur1 n, re G protein ouple reeptors (GPCRs) tht re expresse t exittory synpses in rin n spinl or. GPCRs re often negtively regulte y speifi G protein ouple reeptor kinses n susequent ining of rrestin-like moleules. Here we emonstrte n lterntive mehnism in whih group I mglurs re negtively regulte y proline-irete kinses tht phosphorylte the ining site for the ptor protein Homer, n therey enhne mglur Homer ining to reue signling. This mehnism is epenent on multiomin sffoling protein,, tht ins mglur, Homer n proline-irete kinses n tht is require for their phosphoryltion of mglur t the Homer ining site. Geneti ltion of prevents ynmi phosphoryltion of, n / mie exhiit sustine, -epenent inflmmtory pin tht is linke to enhne mglur signling. retes miroomin for proline-irete kinses with ro sustrte speifiity to phosphorylte mglur n to meite negtive regultion. Group I metotropi glutmte reeptors (mglurs) re enrihe t exittory synpses in the rin, where they re importnt in neurl plstiity 1 n ehviorl responses inluing inflmmtory pin, fer onitioning, rug ition n shizophreni 2 4. Group I mglurs lolize primrily to the lterl mrgin of the postsynpti memrne 5, where they respon to glutmte s it iffuses from relese sites in the entrl synpti region 6. Group I mglurs evoke hnges y moulting memrne ion hnnels 7, intrellulr ion hnnels 8 n signling ses inluing EF2 kinse 9, MAP kinse 1, mtorc1 (ref. 11) n enonninoi synthesis 12. The oupling mehnisms for severl of these outputs involve proteins of n ptor protein fmily, terme Homer, tht in to proline-rih sequene in the C terminus of group I mglurs 13. In n exemplr ssy, Homer ining to group I mglurs expresse in superior ervil gnglion neurons loks oupling to M-type potssium hnnels n voltge sensitive C 2 hnnels 7. Homer ining to group I mglurs lso reues gonist-inepenent tivtion of BK hnnels in grnule ell neurons 14. The Homer ining site (TPPSPF) ontins onsensus site for proline-irete kinses. Proline-irete kinses re tivte y multiple neurotrnsmitter n growth ftor reeptors n re ritil in the ontrol of ellulr proesses inluing ell prolifertion, ifferentition, motility n neuronl plstiity 15. The ility of Homer ining to moify mglur signling egs the question of whether proline-irete kinse meite phosphoryltion of mglur funtions in reeptor signling. Here we report tht group I mglur signling is ynmilly ontrolle y proline-irete kinses ting in onert with multiomin sffoling protein terme. inlues WW, PDZ n FERM omins, n it ws previously reporte to in PSD95 (ref. 16) n to moulte enriti spine morphogenesis. We ientifie in sreen for proteins tht in Homer n etermine tht it lso ins group I mglur n proline-irete kinses inluing CDK5 n ERK. enhnes the ility of proline-irete kinses to in mglur n phosphorylte the Homer ining site in mglur. This tion of ppers nlogous to tht of A-kinse nhoring proteins (AKAPs) 17 to reruit PKA to phosphorylte its sustrtes. -epenent phosphoryltion of mglur enhnes Homer ining n ownregultes mglur signling in ellulr ssys. To ssess this mehnism in vivo, we exmine ehviorl responses in mouse moel of inflmmtory pin, whih is epenent on sustine tivtion of group I mglurs 3. We onfirme tht inflmmtory pin epene on n ws inrese in mouse moels tht interrupt Homer ining to, owing either to geneti eletion of Homer or to geneti knok-in of n point mutnt tht nnot in Homer. Inflmmtory pin ws lso inrese in / mouse, n this ws ouple to inrese tivity. These stuies support moel in whih funtions to filitte 1 Solomon H. Snyer Deprtment of Neurosiene, Johns Hopkins University Shool of Meiine, Bltimore, Mryln, USA. 2 Deprtment of Phrmology n Physiology, University of Rohester Meil Center, Rohester, New York, USA. 3 The Stte Key Lortory of Biotherpy, West Chin Hospitl, Sihun University, Chengu, Chin. 4 Ntionl Institute on Defness n Other Communition Disorers, US Ntionl Institutes of Helth, Bethes, Mryln, USA. 5 Howr Hughes Meil Institute, Johns Hopkins University Shool of Meiine, Bltimore, Mryln, USA. 6 Deprtment of Neurology, Johns Hopkins University Shool of Meiine, Bltimore, Mryln, USA. 7 Present ress: Deprtment of Peitris, University of Cliforni, Sn Frniso, Sn Frniso, Cliforni, USA. Corresponene shoul e resse to P.F.W. (pworley@jhmi.eu). Reeive 16 Ferury; epte 3 April; pulishe online 6 My 212; oi:1.138/nn.313 nture NEUROSCIENCE vne online pulition

2 212 Nture Ameri, In. All rights reserve. proline-irete kinse moultion of Homer ining to mglur n therey ontrols the mplitue n urtion of mglur signling. RESULTS is Homer-ining protein enrihe in rin We use the Homer1 EVH1 omin to perform yest two-hyri sreen of rt rin DNA lirry n ientifie 1.2-k frgment orresponing to the C terminus of KIAA316. The full-length gene ws lone y 5 n 3 rpi mplifition of DNA ens (RACE) n etermine to e homologous to humn FRMPD4 (lso terme PDZD1, PDZK1 n Preso 16 ), n is here terme. The protein ontins severl omins, inluing Homer ining site (Fig. 1), n is onserve from Drosophil melnogster to humn (Supplementry Fig. 1). Puli tses inlue two relte genes tht we term Preso2 (FRMPD3, KIAA1817) n Preso3 (FRMPD1 (ref. 18), FRMD2, KIAA967) (Supplementry Fig. 2). Mouse n humn n Preso2 re ner eh other on the X hromosome, wheres Preso3 is utosoml (humn hromosome 9, mouse hromosome 4). mrnas of ll three Preso genes re expresse roly in rin n spinl or in mouse (Supplementry Fig. 3). Hemgglutinin (HA)-tgge Homer1 (the rin-enrihe Homer1 splie form) 19 immunopreipitte with (Fig. 1). This intertion ws isrupte either y point muttion of the nonil polyproline-ining surfe of the Homer1 EVH1 omin or y point muttion of the preite Homer ining site in (FR; Fig. 1). Thus, the Homer intertion involves onventionl ining properties of Homer, n it seems to e iret. Preso2 enoes onserve Homer ining site, n it immunopreipitte with Homer1 from HEK293T ell lystes, wheres Preso3 lks onserve Homer ining site n i not oimmunopreipitte (Supplementry Fig. 4). To exmine the ssoition of with Homer in vivo, we immunopreipitte from etergent lystes of rt ereellum n onfirme tht Homer3 ws oimmunopreipitte (Fig. 1). mglur1 n omponents of its signling omplex, inluing Shnk, PSD95 n the inositol-1,4,5-trisphosphte reeptor (IP 3 R) lso immunopreipitte with (Fig. 1). immunoretivity ws enrihe in ulture hippompl neurons n showe puntte istriution tht ololize with the exittory synpti mrker PSD95, ut not with the inhiitory synpti mrker glutmi i eroxylse (GAD65) (78.6 ± 4.8% of ololize with PSD95 (ll vlues expresse s ± 95% onfiene intervl), wheres 9.6 ± 2.1% ololize with GAD65; n = 17 2 enrites in 7 or 8 neurons; Fig. 1). immunoretivity lso ololize with in the neurons (68.6 ± 5.1%; Fig. 1). To onfirm tht is synpti protein, we performe immunogol eletron mirosopy in the hippompus. -immunogol lolize to the postsynpti spine, espeilly in the PSD n sujent ytoplsm, overlpping the istriutions of Homer n group I mglurs 5,19 (Fig. 1e). interts with group I mglurs vi its FERM omin We onsiere the possiility tht funtions s sffoling protein for group I mglurs. Consistent with rin oimmunopreipittion t, immunopreipitte with from etergent lystes of HEK293T ells (Fig. 2 n Supplementry Fig. 4). By ontrst, i not immunopreipitte mglur2 or mglur4, whih elong to group II n group III mglurs (Supplementry Fig. 4,). possesses severl protein intertion omins, n we exmine whether might iretly intert with inepenent of Homer. or eletion mutnts were otrnsfete with HA-tgge into HEK293T ells n WW PDZ RA FERM Homer lign PDZ lign HA-: FR HA-Homer1: GN WA 511 PPPGFRD 86 PSD95 Overly ETTV 1312 Figure 1 ins to Homer n lolizes to the postsynpti ensity. () Domin struture of rt protein. F86, Homer ining site. () Vrious n Homer onstruts were trnsfete into HEK293T ells. Anti- immunopreipitte Homer1, n this ws isrupte y the F86R (FR) muttion in, or G89N (GN) or W24A (WA) muttion in Homer1. () Rt ereellum ws lyse n inute with nti-. Homer3, mglur1, IP 3 R, Shnk n PSD95 immunopreipitte with. PI, preimmune serum ontrol; IP, immunopreipittion. () Culture hippompl neurons immunostine with nti- long with nti-psd95 or nti-gad65. ololize with PSD95 ( mrker for exittory synpses), ut not with GAD65 ( mrker for GABAergi inhiitory synpses). Sle rs represent 3 µm in min pnels, 5 µm in the mgnifie enrites (oxe). (e) Immunogol eletron mirosopy leling of in the hilus of the ult hippompus. Gol prtiles (rrows) re enrihe in n sujent to the postsynpti ensity (). p, presynpti terminl. Sle r, 1 nm. Full-length western lots for this figure re shown in Supplementry Figure 9. Homer1 IP: nti- Homer1 Lyste Lyste PI IP Homer3 mglur1 IP 3 R Shnk PSD95 IP: nti- e p GAD65 p Overly Overly p vne online pulition nture NEUROSCIENCE

3 212 Nture Ameri, In. All rights reserve. HA-: My-: /o-ip /Lyste /IP /Lyste FL WW PDZ FERM F86R F86R F1128R IP: nti- ssye for intertion y oimmunopreipittion. ntioy immunopreipitte tht ws expresse with ny of the N-terminl eletion mutnts exept - FERM (Fig. 2). Notly, point mutnts of oth n tht o not in Homer (FR; F1128R (FR)) retine intertion in the oimmunopreipittion ssy (Fig. 2), suggesting tht ining to oes not epen on Homer. These t lso suggest tht the FERM omin is require for ining to. We onfirme tht the isolte FERM omin ws suffiient to immunopreipitte (Fig. 2). We next exmine the region of require for intertion with using oimmunopreipittion ssys with C-terminl mutnts. immunopreipitte 1 12, ut not 1 92 (Fig. 2). As the Homer ining site is t 1128, these t inite tht sequene element(s) in require for ining re t lest 1 mino is remote from the Homer ining site (Fig. 2) inlues preite FERM omin ining site 2 n D-omin implite s ining site for proline-irete kinses in other proteins 21 (Fig. 2). Muttions of the puttive FERM omin ining sites ( Y965A V967A ) resulte in only moest reution of its immunopreipittion with (Fig. 2e). However, the omine point muttions of the puttive FERM-ining site n the Homer ining site ( Y965A V967A F1128R ; YVF) mrkely reue immunopreipittion with (Fig. 2e). The epenene of ining on the Homer ining site in is onsistent with the notion tht ntive Homer proteins in HEK293T ells 22 n link these proteins, n it suggests tht ining to mglur involves ontriutions of oth the FERM omin ining site n the Homer ining site. regultes mglur phosphoryltion n signling Coexpression of inrese ining etween Homer n (172.7 ± 29.4% of ontrol, n = 6, P =.4; Fig. 3). As Homer ining n e inrese y phosphoryltion of t My-FERM: HA-: /o-ip /Lyste FERM/IP FERM/ Lyste IP: nti-my HA-: My-: /o-ip /Lyste /IP /Lyste IP: nti-my Figure 2 ins to the C terminus y mens of its FERM omin. () n progressive N-terminl eletion n point mutnts of were otrnsfete into HEK293T ells. Detergent lystes were inute with nti- n nlyze y western lotting with nti-ha. FL, Full length. WW, PDZ n FERM elete the inite omin from the N terminus. Only the - FERM mutnt file to in. () The FERM omin is suffiient to in. FERM n onstruts were otrnsfete into HEK293T ells, n etergent lystes were inute with nti-my n nlyze y western lotting with nti-ha or nti-my. () Region of etween mino is 92 n 12 is ritil for its intertion with. n vrious mutnts were otrnsfete into HEK293T ells. Detergent lystes were inute with nti-my n nlyze y western lotting with nti-ha or nti-my. () struture showing the mino i sequene etween 92 n 12 n Homer lign. A preite ining site for FERM omin is shown in lue; preite D-omin for ERK ining is shown in green, with ritil mino is itliize. The Homer ining site is shown in yellow, n phosphoryltion site in re. T P P S P F 1126 e YVF YV FR HA-: My-: /o-ip /Lyste /IP /Lyste 92 K S T E N R G P G A A A G G G S G P G V A G A G N A G C T A T G G P E P P D A G P K A L Y D V A E A E E S F P A A A R P R S P S P I S T L S H L A G S A G R T D D D A P S L H S E T A A R S S S S Q G S 12 IP: nti-my (e) n vrious mutnts were otrnsfete into HEK293T ells n ssye for oimmunopreipittion. Muttion of FERM (Y965A, V967A; YV) n Homer (F1128R; FR) ining sites ( YVF) olishe ining. Full-length western lots for this figure re shown in Supplementry Figure 1. the Homer ining site 23, we hypothesize tht might funtion to inrese Homer ining y inresing phosphoryltion. We evelope phospho-speifi ntioy to the Homer ining site of group I mglur (Supplementry Fig. 4e), n foun tht phosphoryltion inrese with expression (198.2 ± 72.8% of ontrol, n = 7; P =.4; Fig. 3). To exlue the possiility tht inreses Homer ining iniretly y mens of the ssoition of with Homer, we onfirme s effet using mutnt (FR) tht oes not in Homer (Fig. 1). Enhne Homer ining is epenent on phosphoryltion, s i not enhne Homer ining to n mglur mutnt (TSAA, ontining TS to AA muttions t Homer ining site) tht nnot e phosphorylte t the Homer site ( wil type (): ± 51.8% of ontrol, n = 6; P =.3; TSAA: ± 49.1% of ontrol, n = 6; P =.58; Fig. 3). These t suggest tht phosphoryltion t its Homer ining site meites s effet of inresing Homer ining to. We next investigte whether regultes group I mglur funtion. In ellulr ssy, group I mglur oupling to voltgesensitive lium hnnels in superior ervil gnglion neurons, whih o not ntively express ny mglurs 24, ws inhiite y Homer ining 7 n ws similrly inhiite y (mglur1: 55 ± 6% in ontrol, n = 15; 16 ± 8% in, n = 11; P =.1; : 38 ± 5% in ontrol, n = 24; 22 ± 6% in, n = 22; P =.4; Fig. 3,). Experiments with mutnts inite tht s tion epene on ining to (sent with YVF), Homer ining to (sent with FR) n phosphoryltion of the Homer ining site in n mglur1 (sent with TSAA n mglur1tsaa) (YVF: 26 ± 1% in ontrol, n = 7; 25 ± 6% in, n = 8, P =.9; FR: 26 ± 1% in ontrol, n = 7; 23 ± 1% in, n = 11, P =.66; TSAA: 16 ± 1% in ontrol, n = 9; 16 ± 6% in, n = 13, P =.98; mglur1tsaa: 43 ± 12% in ontrol, n = 14; 41 ± 16% in, n = 6, P =.88; Fig. 3). tion is speifi for group I mglur, s it i not lter the tivity of mglur2 (64 ± 4% α β γ nture NEUROSCIENCE vne online pulition

4 212 Nture Ameri, In. All rights reserve. Figure 3 enhnes phosphoryltion n Homer ining, n inhiits group I mglur oupling to voltge-gte C 2 hnnels. () HA-tgge Homer1, HA-tgge n HA-tgge onstruts were otrnsfete into HEK293T ells. Lystes were immunopreipitte with nti-homer1 n nlyze y western lotting with nti- HA. Lystes were lso lotte with nti pser-mglur (right). () -epenent enhnement of Homer ining requires phosphoryltion sites of, ut oes not require Homer ining to. HA-tgge Homer1, HA-tgge TSAA n HA-tgge FR onstruts were otrnsfete into HEK293T ells. Sme nlysis s. n = 6 for eh group. (,) Coupling of group I mglurs to voltgegte lium hnnels in superior ervil gnglion neurons. () Smple urrents (inset) n time ourse illustrting reution of inhiition y 1 µm glutmte ut not 1 µm norepinephrine (NE) in superior ervil gnglion neuron expressing with or without. Con, ontrol; Glu, glutmte. in ontrol, n = 7; 57 ± 12% in, n = 5, P =.3; Fig. 3), onsistent with the fining tht seletively oun group I mglurs (Supplementry Fig. 4 ). ins proline-irete kinses Inspetion of the Homer ining site of group I mglur (Fig. 2) suggeste tht the serine phosphoryltion is meite y proline-irete kinses, suh s CDK5 n ERK. We foun tht expression of CDK5 n p35 (CDK5/p35) or onstitutively tive MEK (MEK DD), whih tivtes ERK, inue phosphoryltion in HEK293T ells (CDK5/p35: 563 ± 98% of ontrol, n = 6; P =.8; MEK DD: 1,49 ± 345% of ontrol, n = 6; P =.6; Fig. 4). In ulture neurons, ntive mglur phosphoryltion ws signifintly reue y CDK5 inhiitor or MEK inhiitor (UO126: 57.4 ± 11% of untrete, n = 4; P =.7; purvlnol A: 65.8 ± 9.5% of untrete, n = 4; P =.2; Fig. 4). These t support role for CDK5 n ERK in phosphoryltion of group I mglur t the Homer ining site. We next hypothesize tht ins proline-irete kinses tht phosphorylte group I mglurs. ontins preite D-omin tht is onserve mong Preso fmily memers n ross speies (Supplementry Figs. 1 n 2). As preite, immunopreipitte from HEK293T ells with proline-irete kinses CDK5 n ERK (Fig. 4,). Aitionlly, enhne the ssoition of ERK1 with in HEK293T ells (166.6 ± 4% of ontrol, n = 5; P =.2; Fig. 4e). Enogenous immunopreipitte with CDK5 n with ERK1 n/or ERK2 (ERK1/2) from mouse rin (Fig. 4f,g), suggesting tht n proline-irete kinses re nturlly ssoite in vivo. / mouse genertion We generte onitionl / y inserting loxp sites in flnking introns of exon 3, whih enoes the PDZ omin Homer1: Homer1 Homer1 IP : : Current (na) Current (na) Glu Lyste Glu () Expression of reues mglur1/5-epenent moultion of lium urrent y glutmte, n requires Homer ining n phosphoryltion sites of mglur1/5. Summry of t from ells expressing vrious mglur onstruts with or without : YVF (oes not in Homer or ), FR (oes not in Homer), mglur1/5tsaa (ins Homer ut nnot e phosphorylte t the Homer ining site). i not lter response of mglur2. n = 15, 11, 24, 22, 7, 8, 7, 11, 9, 13, 14, 6, 7, 5, from left to right; P <.1. Error rs, 95% onfiene intervls. Full-length western lots for this figure re shown in Supplementry Figure 11. NE NE Lyste ps-mglur Glu Con Glu Con Homer1: I C inhiition (%) : TSAA: FR: Homer mglur1 Homer1 IP Lyste (Supplementry Fig. 5). ws elete in the germline y mting onitionl knokout mie with CMV-re mie. / mie were ientifie y PCR (t not shown), n mutnt mrna expression ws onfirme y reverse trnsription n PCR of ortex n ereellum (Supplementry Fig. 5). eletion ws further verifie y western lots of etergent lystes of / rin (Supplementry Fig. 5) n ulture ortil neurons (Supplementry Fig. 5). Mle n femle / mie were vile n fertile, n pper similr to mie in their postntl growth. Expression of Homer1/, Homer2, Homer3, NR1, GluA1 n GluA2/3 ws not ltere in 6-week-ol / rin, n expression of ws not ifferent in ortex or spinl or (Supplementry Fig. 5e). Consistent with the hypothesize funtion of, phosphoryltion (pser-mglur) n immunopreipittion with Homer ws reue in / ortex n spinl or ompre those in to littermtes (ining: 76 ± 3.3% of in ortex, n = 5; P =.2; 73.6 ± 8.7% of in spinl or, n = 8; P =.3; phosphoryltion: 57.5 ± 22.2% of in ortex, n = 5; P =.4; 79.1 ± 1.6% of in spinl or, n = 5; P =.3; Fig. 5). Aitionlly, immunopreipittion of ERK1/2 with ws reue in / rin (64.4 ± 1.9% of, n = 5; P =.4; Fig. 5). is require for ynmi Homer ining We exmine the hypothesis tht is require for tivityepenent phosphoryltion of n enhne Homer ining. In neurons, the group I mglur gonist 3,5-ihyroxyphenylglyine (DHPG) resulte in n inrese of phosphoryltion n Homer ining to fter 3 min (ining: 157 ± 33.8% of untrete, n = 5; P =.1; phosphoryltion: 149 ± 36% of untrete, n = 5; P =.2; Fig. 5). By ontrst, phosphoryltion n Homer ining were reue in / neurons n were not FR YVF Homer1 IP Lyste TSAA mglur1 TSAA mglur2 vne online pulition nture NEUROSCIENCE

5 HA-: HA-CDK5/p35: HA-MEK DD: pser-mglur MEK DD p35 CDK5 HA-: HA-ERK1: ERK1 IP: nti- Lyste 28 kd pser- (% of ontrol) 1,4 1,2 1, e CDK5/p35 MEK DD HA-: HA-: HA-ERK1: imer monomer ERK1 pser-mglur IP: nti- Lyste UO126 Purvlnol A pser- (% of ontrol) f CDK Lyste PI UO126 Purvlnol A IP IP: nti-cdk5 28 kd HA-: HA-CDK5: CDK5 g IP: nti- Lyste ERK1/2 Lyste PI IP IP: nti-erk 28 kd 212 Nture Ameri, In. All rights reserve. Figure 4 ins proline-irete kinses n enhnes kinse- ining. () CDK5/p35 or MEK DD expression inreses phosphoryltion. HA-tgge n HA-CDK5/HA-p35 or HA-MEK DD (onstitutively tive) were otrnsfete into HEK293T ells. Lystes were lotte with nti-, nti pser-mglur n nti-ha. Quntittion (elow): P <.1, n = 3 eh group. () Inhiitors of MEK (UO126, 4 µm) or CDK5 (purvlnol A, 2 µm) for 3 min reue sl phosphoryltion of in ortil neurons t 14 ys in vitro (DIV). ws immunopreipitte n lotte with or pser-mglur. Quntittion (,, elow): P <.5, P <.1, n = 4 for eh group. () CDK5 immunopreipittes with from HEK293T ells. () ERK1 immunopreipittes with from HEK293T ells. (e) inreses immunopreipittion of ERK1 with. HA-tgge n HA-tgge were otrnsfete into HEK293T ells, n lystes were preipitte with nti- n lotte with nti-ha n nti-erk1. (f) immunopreipittes with CDK5 from mouse rin etergent lyste. (g) immunopreipittes with ERK1/2 from mouse rin etergent lyste. PI, preimmune; IP, immunopreipittion. Error rs, 95% onfiene intervls. Full-length western lots for this figure re shown in Supplementry Figure 12. hnge fter DHPG stimultion (ining: 47 ± 7.7% in untrete /, n = 5; P =.7; 54 ± 12.9% in trete /, n = 3; P =.3; phosphoryltion: 71.7 ± 22.4% in untrete /, n = 5; P =.4; 67.3 ± 14.8% in trete /, n = 5; P =.76; Fig. 5). This is onsistent with tivtion of CDK5 n ERK 25, whih then phosphorylte the reeptor to meite enhne Homer ining in reeptor homologous feek pthwy. We lso exmine the effet of BDNF, whih tivtes TrkB to inrese ERK 26, n foun similr inreses of phosphoryltion n Homer ining in ut not / neurons (ining: ± 14.5% in trete, n = 4; P =.1; 65.3 ± 18% in untrete /, n = 4; P =.2; 69.8 ± 22.9% in trete /, n = 4; P =.77; phosphoryltion: ± 15.1% of untrete, n = 5; P =.2; 66.8 ± 9.5% in untrete /, n = 4; P =.2; 79.7 ± 8.3% in trete /, n = 4; P =.9; Fig. 5). These oservtions suggest tht is require for ynmi regultion of phosphoryltion n Homer ining in oth homologous n heterologous reeptor signling pthwys. Tmlin 27, lineurin inhiitor protein (CAIN) 28 n Norin 29 in group I mglurs n moify signling y hnging expression of mglur on the ell surfe. expression on the surfe of neurons in ulture ws not ifferent etween n / (115.6 ± 21.6% of, n = 6; P =.14; Fig. 5). Furthermore, DHPG inue ientil ereses of surfe (65.4 ± 16.7% in, n = 4; P =.2; 65.2 ± 1.1% in /, n = 3; P =.4; Fig. 5), suggesting tht oes not funtion y regulting surfe expression. This is onsistent with the oserve sene of n effet of Homer1 / Homer2 / Homer3 / genotype on surfe expression 3. Pin response is inrese in / mie Formlin injetion into the mouse hin pw results in n initil, trnsient ehviorl pin response tht is followe y seon, sustine response tht is ssoite with tissue inflmmtion n is terme inflmmtory pin 31. This moel is wiely use in pin reserh, n group I mglurs hve een implite in inflmmtory pin y mens of phrmologil gonists n ntgonists 3. To further vlite the role of, we exmine Grm5 / mie n onfirme erese pin responses in the seon phse, ut not in the first phse, fter formlin injetion ompre with responses in their littermtes (Supplementry Fig. 6,). Furthermore, 2-methyl-6-(phenylethynyl)-pyriine (MPEP), n speifi ntgonist, sustntilly reue the formlininue inflmmtory pin response in mie, ut not in Grm5 / mie (Supplementry Fig. 6,). These results onfirm role for in formlin-inue inflmmtory pin. We next ske whether formlin-inue inflmmtory pin is ltere in / mie. / mie exhiite greter ehviorl pin responses uring the seon phse, ut not the first phse, ompre with their littermtes (first phse: 94.6 ± 24.2 s in, n = 9; 88 ± 25 s in /, n = 8; P =.72; seon phse: ± 52.8 s in, n = 9; ± s in /, n = 8; P =.2; Fig. 6,). Notly, MPEP (3 mg per kilogrm oy weight, intrperitonelly) erese pin responses uring the seon phse to the sme egree in / n mie (first phse: 68.7 ± 11.7 s in trete, n = 6; P =.13; 76.8 ± 24.7 s in trete /, n = 6; P =.56; seon phse: 27 ± 33.5 s in trete, n = 6; P =.2; ± 47.3 s in trete /, n = 6; P =.2; Fig. 6,). These t inite tht the inrese inflmmtory pin response in / is meite y. Beuse ongoing sensory inputs ontriute to formlin-inue pin 32, we mesure the exitility of orsl root gnglion neurons from n / mie n foun similr exitilities (Supplementry Fig. 7). To ssess the notion tht Homer ining to is importnt to limit the intensity of the inflmmtory pin response, we onfirme tht inflmmtory pin ehvior ws inrese in Homer2 / Homer3 / mie, n in n mutnt nture NEUROSCIENCE vne online pulition

6 212 Nture Ameri, In. All rights reserve. Figure 5 is require for tivityepenent inrese of phosphoryltion n Homer ining. () mglur phosphoryltion n Homer oimmunopreipittion re erese in ortex n spinl or of / mie. Cortex (Cx) n spinl or (S) lystes from n / mie were lotte with nti pser-mglur or immunopreipitte with pn-nti-homer n lotte with inite ntioies. P <.5, P <.1, n = 5 8 for eh group. () ERK1/2 immunopreipittion with is erese in / mie. Forerin lystes from n / mie were immunopreipitte with nti-, n the lystes (ottom pnels) n immunopreipittes were lotte with nti- ERK or nti-. () Ativtion of or BDNF reeptor TrkB inreses serine phosphoryltion n Homer oimmunopreipittion in ut not / DIV 14 ortil neurons. Cultures were trete with DHPG (DH; 1 µm) or BDNF (BD; 2 ng ml 1 ) for 3 min n lyse for pn-homer immunopreipittion n western lot. Ctl, ontrol. Quntittion (right): P <.5, P <.1, n = 3 8 for eh group. () Surfe n totl expression of is not ifferent etween n / DIV 14 ortil neurons. Cultures were trete with DHPG (1 µm) for 5 min or 3 min n proesse for surfe iotinyltion n western lotting. Quntittion (right): P <.5, n = 3 6 for eh group. D5, DHPG 5 min; D3, DHPG 3 min. Error rs, 95% onfiene intervls. Full-length western lots for this figure re shown in Supplementry Figure 13. knok-in mouse in whih Homer nnot in the reeptor (Grm5 R/R or FR mie 33 Atin/Totl ) Atin/Surfe (Supplementry Fig. 8 ). Furthermore, we foun tht inflmmtory pin ehvior ws erese in mie with seletive eletion of the Homer1 isoform of the Homer1 gene (Homer1 / mie) 3 (Supplementry Fig. 8e,f ), onsistent with mehnism wherey eletion of Homer1 inreses Homer ining with. -Fos expression is inue in neurons of the lumr orsl spinl or y formlin injetion into the hin pw, n it ientifies neurons tivte in the pin iruit tht inrese ellulr [C 2 ] 34. We performe -Fos immunostining in lumr level L4 L5 spinl or from n / mie fter formlin injetion n foun more -Fos positive neurons in the / spinl or thn in spinl or (lmin 1 2: 19.6 ± 2.7 in, n = 25; 26.6 ± 4.1 in /, n = 22; P =.7; lmin 3 4: 6.1 ± 1. in, n = 25; 8.7 ± 2.1 in /, n = 18; P =.3; lmin 5 6: 5.8 ± 1.1 in, n = 25; 11.1 ± 2.4 in /, n = 18; P =.1; Fig. 6,). Furthermore, preinjetion with MPEP reue the numer of -Fos positive neurons in the n / spinl or to the sme egree (lmin 1 2: 14.9 ± 2.8 in trete, n = 18; P =.3; 13.6 ± 2.22 in trete /, n = 18; P =.9; lmin 3 4: 3.7 ±.7 in trete, n = 18; P =.1; 5.2 ±.8 in trete /, n = 18; P =.9; lmin 5 6: 6.2 ± 1.2 in trete, n = 18; P =.62; 7.8 ± 1.2 in trete /, n = 18; P =.3; Fig. 6,). These finings orroorte ehviorl pin ssys n suggest tht / inreses the numer of neurons in the spinl or pin pthwy tht reh ritil level of [C 2 ] for -Fos inution, through -epenent mehnism. /Lyste /o-ip pser-mglur Homer/IP /Lyste /Lyste /o-ip ps-mglur Atin/Lyste Homer/IP /Totl /Surfe Cx S / / / / Ctl DH Ctl DH Ctl BD Ctl BD / / Ctl D5 Ctl D5 Ctl D3 Ctl D3 Homer ining (% of ) pser-mglur (% of ) / Co-IP/lyste (% of tl) Co-IP/lyste (% of tl) Surfe/totl (% of tl) Cx Cx S / / Ctl BD 16 We lso teste pin responses in the omplete Freun s juvnt (CFA) hroni pin moel. / mie were not ifferent from littermtes in their sl therml pin responses, n they showe n ientil initil pin response (: 13.6 ± 2.2 s, n = 9; / : 13.6 ± 3.3 s, n = 9; P =.96; Fig. 6e). However, / mie exhiite inrese therml pin responses eginning 3 fter CFA injetion n persisting through 5 (y 1: 6.9 ± 2.1 s in, n = 9; 6.6 ± 2.6 s in /, n = 9; P =.83; y 2: 1.2 ± 5. s in, n = 9; 7.3 ± 5.5 s in /, n = 9; P =.26; y 3: 11.7 ± 3.6 s in, n = 9; 7.5 ± 3.3 s in /, n = 9; P =.2; y 4: 11.1 ± 2.9 s in, n = 9; 2.6 ± 2.4 s in /, n = 9; P =.3; y 5: 13.6 ± 4.2 s in, n = 9; 9.9 ± 2.2 s in /, n = 9; P =.3; Fig. 6e). Enhne mglur-meite C 2 response in / neurons is expresse in the orsl spinl or (Supplementry Fig. 3) with n expression pttern similr to 35. On the sis of the inrese ehviorl pin n -Fos expression in spinl or of / mie, we preite tht group I mglur funtion woul e enhne in / orsl spinl or neurons. We ulture these neurons from n / mie n performe lium imging ssys. Glutmte (1 µm) omine with APV (1 µm), n NMDA reeptor loker, n NBQX (1 µm), n AMPA reeptor loker, were use to stimulte mglur. This oktil ws foun to e more onsistent thn seletive gonist for (DHPG), s is onsistent S Ctl D5 ERK1/2/o-IP /IP ERK1/2/Lyste /Lyste ps-mglur (% of tl) ps-mglur (% of tl) Ctl DH / / Surfe/totl (% of tl) / Ctl D3 / / vne online pulition nture NEUROSCIENCE

7 212 Nture Ameri, In. All rights reserve. Durtion of response (s) Con / / MPEP / / MPEP / / MPEP / / MPEP Time fter formlin injetion (min) Ip Numer of -Fos positive ells per setion fter formlin injetion e Response lteny (s) with the oservtion tht mglur1 signling is enhne y preeing epolriztion 36. Stimultion inue rise of [C 2 ] in oth n / neurons tht persiste until wshout (Fig. 7). The lium response in / neurons ws signifintly greter thn tht in neurons (: 3.28 ±.33 fol inrese over sl C 2, n = 9; / : 4.47 ±.44 fol inrese over sl C 2, n = 74; P =.3; Durtion of response (s) Lmin 1 2 / / MPEP / / MPEP 1 min 1 6 min Bsl Dy 1 Dy 2 / / MPEP / / MPEP Lmin 3 4 / Dy 3 Dy 4 Dy 5 Lmin 5 6 Figure 6 Inrese pin response to formlin or omplete Freun s juvnt in / mie. (,) Formlin-inue inflmmtory pin is inrese in / mie n is epenent on. () The urtion of ehviorl responses to hin pw formlin injetion in 5-min intervls ompring littermte n / mie with or without MPEP efore injetion. () Results from groupe into two phses. P <.5, P <.1, n = 6 9 for eh group. (,) Formlin-inue -Fos expression in / mie. () -Fos immunohistohemistry of L4 L5 of spinl or 9 min fter formlin. Boxe regions re mgnifie t right. Sle rs, 5 µm t left, 2 µm t right. Con, ontrlterl; Ip, ipsilterl. () Sttistil t from. n = setions from 3 5 mie in eh group. P <.5, P <.1. (e) Pin response to omplete Freun s juvnt is inrese in / mie. Lteny to withrwl ws monitore efore n for 5 fter injetion of omplete Freun s juvnt into hin pw of littermte n / mie. P <.5, n = 9 for eh group. Error rs, 95% onfiene intervls. Fig. 7). Pretretment with MPEP (1 µm) n with By (2 µm), n mglur1 ntgonist, loke the sustine lium response (Fig. 7), onfirming tht it is epenent on group I mglur. We next ske whether the enhne mglur-meite lium response in / spinl or neurons woul e resue y expressing trnsgene. GFP or GFP- onstruts were eletroporte into orsl spinl or neurons from / mie on the y of ulture initition n use for lium imging fter 2. GFP- expressing neurons showe mrkely reue mglur-meite lium response ompre to GFP expressing neurons (GFP: 2.38 ±.36 fol inrese over sl C 2, n = 23; GFP-:.46 ±.35 fol inrese over sl C 2, n = 19; P = ; Fig. 7), initing tht inrese C 2 responses in / neurons n e reverse y. Our moel preits tht filittes proline-irete kinses to inrese mglur phosphoryltion t the Homer ining site, n inhiits sustine mglur responses (Fig. 4). Aoringly, we ske whether the mglur-epenent C 2 response in spinl or neurons is ltere y proline-irete kinse inhiitors. We oserve tht the C 2 response to sustine tretment with glutmte, APV n NBQX erese over n intervl of 2 9 min in most neurons, ut [C 2 ] gin inrese until wshout in some neurons (Fig. 7). The perentge of neurons with C 2 rise ws signifintly higher in / thn tht in (: 11.5 ± 4.3%, n = 39; / : 22.3 ± 7.4%, 34 nm/38 nm rtio Stimulte Wsh out / Time (s) 34 nm/38 nm rtio Stimulte Wsh out Time (s) GFP GFP- 34 nm/38 nm rtio Kinse inhiitor Stimulte Without C 2 rise With C 2 rise Wsh out Time (s) Neurons with C 2 rise (%) UO126 Purvlnol A Figure 7 Enhne group I mglur-meite lium response in the spinl or neurons of / mie. () Representtive tres (left) n popultion (right) pek C 2 responses of n / DIV 14 orsl spinl or neurons stimulte with glutmte (1 µm), APV (1 µm) n NBQX (1 µm). Pretretment with group I mglur ntgonists By (mglur1 ntgonist, 2 µm) n MPEP ( ntgonist, 1 µm) loke the C 2 response. Clium onentrtion ws mesure y Fur-2 34 nm/38 nm rtio. n = 9 for neurons; n = 74 for / neurons. () A trnsgene reues C 2 response in DIV 2 / orsl spinl or neurons. Representtive tres n popultion responses of / neurons expressing GFP or GFP-. Sme ssy s in ; n = 23 for GFP neurons, n = 19 for GFP- neurons. () MEK or CDK5 inhiitors inrese the perentge of neurons showing elye C 2 inrese in ut not / orsl spinl or neurons. Neurons were stimulte s in, n fter 2 min, kinse inhiitors UO126 (4 µm) or purvlnol A (5 µm) or vehile (ontrol) were e, followe y wshout t 1 min. Left pnel shows tres of C 2 responses from neurons, with exmple of elye rise of [C 2 ] tht reverses on wshout. For neurons, n = 39 neurons in 8 experiments, 42 neurons in 9 experiments n 33 neurons in 1 experiments for vehile, UO126 n purvlnol A, respetively. For / neurons, n = 36 neurons in 8 experiments, 27 neurons in 7 experiments n 19 neurons in 6 experiments for vehile, UO126 n purvlnol A onitions, respetively. P <.5, P <.1. Error rs, 95% onfiene intervls. / nture NEUROSCIENCE vne online pulition

8 212 Nture Ameri, In. All rights reserve. n = 36; P =.3; Fig. 7). The reversl y wshout n susequent ell viility ssesse y Nomrski mirosopy inite tht the elye response ws not ue to ell eth. Aition of UO126 (4 µm), or purvlnol A (5 µm) 2 min fter onset of stimultion resulte in two- to threefol inrese of the perentge of neurons exhiiting elye rise of [C 2 ] (UO126: 28.3 ± 5.7%, n = 42; P =.4; purvlnol A: 23.8 ± 5.6%, n = 33; P =.5; Fig. 7). By ontrst, kinse inhiitors i not hnge the perentge of / spinl or neurons showing this elye response (UO126: 27.6 ± 6.6%, n = 27; P =.32; purvlnol A: 25. ± 5.3%, n = 19; P =.6; Fig. 7). Although kinse inhiitors re likely to lter multiple signling pthwys, their ute tion in loking mglur-epenent inreses of [C 2 ] is onsistent with our moel of funtion. DISCUSSION The present stuy inites tht funtions s n essentil prt of the group I mglur signling omplex, n supports its role s n nhoring protein for proline-irete kinses tht meite tivityepenent, negtive regultion of mglur. The prolineirete kinse Homer mehnism is highly ynmi, n finings support onsistent moel ross iohemil n ehviorl phenotypes in severl geneti moels tht moify Homer expression or Homer ining to mglur. This mehnism is istint from the nonil mehnism of GPCR esensitiztion meite y G protein ouple reeptor kinses n β-rrestin pthwy 37 in severl wys. First, n ynmi Homer ining o not influene the level of surfe mglur expression either in stey stte onitions or fter tivtion of the mglur reeptor. Inhiition of mglur signling ppers to result s iret onsequene of Homer ining rther thn of removl from the plsm memrne. Seon, the prolineirete kinses tht meite this response re not speifi for the reeptor, ut rther re tivte y mny signling pthwys. This ffors the possiility of reeptor rosstlk, ut it lso highlights the importne of n nhoring protein for mglur tht n onfer speifiity to ro-sustrte proline-irete kinses n therey mimi the intrinsi speifiity of G protein ouple reeptor kinses. Group I mglurs reportely esensitize in response to G protein ouple reeptor kinses 38, ut the nturl relevne of the suggeste pthwys remins unknown. The mehnism is istint from other proteins tht in group I mglur n moify signling y hnging expression of mglur on the ell surfe, inluing Tmlin 27, lineurin inhiitor protein (CAIN) 28 n Norin 29. ppers to limit the mplitue n urtion of tivtion tht unerlies inflmmtory pin in the formlin moel n hroni pin in the CFA moel. -epenent inhiition of my our normlly s prt of homologous reeptor ownregultion ue to inrese tivtion y glutmte, or s heterologous reeptor response ue to inrese proline-irete kinse tivtion y reeptors suh s TrkB 26 or the rykinin reeptor 39. Phosphorylte ERK is upregulte uring inflmmtory pin n hs een implite s meitor of neuronl exitility ue to phosphoryltion of potssium hnnel 4. CDK5 expression n tivity is lso elevte uring inflmmtion n regultes pin signling 41. Previous stuies hve emonstrte tht group I mglur signling eomes gonist inepenent if Homer oes not in n rosslink the reeptor 14. This mehnism unerlies the effet of Homer1 in riving homeostti sling of AMPA reeptors 3 n hs een suggeste to ontriute to stress effets in fer onitioning 42. Aoringly, gonistinepenent signling my ontriute to inflmmtory pin. Pin inue y inflmmtion lso uses entrl sensitiztion n hypersensitivity 43, n reent stuies suggest speil role for the mygl 44. Hene, it is possile tht lso ontriutes to entrl sensitiztion through regultion of mglur funtion. Group I mglurs re importnt trgets for therpies of ognitive iseses inluing frgile X mentl retrtion synrome 45 n Alzheimer s isese 5, n re notle for the ility of phrmologil gents to ifferentilly lter signling outputs (ise ligns) 46. It is possile tht ontriutes to this istintive phrmology. Although n Homer ining uniformly inhiite the responses of group I mglurs monitore here, group I mglurs signl in severl output pthwys (see introution), n Homer ining to oes not uniformly inhiit ll outputs. For exmple, mglur1 oupling to intrellulr IP 3 reeptors is inrese y Homer rosslinking 47. Aoringly, the effet of on the signling of group I mglurs oul e output speifi. The yest protein STE5 provies preeent for proline-irete kinse nhoring protein 48, ut ppers to e the first exmple of proline-irete kinse nhoring protein for higher orgnisms. This tion is similr to tht of AKAPs, whih oorinte signl trnsution omplexes y reruiting multiple signling enzymes ner potentil sustrtes, inluing G protein ouple reeptors 49. Consistent with the notion tht funtions s n nhoring protein, inlues omins tht n intert with the ytoskeleton, inluing the R-tivtion omin, n hs een reporte to intert with β-pix 16. Mny GPCRs enoe preite FERM omin ining sites n D-omins (GPCR tses; 7tm/), suggesting tht Preso fmily memers my hve roer role in GPCR signling. However, only group I mglurs ontin Homer ining site tht is lso onsensus for proline-irete kinses, whih preits tht Presos uniquely oorinte ynmi hnges of Homer ining to group I mglur. Preso2 n Preso3 mrnas re roly expresse in the nervous system, ut they hve not een exmine for their role in regulting GPCR signling. Presos re lso expresse outsie of the entrl nervous system 18, n they will e importnt for omprehensive unerstning of GPCR signling. Methos Methos n ny ssoite referenes re ville in the online version of the pper. Note: Supplementry informtion is ville in the online version of the pper. Aknowlegments We thnk J. Roer of University of Toronto for Grm5 / mie, J. Worley for help with ehviorl experiments n Y.-X. Wng for help with the immunogol. This work ws supporte y US Ntionl Institutes of Helth grnts from the Ntionl Institute on Drug Ause (DA139), Ntionl Institute of Mentl Helth (MH842) n Ntionl Institute of Neurologil Disorers n Stroke (NS5274 (P.F.W.); NS54791 n GM87369 (X.D.)); Ntionl 973 Bsi Reserh Progrm of Chin (29CB9414; B.X.); n the Ntionl Institute on Defness n Other Communition Disorers Intrmurl Reserh Progrm (R.S.P.). AUTHOR CONTRIBUTIONS J.-H.H. esigne, performe n nlyze experiments inlue in Figures 1 7 n Supplementry Figures 1 6 n 8, n wrote first rft of the mnusript. L.Y. performe n nlyze experiments in Figures 1 n 2 n Supplementry Figure 4. P.J.K. performe n nlyze eletrophysiology experiments in Figure 3. C.G.M. generte n ientifie the phospho-mglur ntioy in Supplementry Figure 4. P.R.B. performe the yest-two hyri sreen tht ientifie s Homer ining protein. J.T. n B.X. lone n i initil hrteriztions. S.Y. generte the ntioy. R.S.P. performe the experiments in Figure 1. Z.L. performe n nlyze experiments in Supplementry Figure 7. P.-W.Z. generte Grm5 R/R mie. J.M.P. performe eletrophysiologil experiments. X.D. provie intelletul input n tehnil support on the ehviorl experiments n orsl root gnglion neuron reorings. B.X. generte ntioies, esigne n performe experiments in Figure 1 n provie intelletul input n tehnil support for / vne online pulition nture NEUROSCIENCE

9 212 Nture Ameri, In. All rights reserve. mouse genertion. P.F.W. supervise the overll projet, esigne experiments n wrote the finl version of the mnusript. COMPETING FINANCIAL INTERESTS The uthors elre no ompeting finnil interests. Pulishe online t Reprints n permissions informtion is ville online t reprints/inex.html. 1. Lüsher, C. & Huer, K.M. Group 1 mglur-epenent synpti long-term epression: mehnisms n implitions for iruitry n isese. Neuron 65, (21). 2. Chimuler, C. et l. Reinforing n loomotor stimulnt effets of oine re sent in null mutnt mie. Nt. Neurosi. 4, (21). 3. Bhve, G., Krim, F., Crlton, S.M. & Gereu, R.W.t. Peripherl group I metotropi glutmte reeptors moulte noieption in mie. Nt. Neurosi. 4, (21). 4. Niswener, C.M. & Conn, P.J. Metotropi glutmte reeptors: physiology, phrmology, n isese. Annu. Rev. Phrmol. Toxiol. 5, (21). 5. Lujn, R., Nusser, Z., Roerts, J.D., Shigemoto, R. & Somogyi, P. Perisynpti lotion of metotropi glutmte reeptors mglur1 n on enrites n enriti spines in the rt hippompus. Eur. J. Neurosi. 8, (1996). 6. Torres, G.E. & Amr, S.G. Glutmte n monomine trnsporters: new visions of form n funtion. Curr. Opin. Neuroiol. 17, (27). 7. Kmmermeier, P.J., Xio, B., Tu, J.C., Worley, P.F. & Ike, S.R. Homer proteins regulte oupling of group I metotropi glutmte reeptors to N-type lium n M-type potssium hnnels. J. Neurosi. 2, (2). 8. Tu, J.C. et l. Homer ins novel proline-rih motif n links group 1 metotropi glutmte reeptors with IP3 reeptors. Neuron 21, (1998). 9. Prk, S. et l. Elongtion ftor 2 n frgile X mentl retrtion protein ontrol the ynmi trnsltion of Ar/Arg3.1 essentil for mglur-ltd. Neuron 59, 7 83 (28). 1. Wng, J.Q., Fiuh, E.E. & Mo, L. Regultion of mitogen-tivte protein kinses y glutmte reeptors. J. Neurohem. 1, 1 11 (27). 11. Hou, L. & Klnn, E. Ativtion of the phosphoinositie 3-kinse-Akt-mmmlin trget of rpmyin signling pthwy is require for metotropi glutmte reeptor-epenent long-term epression. J. Neurosi. 24, (24). 12. Glnte, M. & Din, M.A. Group I metotropi glutmte reeptors inhiit GABA relese t interneuron-purkinje ell synpses through enonninoi proution. J. Neurosi. 24, (24). 13. Brkemn, P.R. et l. Homer: protein tht seletively ins metotropi glutmte reeptors. Nture 386, (1997). 14. Ango, F. et l. Agonist-inepenent tivtion of metotropi glutmte reeptors y the intrellulr protein Homer. Nture 411, (21). 15. Dhvn, R. & Tsi, L.H. A ee of CDK5. Nt. Rev. Mol. Cell Biol. 2, (21). 16. Lee, H.W. et l. Preso, novel PSD-95-interting FERM n PDZ omin protein tht regultes enriti spine morphogenesis. J. Neurosi. 28, (28). 17. Zhong, H. et l. Suellulr ynmis of type II PKA in neurons. Neuron 62, (29). 18. An, N., Blumer, J.B., Bernr, M.L. & Lnier, S.M. The PDZ n n 4.1 ontining protein Frmp1 regultes the suellulr lotion of tivtor of G-protein signling 3 n its intertion with G-proteins. J. Biol. Chem. 283, (28). 19. Xio, B. et l. Homer regultes the ssoition of group 1 metotropi glutmte reeptors with multivlent omplexes of homer-relte, synpti proteins. Neuron 21, (1998). 2. Hoover, K.B. & Brynt, P.J. The genetis of the protein 4.1 fmily: orgnizers of the memrne n ytoskeleton. Curr. Opin. Cell Biol. 12, (2). 21. Shrroks, A.D., Yng, S.H. & Glnis, A. Doking omins n sustrte-speifiity etermintion for MAP kinses. Trens Biohem. Si. 25, (2). 22. Hung, G.N. et l. NFAT ining n regultion of T ell tivtion y the ytoplsmi sffoling Homer proteins. Siene 319, (28). 23. Orlno, L.R. et l. Phosphoryltion of the homer-ining omin of group I metotropi glutmte reeptors y ylin-epenent kinse 5. J. Neurohem. 11, (29). 24. Kmmermeier, P.J. & Ike, S.R. Expression of RGS2 lters the oupling of metotropi glutmte reeptor 1 to M-type K n N-type C2 hnnels. Neuron 22, (1999). 25. Gllgher, S.M., Dly, C.A., Ber, M.F. & Huer, K.M. Extrellulr signl-regulte protein kinse tivtion is require for metotropi glutmte reeptor-epenent long-term epression in hippompl re CA1. J. Neurosi. 24, (24). 26. Segl, R.A. & Greenerg, M.E. Intrellulr signling pthwys tivte y neurotrophi ftors. Annu. Rev. Neurosi. 19, (1996). 27. Kitno, J. et l. Tmlin, PDZ omin-ontining protein, links protein omplex formtion of group 1 metotropi glutmte reeptors n the gunine nuleotie exhnge ftor ytohesins. J. Neurosi. 22, (22). 28. Ferreir, L.T. et l. Clineurin inhiitor protein (CAIN) ttenutes Group I metotropi glutmte reeptor enoytosis n signling. J. Biol. Chem. 284, (29). 29. Wng, H. et l. Norin is n enogenous regultor of metotropi glutmte reeptor 5 signling. Siene 326, (29). 3. Hu, J.H. et l. Homeostti sling requires group I mglur tivtion meite y Homer1. Neuron 68, (21). 31. Hunskr, S. & Hole, K. The formlin test in mie: issoition etween inflmmtory n non-inflmmtory pin. Pin 3, (1987). 32. Puig, S. & Sorkin, L.S. Formlin-evoke tivity in ientifie primry fferent fiers: systemi lioine suppresses phse-2 tivity. Pin 64, (1996). 33. Cozzoli, D.K. et l. Binge rinking upregultes umens -Homer2 PI3K signling: funtionl implitions for loholism. J. Neurosi. 29, (29). 34. Sheng, M., MFen, G. & Greenerg, M.E. Memrne epolriztion n lium inue -fos trnsription vi phosphoryltion of trnsription ftor CREB. Neuron 4, (199). 35. Alvrez, F.J., Villl, R.M., Crr, P.A., Grnes, P. & Somohno, P.M. Differentil istriution of metotropi glutmte reeptors 1, 1, n 5 in the rt spinl or. J. Comp. Neurol. 422, (2). 36. Kim, S.J. et l. Trnsient upregultion of postsynpti IP3-gte C relese unerlies short-term potentition of metotropi glutmte reeptor 1 signling in ereellr Purkinje ells. J. Neurosi. 28, (28). 37. Ginetinov, R.R., Premont, R.T., Bohn, L.M., Lefkowitz, R.J. & Cron, M.G. Desensitiztion of G protein-ouple reeptors n neuronl funtions. Annu. Rev. Neurosi. 27, (24). 38. Dhmi, G.K. & Ferguson, S.S. Regultion of metotropi glutmte reeptor signling, esensitiztion n enoytosis. Phrmol. Ther. 111, (26). 39. Bernier, S.G., Hlr, S. & Mihel, T. Brykinin-regulte intertions of the mitogen-tivte protein kinse pthwy with the enothelil nitri-oxie synthse. J. Biol. Chem. 275, (2). 4. Hu, H.J., Alter, B.J., Crrsquillo, Y., Qiu, C.S. & Gereu, R.W. Metotropi glutmte reeptor 5 moultes noieptive plstiity vi extrellulr signl-regulte kinse- Kv4.2 signling in spinl or orsl horn neurons. J. Neurosi. 27, (27). 41. Utrers, E., Futtsugi, A., Preek, T.K. & Kulkrni, A.B. Moleulr Roles of Ck5 in Pin Signling. Drug Disov. Toy Ther. Strteg. 6, (29). 42. Tronson, N.C. et l. Metotropi glutmte reeptor 5/Homer intertions unerlie stress effets on fer. Biol. Psyhitry 68, (21). 43. Ltremoliere, A. & Woolf, C.J. Centrl sensitiztion: genertor of pin hypersensitivity y entrl neurl plstiity. J. Pin 1, (29). 44. Neugeuer, V., Li, W., Bir, G.C., Bhve, G. & Gereu, R.W.t. Synpti plstiity in the mygl in moel of rthriti pin: ifferentil roles of metotropi glutmte reeptors 1 n 5. J. Neurosi. 23, (23). 45. Ber, M.F. Therpeuti implitions of the mglur theory of frgile X mentl retrtion. Genes Brin Behv. 4, (25). 46. Roher, J.P. et l. negtive llosteri moultors overview: meiinl hemistry pproh towrs series of novel therpeuti gents. Curr. Top. Me. Chem. 11, (211). 47. Yun, J.P. et l. Homer ins TRPC fmily hnnels n is require for gting of TRPC1 y IP3 reeptors. Cell 114, (23). 48. Choi, K.Y., Sttererg, B., Lyons, D.M. & Elion, E.A. Ste5 tethers multiple protein kinses in the MAP kinse se require for mting in S. erevisie. Cell 78, (1994). 49. Wong, W. & Sott, J.D. AKAP signlling omplexes: fol points in spe n time. Nt. Rev. Mol. Cell Biol. 5, (24). nture NEUROSCIENCE vne online pulition

10 212 Nture Ameri, In. All rights reserve. ONLINE METHODS Expression onstruts. All the expression onstruts were me y PCR. Internl eletions n point muttions were me either using QuikChnge Site-Direte Mutgenesis Kit (Strtgene) or y megprimer metho. The sequene for eh mutnt will e supplie upon request. PCR prouts were lone into expression vetors pegfpc1 (Clonteh), pgex 4T2 (Phrmi), n prk5 (Genenteh), with My or HA tgs. All onstruts were verifie y sequening. Antioies. ntioies were generte in rits using the rt C terminl 2 or 33 s ntigens. The ntioy from 2- ntigen ws use for immunoeletron mirosopy (1:1), n the ntioy from 33- ntigen ws use for western lot (1:2,) n immunostining (1:1). Group I mglur phospho-ser ntioy ws generte in two femle rits using rt peptie (ELVALTPPpSPFRD) s ntigen (1:3, for western lot). These rit ntioies were generte y Covne, exept the ntioy ginst the 33- ntigen, whih ws generte t Sihun University uner rit protool pprove y Animl Cre n Use Committee of Sihun University. All other ntioies were previously esrie or were quire ommerilly, n their ilutions re s follows: (Upstte, 6-451) t 1:1,, mglur1 (BD, 61965) t 1:1,, IP 3 R (gift from Aln Shrp (Johns Hopkins University Shool of Meiine)) t 1:1,, Shnk (Neurom, lone N23B/49) t 1:2, PSD95 (Neurom, lone K28/43) t 1:5,, GluA1 (JH171) t 1:1, GluA2 (JH177) t 1:5, NR1 (Millipore, 5-432) t 1:1,, pn-homer (Snt Cruz, s-17842) for immunopreipittion, Homer1 (JH2621) t 1:5,, Homer2 (JH2623) t 1:5,, Homer3 (JH2793) t 1:5,, ERK (Cell Signling, 912) t 1:1,, CDK5 (Snt Cruz, s-173) t 1:1,, tin (Sigm-Alrih, lone AC-74) t 1:1,, -Fos (Cliohem, PC38) t 1:4,, HA-HRP (Rohe, lone 3F1) t 1:4,, My-HRP (Snt Cruz, lone 9E1) t 1:3,. Mouse moels. / mie were generte s follows. A 13-k NheI BAC frgment ontining exon 3 of ws sulone into the pbs vetor. Exon 3 ws relese y Bsu36I n BstBI n sulone into loxp/pgk-neo vetor. The trgeting onstrut ws generte y inserting the loxp/pgkneo/exon 3 k into Bsu36I n BstBI sites of the 13-k BAC frgment of the mouse gene (Supplementry Fig. 7). The resulting trgeting onstrut ws linerize n eletroporte into ES ells. ES ells were selete with G-418, n lones were pike, sreene y PCR, n onfirme y Southern lotting for homologous reomintion. Corretly trgete ES ell lones were injete into lstoysts, n himers were mte to C57BL/6 mie to proue floxe heterozygotes. These floxe heterozygotes were mte to CMV-re mie to proue / mie, whih were rosse to generte n / mie. Grm5 / ( / ) mie were otine from John Roer 5. Grm5 R/R (FR) mie reple the gene with mutnt tht oes not in Homer, n were esrie previously 33. Homer2 / Homer3 / mie were generte s previously reporte 22, n re helthy, fertile n not ifferent from mie in size or ehvior y sul oservtion. Mie were group house in plsti mouse ges with free ess to stnr roent how n wter. The olony room ws mintine t 22 ± 2 C with 12 h:12 h light:rk yle. / mie were krosse t lest five genertions onto C57/Bl6J mie, n ll the experiments were performe on littermte ontrols n lin to mouse genotype. Grm5 / mie, Grm5 R/R mie or Homer2 / Homer3 / mie n their ge-mthe, sme-kgroun mie were use in ll the experiments. Only ult mle mie were use for ehviorl experiments, n totl 25 mie were use for ll the experiments. All experiments were performe uner protools pprove y the Animl Cre n Use Committee of Johns Hopkins University Shool of Meiine for mie n rts. Cell ulture n trnsfetion. HEK293T ells were ulture in DMEM meium with 1% FBS. Trnsfetions were performe with Fugene 6 to mnufture s speifitions. Cells were hrveste 2 fter trnsfetion. Neuronl ulture n eletroportion. Neuronl ortil ultures from emryoni y 18 pups were prepre s reporte previously 3. Dorsl spinl or neurons were prepre from emryoni y 15 pups neurons were e to eh well of 12-well plte (Corning) with over slips ote with poly-l-lysine. Growth meium onsiste of MEM (Invitrogen) supplemente with 5% horse serum (Hylone), 2% B27, 1% glutmine (Invitrogen), 1 U/ml peniillin, n 1 U/ml streptomyin (Invitrogen). Neurons were fe twie per week with glionitione growth meium. DIV 14 neurons were use for lium imging. For the resue experiment, eletroportion ws performe using Nuleofetor kit (Amx) fter neuron issoition. Clium imging ws performe fter 2. Coimmunopreipittion ssys. Mouse rin tissues or HEK293T ells were use for the oimmunopreipittion ssy s previous reporte 3. Briefly, 5 miroliters of immunopreipittion uffer (PBS, ph 7.4, with 5 mm EDTA, 5 mm EGTA, 1 mm N 3 VO 4, 1 mm soium pyrophosphte, 5 mm NF, n 1% Triton X-1) ontining Complete EDTA-Free protese inhiitors (Rohe) ws e n smples were sonite. After entrifugtion, the superntnt (3 µl) ws then mixe with.5 2 µg of the pproprite ntioy for 3 h t 4 C. Then 5 µl of 1:1 protein A or protein G Sephrose slurry (Amershm-Phrmi Bioteh) ws e for n itionl 1 h. The protein es were wshe three times with immunopreipittion uffer ontining 1% Triton X-1. The protein smples were elute with SDS loing uffer n nlyze y gel eletrophoresis n western lotting. Surfe iotinyltion ssy. DIV 14 ortil neurons were use for surfe iotinyltion ssy s previously reporte 3. Briefly, ortil neurons were oole on ie, wshe twie with ie-ol PBS (PBS, 1 mm CCl 2,.5 mm MgCl 2 ) n then inute with PBS ontining 1 mg/ml sulfo-nhs-ss-iotin (Piere) for 3 min t 4 C. Unrete iotin ws quenhe y wshing ells three times with PBS ontining 1 mm glyine (ph 7.4) (riefly one n for 5 min twie). Cultures were hrveste in RIPA uffer n sonite. Homogentes were entrifuge t 13,2 r.p.m. (16,1g) for 2 min t 4 C. Fifteen perent of the superntnt ws sve s the totl protein. The remining 85% of the homogente ws rotte with streptviin es (Piere) for 2 h. Preipittes were wshe with RIPA uffer three times (5 min eh time). All proeures were one t 4 C. Immunoeletron mirosopy. Immunoeletron mirosopy ws performe y postfixtion immunogol leling s esrie 51, using rit ntioy ginst the C-terminl 2. Immunohistohemistry. Mouse spinl or L4 5 setions were wshe in PBS n inute with.3% H 2 O 2 in PBS for 3 min. After wshing with PBS, setions were then loke for 2 h t room temperture in loking solution. -Fos ntioy ws ilute in loking solution (1:4,) n inute with setions overnight t 4 C. After wshing with PBS six times, setions were proesse using n ABC kit (Vetor) oring to the mnufturer s instrutions. Slies were mounte n photogrphe with Nikon mirosope. Clium imging. HEK293T ells were loe t 37 C in CSF uffer with 1 µm Fur-2/AM for 45 min. Dorsl spinl or neurons were loe t room temperture in CSF uffer with 1 µm Fur-2/AM for 3 min. Rtiometri C 2 imging ws performe t 34 n 38 nm in 2 mm C 2 solution with Nikon Elipse 2-U inverte mirosope equippe with fluoresene r lmp, exittion filter wheel, n Hmmtsu Or CCD mer. Imges were ollete with Openl (Improvision) n nlyze with Igor Pro. Eletrophysiology. Group I mglur ouple lium urrent in the SCG neurons ws mesure s previously reporte 7. Briefly, oth gngli were remove from ult mle Wistr rts ( g, totl 14 rts) following CO 2 euthnsi n epittion, enzymtilly n mehnilly issoite, n ulture overnight t 37 C. Pth lmp experiments were performe the following y. All mglur1 n onstruts were injete t 1 13 ng µl 1 (pcdna3.1; Invitrogen); ws injete t 1 ng/µl where inite. All neurons were oinjete with enhne green fluoresent protein DNA (.2 µg / µl; pegfpn1; BD Biosienes-Clonteh, Plo Alto, CA) for ientifition of expressing ells. Whole-ell urrent-lmp reorings were me using ulture DRG neurons. DRG neurons were ulture on over slips for 1, n were trnsferre into hmer with meium (the extrellulr solution: ECS) of the following omposition (in mm): NCl 14, KCl 4, CCl 2 2, MgCl 2 2, HEPES 1, gluose 5, with ph juste to 7.39 using NOH n osmolrity juste to 31 mosm with surose. The intrellulr pipette solution (ICS) ontine (in mm): KCl 135, Mg-ATP 3, N 2 -ATP.5, CCl 2 1.1, EGTA 2, HEPES 1, gluose 5, with ph juste to nture NEUROSCIENCE oi:1.138\nn.313