© 2012 Nature America, Inc. All rights reserved. should should be addressed to P.F.W. ([email protected]). Baltimore, Maryland, USA. Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Chengdu, China. Physiology, University of Rochester Medical Center, Rochester, New York, USA. 1 the begs signaling plasticity mGluR modify to neuronal binding Homer of and ability The motility cell including differentiation, processes proliferation, cellular of control receptors the in factor acti critical growth are are and and kinases neurotransmitter multiple by Proline-directed vated kinases. proline-directed for neurons cell granule in of BK channels activation agonist-independent reduces also mGluRs Ca sensitive voltage and nels chan potassium M-type to coupling blocks neurons ganglion vical cer superior in expressed mGluRs I group to binding assay,Homer mGluRs I group of terminus C the in sequence Homer, family, an protein adaptor termed to bind that a proline-rich of outputs involve proteins of these for several mechanisms coupling kinase channels ion ion channels membrane by changes evoke modulating region synaptic central the in sites release from membrane synaptic post of the margin lateral the to primarily localize I mGluRs Group schizophrenia and addiction drug conditioning, fear pain, tory in important are they plasticity where neural brain, the in synapses excitatory at enriched are (mGluRs) receptors glutamate metabotropic I Group regulation. negative mediate and to mGluR to phosphorylate specificity substrate broad with kinases for proline-directed a microdomain creates Preso1 and of mGluR5, phosphorylation dynamic prevents of Preso1 ablation Genetic site. of at binding mGluR the Homer phosphorylation for their and is that required kinases and proline-directed Homer mGluR, binds that Preso1, protein, scaffolding a multidomain on is dependent mechanism This signaling. to reduce binding Homer, mGluR–Homer protein enhance the and adaptor thereby for site the binding phosphorylate that kinases by proline-directed regulated I are negatively in group mGluRs which mechanism an alternative we Here demonstrate molecules. of arrestin-like binding and subsequent kinases receptor G protein–coupled by specific regulated negatively are often GPCRs cord. in and spinal brain synapses at excitatory are that expressed (GPCRs) receptors are G and protein–coupled mGluR5, mGluR1 including (mGluRs), receptors glutamate I Group metabotropic Paul F Worley Shouyang Yu Jia-Hua Hu glutamate receptors Preso1 dynamically regulates group I metabotropic nature nature Received 16 February; accepted 3 April; published online 6 May 2012; Solomon Solomon H. Snyder Department of Johns Neuroscience, Hopkins University School of Medicine, Baltimore, Maryland, USA. The Homer binding site (TPPSPF) contains a consensus site site consensus a contains (TPPSPF) site binding Homer The Preso1 1 0 , mTORC1 (ref. (ref. mTORC1 , NEUR −/− 8 and signaling cascades including EF2 kinase EF2 including cascades signaling and 1 OSCI 4 mice exhibit sustained, mGluR5-dependent inflammatory pain that is linked to enhanced mGluR signaling. signaling. mGluR to is that enhanced pain linked inflammatory mGluR5-dependent sustained, exhibit mice , Linlin , YangLinlin National National Institute on Deafness and Other Disorders, Communication US National Institutes of Health, Bethesda, Maryland, USA. 3 1 , , Ronald S Petralia 1 n bhvoa rsoss nldn inflamma including responses behavioral and , 6 EN 5 , where they respond to glutamate as it diffuses diffuses it as glutamate to respond they where , C 7 1 E Present Present address: Department of Pediatrics, University of California, San Francisco, San Francisco, California, USA. Correspondence 4

11) and endocannabinoid synthesis endocannabinoid and 11) . advance online publication online advance 2+ channels 1 , , Paul J Kammermeier 4 7 . Homer binding to group I group to binding . Homer , , Zhe Li 6 1 . Group I mGluRs mGluRs I Group . 3 1 . In an exemplar exemplar an In . , , Ping-Wu Zhang 7 , intracellular , intracellular

2 doi:10.1038/nn.310 , , Chester G Moore 9 1 , MAP MAP , 2 . The The . 2– 1 3 5 4 - - - - - The The State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, . .

These studies support a model in which Preso1 functions to a support facilitate in studies functions Preso1 model These which Preso1 a in increased also was pain Homer. Inflammatory bind cannot that genetic deletion of to Homer or to of genetic knock-in either an mGluR5 point mutant owing mGluR5, to binding Homer interrupt that models mouse in increased was and on mGluR5 depended pain tory of I group mGluRs activation tained on sus dependent is which pain, of inflammatory model a in mouse To mechanism this assess Homer binding and downregulates mGluR signaling in enhances cellular assays.mGluR of phosphorylation Preso1-dependent substrates. (AKAPs) proteins anchoring A-kinase of that to analogous appears Preso1 of action This mGluR. Homer the site in binding to bind and mGluR phosphorylate kinases proline-directed of ability the enhances Preso1 ERK. and CDK5 ing that it also binds group I includ mGluR kinases and proline-directed for that bind Homer in a proteins and determined Preso1 screen fied (ref. PSD95 bind to reported previously was it and domains, FERM and multi a with domain scaffolding protein concert termed Preso1. Preso1in includes WW,acting PDZ kinases proline-directed by trolled signaling. receptor in functions mGluR of phosphor ylation kinase–mediated proline-directed whether of question 1 Here we report that group I mGluR signaling is dynamically con dynamically is signaling mGluR I group that report we Here , , Joo Min Park 16) and to modulate dendritic spine morphogenesis. We identi morphogenesis. spine dendritic to and modulate 16) 6 −/− 3 Department Department of Neurology, Johns Hopkins University School of Medicine, mouse, and this was coupled to increased mGluR5 activity. mGluR5 to and was coupled increased this mouse, 1 , , Paul R Brakeman 1 , , Xinzhong Dong in in vivo 1 7 to recruit PKA to phosphorylate its its phosphorylate to PKA recruit to , we examined behavioral responses responses behavioral , we examined 2 Department Department of Pharmacology and 1 3 , . We confirmed that . inflamma Wethat confirmed 7 , , Jiancheng Tu 1 , 5 , , Bo Xiao t r a 5 Howard Howard Hughes 1 1 , C I , 3 & e l s  ------­ © 2012 Nature America, Inc. All rights reserved. Scale Scale bar, blots for 100 western nm. Full-length (*). terminal. density p, presynaptic postsynaptic are in (arrows) enriched to and subjacent the hilus of Gold particles the adult hippocampus. of labeling in Preso1 microscopy the electron ( (boxed). dendrites magnified 30 represent Scale bars synapses). inhibitory GABAergic but not synapses), with GAD65 (a for marker with PSD95 (a colocalized for marker excitatory Preso1 or along with anti-GAD65. anti-PSD95 with anti-Preso1 immunostained neurons ( immunoprecipitation. IP, control; serum preimmune PI, with Preso1. IP mGluR1, Homer3, with anti-Preso1. incubated in ( Homer1c. or Preso1, G89N (GN) or W24A (WA) mutation by was disrupted the F806R in (FR) mutation and Homer1c, this immunoprecipitated Preso1 into Anti- cells. HEK293T were transfected site. ( of rat F806, Homer binding protein. Preso1 density. the postsynaptic ( 1 Figure ( munoprecipitated coim was Homer3 that confirmed and lum cerebel rat of lysates detergent from Preso1 Homer precipitate ( coimmuno not did and site binding Homer cell lysates, whereas Preso3 lacks a conserved noprecipitated with Homer1c from HEK293T a conserved Homer binding site, and it immu binding properties of Homer, and it seems to be direct. Preso2 encodes 1 Fig. (Preso1FR; Preso1 in site binding Homer predicted the of mutation ­polyproline-binding surface of the Homer1 EVH1 domain or by point canonical the of mutation point by either disrupted was interaction form) splice ( mouse in cord spinal and brain three all of mRNAs 4). some whereas human (ref. term we that ( site ( binding Homer a including domains, several contains protein PDZD10 human to homologous be to determined and 5 by cloned was of terminus C the to corresponding fragment 1.2-kb a identified and library cDNA brain rat a of screen two-hybrid yeast a perform to domain EVH1 Homer1 the Weused Preso1 is a Homer-binding protein enriched in brain RESULTS signaling. mGluR of duration and amplitude the controls thereby of modulation kinase Homer binding to proline-directed mGluR and t r a  this are figure shown in Supplementary Fig. 1 Fig. Supplementary Fig. 1 Fig.

3 To examine the association of Preso1 with with of Preso1 To association the examine Hemagglutinin (HA)-tagged Homer1c (the brain-enriched Homer1 R, Shank and PSD95 immunoprecipitated R, Shank and PSD95 immunoprecipitated

18), 18), b b ) ) Various and Preso1 Homer constructs a ). Thus, the Homer–Preso1 inte Homer–Preso1 the Thus, ).

Preso1 C I ), and is conserved from from conserved is and ), Preso1 binds Preso1 to Homer to and localizes Preso3 , n vivo in FRMD2 PDZK10 Supplementary Fig. 4a Fig. Supplementary µ e l c 1 m m in main 5 panels, ) Rat cerebellum ) was Rat lysed and cerebellum 9 and Preso2 muorcpttd ih rs1 ( Preso1 with immunoprecipitated is autosomal (human chromosome 9, mouse chromo ′ s , we immunoprecipitated immunoprecipitated we , and 3 and , , and KIAA0967 Preso2 Fig. ( d Supplementary Figure 9 Figure Supplementary ) Cultured hippocampal ) hippocampal Cultured FRMPD3 Preso ′ ). Public databases include two related genes genes related two include databases Public ). a rapid amplification of cDNA ends (RACE) (RACE) ends cDNA of amplification rapid ) Domain structure ) structure Domain

1 are near each other on the X chromosome, on X the other are chromosome, each near c e ). mGluR1 and and mGluR1 ). 1 ) Immunogold ) Immunogold 6 ) ( ) ), and is here termed Preso Supplementary Fig. 2 Fig. Supplementary µ , , Supplementary Fig. 3 Fig. Supplementary m m in the Drosophila melanogaster Drosophila KIAA1817 ). genes are expressed broadly in in broadly expressed are genes KIAA0316 r action involves conventional conventional involves action . - - - - ) and and ) FRMPD4 . The full-length gene gene full-length The . b a c HA-Homer1c: Preso1 Preso3 HA-Preso1: mGluR1 Homer3 Preso1 PSD95 Preso1 Shank i. 1 Fig. Homer1 Homer1 IP ). Mouse and and Mouse ). (also termed termed (also Preso1 Preso1 ). 3 . The Preso1 R ( to human human to Lysat IP: anti-Preso FRMPD1 25 c c b WW ). This This ). 58 P e IP: anti-Preso1 70 + + F + PDZ I I Ly + R 147 ­ sate

P 1 GN 194 which belong to group II and group III mGluRs ( mGluRs III group and II group to belong mGluR4, or which mGluR2 immunoprecipitate not did Preso1 contrast, ( cells HEK293T of lysates tation data, Preso1 immunoprecipitated with mGluR5 from detergent protein for group I mGluRs. Consistent with brain coimmunoprecipi scaffolding a as functions Preso1 that possibility the Weconsidered domain FERM its via I mGluRs group with interacts Preso1 mGluRs I group and Homer of the overlapping distributions in the PSD cytoplasm, and subjacent especially spine, postsynaptic the to localized Preso1-immunogold we performed immunogold electron microscopy in the hippocampus. neurons the in mGluR5 (68.6 with colocalized also immunoreactivity GAD65; as (78.6 the inhibitory synaptic marker glutamic (GAD65) acid decarboxylase but with not PSD95, marker synaptic excitatory the with colocalized that distribution a and punctate showed neurons hippocampal tured ( Preso1 with inositol-1,4,5-trisphosphate receptor (IP components of its signaling complex, including Shank, PSD95 and the cotransfected with HA-tagged mGluR5 into HEK293T cells and and cells HEK293T into mGluR5 HA-tagged with with were interact cotransfected mutants deletion directly or Preso1 Homer. might of independent Preso1 mGluR5 whether examined we and 4c Fig. 188 kD 98 kD 188 kD 188 kD 188 kD 49 kD WA +

RA

± 95% confidence interval), whereas 9.6 9.6 whereas interval), confidence 95% a a a a a a ± 49 kD 98 kD 49 kD 98 kD ± FERM 4.8% of Preso1 colocalized with PSD95 (all values expressed expressed values (all PSD95 with colocalized Preso1 of 4.8% 5.1%; 5.1%; , d ). Preso1 possesses several protein interaction domains, domains, interaction protein several possesses Preso1 ). n a a a a advance online publication online advance = 17–20 dendrites in 7 or 8 neurons; neurons; 8 or 7 in dendrites 17–20 = e Fig. 1 Fig. Fig. 1 Fig. d 511 p c Preso Preso Preso * d ). Preso1 immun ). Preso1 ). To confirm that Preso1 is a synaptic protein, protein, synaptic a is Preso1 that To ). confirm 1 1 1 Homer ligand Fig. 2 Fig. PPPG 5 , 806 1 9 ( F RD Fig. 1 Fig. a and and PSD95 GAD6 mGluR5 p o * reactivity was enriched in cul enriched was reactivity e 5 Supplementary Fig. 4b Fig. Supplementary 3 ). R) R) also immunoprecipitated

nature nature ± 2.1% colocalized with with colocalized 2.1% O O O verla verla verlay Fig. Supplementary Supplementary NEUR y y PDZ ligand

ETTV 1312 1 d OSCI p * ). Preso1 Preso1 ). EN ). By By ). C E - -

© 2012 Nature America, Inc. All rights reserved. Coexpression of Preso1 increased binding between Homer and and Homer between binding (172.7 mGluR5 increased Preso1 of Coexpression signaling and phosphorylation mGluR regulates Preso1 site. binding Homer the and site domain–binding FERM the both of contributions involves mGluR to binding Preso1 cells HEK293T in proteins Homer native that notion the with consistent is mGluR5 in site binding Homer the on binding mGluR5–Preso1 of dependence ( Preso1 with immunoprecipitation mGluR5 reduced (mGluR5 site binding Homer the and site FERM-binding putative the of mutations point ( Preso1 with immunoprecipitation (mGluR5 proteins other in kinases ( proline-directed for site binding a as site binding domain FERM dicted ( the site from remote binding acids amino Homer 100 least at are binding required Preso1 mGluR5 in for element(s) sequence that indicate data these mGluR5 not mGluR5 immunoprecipitated Preso1 mutants. C-terminal mGluR5 with assays coimmunoprecipitation using Preso1 with tion ( mGluR5 immunoprecipitate to sufficient was domain FERM Preso1 isolated the that We confirmed gest that the FERM domain is required for Preso1 binding to mGluR5. on Homer.not sug depend does to also data mGluR5 binding These assay ( in the coimmunoprecipitation Homer mGluR5 (Preso1FR; Preso1- except not bind do that mGluR5 and Notably, Preso1 of mutants both point mutants deletion N-terminal immun antibody Preso1 coimmunoprecipitation. by interaction for assayed in shown are figure this for blots western Full-length binding. Preso1 abolished YVF) (mGluR5 sites binding FR) (F1128R; Homer mGluR5 and YV) V967A; (Y965A, ( red. in site phosphorylation and yellow, in shown is site binding Homer The italicized. acids amino critical with green, in shown is binding ERK for D-domain a predicted blue; in shown is domain FERM Preso1 for site binding A predicted ligand. Homer and 1020 and 920 between sequence acid amino the showing structure ( anti-Myc. or anti-HA with blotting western by analyzed and anti-Myc with incubated were lysates Detergent cells. HEK293T into cotransfected were mutants mGluR5 various and Preso1 Preso1. with interaction ( anti-Myc. or anti-HA with blotting western by analyzed and anti-Myc with incubated were lysates detergent and cells, HEK293T into cotransfected ( ∆ length. Full FL, anti-HA. with blotting western by analyzed and anti-Preso1 with incubated were lysates Detergent cells. HEK293T into cotransfected were Preso1 of mutants point and deletion N-terminal 2 Figure nature nature at mGluR5 of phosphorylation by increased be can binding Homer mGluR5/Lysate a b e Fig. 2 Fig. FERM delete the indicated domain from the N terminus. Only the Preso1- the Only N terminus. the from domain indicated the delete FERM Preso1/Lysate mGluR5/co-IP ) Preso1 and various mGluR5 mutants were cotransfected into HEK293T cells and assayed for coimmunoprecipitation. Mutation of mGluR5 FERM FERM mGluR5 of Mutation coimmunoprecipitation. for assayed and cells HEK293T into cotransfected were mutants mGluR5 various and ) Preso1 ) The Preso1 FERM domain is sufficient to bind mGluR5. Preso1FERM and mGluR5 constructs were were constructs mGluR5 and Preso1FERM mGluR5. bind to sufficient is domain FERM Preso1 ) The Myc-mGluR5: HA-Preso1: e et xmnd h rgo o mlR rqie fr interac for required mGluR5 of region the examined next We Preso1/IP d o

NEUR ). Mutations of the putative FERM domain binding sites sites binding domain FERM putative the of Mutations ). Preso1 binds to the mGluR5 C terminus by means of its FERM domain. ( domain. FERM its of means by C terminus mGluR5 the to binds Preso1 precipitated mGluR5 that was expressed with any of the the of any with expressed was that mGluR5 precipitated 95 V967A Y965A Supplementary Figure 10 Figure Supplementary 1–920 + + OSCI ±

( FL 29.4% of control, control, of 29.4% IP: anti-Preso1 2 EN Fig. 2 Fig. + + 2

can link these proteins, and it suggests that that suggests it and proteins, these link can ∆

) resulted in only a modest reduction of its its of reduction modest a only in resulted ) WW C 95 V6A F1128R V967A Y965A E i. 2 Fig. ∆

PDZ c + + advance online publication online advance ). As the Homer binding site is at 1128, 1128, at is site binding Homer the As ). ∆ FERM F1128R

d F806R F1128R . mGluR5 ). c Fig. 2 Fig. ) Region of mGluR5 between amino acids 920 and 1020 is critical for its its for critical is 1020 and 920 acids amino between mGluR5 of ) Region

(mGluR5FR)) retained interaction interaction retained (mGluR5FR)) F806R . Fig. 2 Fig. 188 kDa 188 kDa 98 kDa 98 kDa 2 Fig. Fig. 2 0 n and a D-domain implicated implicated D-domain a and b = 6, 6, = ). e ; mGluR5YVF) markedly markedly mGluR5YVF) ; ). However, the combined ). However, combined the a b 920–1020 Preso1FERM/IP HA-mGluR5: Myc-Preso1FERM: mGluR5/Lysate ), suggesting that Preso1 ), suggesting P Preso1FERM/ mGluR5/co-IP = 0.004; 0.004; = ∆ EM ( FERM Lysate nlds pre a includes IP: anti-Myc Fig. 2 Fig. Fig. 3 Fig. + + 1–1020

i. 2 Fig. + e ). The The ). a 188 kD 188 kD 38 kD 38 kD ). As ). , , but a 2 ). ). a a 1 - - - a a

∆ mGluR5/Lysate c FERM mutant failed to bind mGluR5. mGluR5. bind to failed mutant FERM Preso1/Lysate mGluR5/co-IP HA-mGluR5: Myc-Preso1: group I mGluR, as it did not alter the activity of mGluR2 (64 (64 mGluR2 of activity the alter not did it as mGluR, I group Preso1, in P 16 mGluR5TSAA: 26 control, in 26 (mGluR5YVF: mGluR1TSAA) and mGluR5TSAA with (absent mGluR1 and mGluR5 in site binding Homer the of rylation phospho and mGluR5FR) with (absent mGluR5 to binding Homer mGluR5YVF), on with to binding (absent mGluR5 Preso1 depended action Preso1’s that indicated mutants mGluR5 with Experiments 5% in control, control, binding neurons, ganglion which do not natively express any cervical mGluRs superior in voltage- channels to calcium coupling sensitive mGluR I group assay, cellular a In function. mGluR5. to binding Homer increasing of effect Preso1’s mediates site binding Homer its at phosphorylation control, control, of 51.8% 183.7 (WT): type wild (mGluR5 site Homer at the phosphorylated be cannot that site) binding Homer at mutations AA to TS taining con (mGluR5TSAA, mutant mGluR an to binding Homer enhance not did Preso1 as phosphorylation, on dependent is binding Homer ( Homer bind not does that (Preso1FR) mutant a of association Preso1 with Homer, we Preso1’sconfirmed using effect by means of Homer–mGluR5 indirectly binding the increases Preso1 control, of (198.2 expression Preso1 with increased phosphorylation of group I ( mGluR We antibody to a developed the phospho-specific Homer binding site phosphorylation. mGluR5 increasing by binding Homer increase to the Homer binding site = 0.98; mGluR1TSAA: 43 43 mGluR1TSAA: 0.98; = We next investigated whether Preso1 regulates group I mGluR mGluR I group regulates Preso1 whether investigated next We Preso1/IP ± a 10% in control, control, in 10% ) mGluR5 and progressive progressive and ) mGluR5 7 n n and was similarly inhibited by Preso1 (mGluR1: 55 (mGluR1: by Preso1 inhibited similarly and was = 15; 16 16 = 15; = 6; 6; = IP: anti-Myc n n n WT = 7; 7; = = 6, 6, = = 7; 25 25 7; = n + + 1–1020 P = 24; 22 = 0.58; 0.58; = ∆ d P WW, n 1–920 P ± ± Supplementary Fig. 4e Fig. Supplementary ) mGluR5 ) mGluR5 = 0.04; 0.04; = = 6; 6; = = 0.88; 0.88; = 8% in Preso1, Preso1, in 8% 10% in control, in control, 10% 188 kDa 188 kDa 98 kDa 188 kDa 98 kDa 188 kDa ± n 2 ∆ 6% in Preso1, Preso1, in 6%

3 = 7; 23 23 7; = PDZ and and PDZ ± , we hypothesized that Preso1 might function

P Fig. 3 Fig. 6% in Preso1, = 0.03; mGluR5TSAA: 122.6 122.6 mGluR5TSAA: 0.03; =

Fig. 3 Fig.

Fig. 3 Fig.

d ±

b 12% in control, control, in 12%

±

). These data suggest that mGluR5 mGluR5 that suggest data These ). P T e 10% in Preso1, Preso1, in 10%

a mGluR5/Lysate n ). To exclude the possibility that that possibility the Toexclude ). d Preso1/Lysate mGluR5/co-IP = 11; = 11; n α ). Preso1 action is specific for for specific is action Preso1 ). HA-mGluR5: Myc-Preso1: P 1126 = 9; 16 β γ S Preso1/IP n n = 8, 8, = P mGluR5 = 22; 2 P ), ), and that found mGluR5 4 F , , was inhibited by Homer = 0.0001; mGluR5: 38 38 mGluR5: = 0.0001; ± P P 6% in Preso1, 6% in Preso1, = 0.90; mGluR5FR: mGluR5FR: 0.90; = + + = 0.0004;

IP: anti-Myc YVF Fig. 1 Fig. n t r a n 920 = 14; 41 41 14; = S S R A H A L S P A D D G A L H A A R A P F S E E D P P P E P G G T A N G A G G A P G S G G G A A E T S K = 11, 11, = YV Y L A K P S + + b FR ). Enhanced Enhanced ). P S ± G P G R N C I S G Q S S S D T R G A S

Fig. Fig. 3c WT 49.1% of of 49.1% P D I ± = 0.66; 0.66; = E A V T S A T C G ± 188 kDa 188 kDa 188 kDa 188 kDa 72.8% 72.8% n ± ± e l 6% in = 13, 16% 16% 10% 10% ± G A R T E L 1020 4% 4% , A A V S P d

s ). ). ± ±  - -

© 2012 Nature America, Inc. All rights reserved. We generated a conditional conditional a generated We Preso1 associated naturally are kinases ( brain mouse from (ERK1/2) ERK2 and/or ERK1 with and CDK5 with cipitated of control, the association of ERK1 with mGluR5 in HEK293T cells (166.6 proline-directed ( with ERK and CDK5 kinases cells HEK293T from and immunoprecipitated members family Preso ( species among across conserved is predicted a that contains D-domain Preso1 mGluRs. I group phosphorylate that site. binding Homer at the mGluR I group of phosphorylation in ERK and CDK5 for role a support data purvalanol A: 65.8 57.4 (UO126: inhibitor or MEK mGluR phosphorylation was significantly reduced by CDK5 inhibitor 345% of control, (CDK5/p35: 563 cells HEK293T in phosphorylation mGluR5 induced ERK, activates and p35 or (CDK5/p35) constitutively active MEK (MEK DD), which Weof CDK5 ERK. and CDK5 as expression such that found kinases, gested that the serine phosphorylation is mediated by proline-directed of Inspection the Homer binding site of group I mGluR ( kinases proline-directed binds Preso1 I group bound ( mGluRs selectively Preso1 that finding the with ­consistent in in shown are figure this for blots western Full-length mGluR2. of response alter not did Preso1 site). binding Homer the at phosphorylated be cannot but Homer (binds mGluR1/5TSAA Homer), bind not (does mGluR5FR Preso1), or Homer bind not (does mGluR5YVF Preso1: without or with constructs mGluR various expressing cells from data of Summary mGluR1/5. of sites ( glutamate. Glu, control; Con, Preso1. without or with mGluR5 expressing neuron ganglion cervical a superior in (NE) norepinephrine 100 by inhibition of reduction illustrating course time and ( neurons. ganglion cervical superior in channels calcium gated ( as analysis Same cells. HEK293T into cotransfected were constructs Preso1FR HA-tagged and mGluR5TSAA HA-tagged Homer1c, HA-tagged Preso1. to binding Homer require not does but mGluR5, of sites phosphorylation requires binding mGluR5–Homer of enhancement ( (right). pSer-mGluR anti– with blotted also were Lysates HA. anti- with blotting western by analyzed and anti-Homer1c with immunoprecipitated were Lysates cells. HEK293T into cotransfected were constructs Preso1 HA-tagged and ( Ca voltage-gated to coupling mGluR I group inhibits and binding, Homer–mGluR5 and phosphorylation 3 Figure t r a  domain PDZ the encodes which 3, exon of introns flanking in d c a

, mGluR5 HA-tagged Homer1c, HA-tagged ) ) Expression of Preso1 reduces mGluR1/5-dependent modulation of calcium current by glutamate, and requires Homer binding and phosphorylation phosphorylation and binding Homer requires and glutamate, by current calcium of modulation mGluR1/5-dependent reduces Preso1 of ) Expression

We next hypothesized that Preso1 binds proline-directed kinases kinases proline-directed binds Preso1 that hypothesized Wenext d control, control, ) Coupling of group I mGluRs to voltage- to mGluRs I group of Coupling ) −/− C I

Preso1 enhances mGluR5 mGluR5 enhances Preso1 Supplementary Fig. 4b Fig. Supplementary mouse generation mouse a n n . . = 5; e l n = 7; 57 57 7; = Fig. 4f Fig. = 6 for each group. group. each for 6 = Supplementary Figs. 1 1 Figs. Supplementary s n ± P µ = 6; 98% of control, c M glutamate but not 10 not but glutamate M = 0.02; = 0.02; ± ) Sample currents (inset) (inset) currents Sample ) 9.5% of untreated, b , ) Preso1-dependent Preso1-dependent ) g ), suggesting that Preso1 and proline-directed proline-directed and Preso1 that suggesting ), ± P 12% in Preso1, Preso1, in 12% = 0.006; n = 15, 11, 24, 22, 7, 8, 7, 11, 9, 13, 14, 6, 7, 5, from left to right; ** right; to left from 5, 7, 6, 14, 13, 9, 11, 7, 8, 7, 22, 24, 11, = 15, Fig. 4c Fig. Fig. 4 Fig. 2+ channels. channels. e in vivo in ± Preso1 Fig. Fig. 4 , ). Endogenous Preso1 immunopre Preso1 ). Endogenous 11% of untreated, of untreated, 11% d n

–

). Additionally, Preso1 enhanced enhanced Preso1 Additionally, ). = 6; d

). a n . −/−

P ). ). In cultured neurons, native and = 4;

= 0.0008; MEK DD: 1,049 µ n by inserting inserting by M = 5, 5, = 2 P ). As predicted, Preso1 Preso1 predicted, As ). = 0.02; Supplementary Figure 11 Figure Supplementary P c a mGluR5 WT Homer1c: Preso1: = 0.30; 0.30; =

Current (nA) Current (nA) Homer1c n mGluR –1.2 –0.8 –0.4 –1.5 –1.0 –0.5 Preso1 Fig. = 4; 0 0 Fig. Fig. 2

5 mGluR5 +Preso1 mGluR lox 4 P Homer1c : b Fig. 3 Fig. = 0.007; = 0.007; + + ). ). These P sites sites P d IP Glu ± ) ) sug 5 + 40% + + Glu d ), ), ± Lysate - - + +

NE + + + Homer binding were reduced in in reduced were binding Homer n of untreated, 157 (binding: min 30 after mGluR5 to binding Homer and phosphorylation mGluR5 of increase an in resulted (DHPG) glycine 3,5-dihydroxyphenyl agonist mGluR I group the neurons, WT In activity- for required dependent is phosphorylation Preso1 of mGluR5 and that enhanced Homer binding. hypothesis the examined We binding Homer–mGluR5 dynamic for required is Preso1 in reduced was mGluR5 with Preso1 ERK1/2 of immunoprecipitation cord, spinal in WT of 10.6% 57.5 phosphorylation: n pared those in to WT littermates (binding: 76 in reduced was Homer with immunoprecipitation mGluR5 and (pSer-mGluR) phosphorylation ( not was of mGluR5 expression and GluA1 and GluA2/3 was not altered in 6-week-old 6-week-old in altered not NR1, was GluA2/3 Homer3, and GluA1 Homer2, Homer1b/c, of Expression growth. natal post in their mice to WT similar appear and fertile, and viable were ( neurons lysates detergent of of blots western by verified further was deletion mRNA expression was confirmed by reverse transcription and and ( transcription cerebellum and reverse cortex by of PCR confirmed was expression mRNA Preso1 mutant and shown), not (data PCR by identified were mice with mice knockout conditional mating ( NE . Supplementary Fig. 5e Fig. Supplementary Supplementary Fig. 5a Fig. Supplementary = 5; 5; = = 5; 5; = Consistent with the hypothesized function of Preso1, mGluR5 mGluR5 Preso1, of function hypothesized the with Consistent Lysate Preso1 pS-mGluR + + 98 kDa 38 kDa + + + −/− P P = 0.002; 73.6 73.6 0.002; = = 0.02; 0.02; = Con Glu Con Glu brain (64.4 (64.4 brain −/− Supplementary Fig. 5d Fig. Supplementary advance online publication online advance 188 kDa brain ( brain n = 5; Fig. 5 Fig. P d b P Preso1FR: mGluR5TSAA: mGluR5 WT Homer1c: = 0.01; phosphorylation: 149 Supplementary Fig. 5c Fig. Supplementary ICa inhibition (%) < 0.01. Error bars, 95% confidence intervals. intervals. confidence 95% bars, Error < 0.01. ± c ± 10 20 30 40 50 60 70 ± Homer1c 10.9% of WT,of 10.9% 0 ). By contrast, mGluR5 phosphorylation and phosphorylation mGluR5 contrast, By ). mGluR5 22.2% of WT in cortex, 8.7% of WT in spinal cord, cord, spinal in WT of 8.7% Preso1

). mGluR1 ). Preso1 was deleted in the germline by by germline the in deleted was Preso1 ). ** : Preso1 n

mGluR5 Homer1c = 5; 5; = + + IP ** ). Male and female female and Male ). Supplementary Fig. 5b Fig. Supplementary mGluR5 + + + Preso1

different in cortex or spinal cord or spinal cortex in different −/− P n = 0.03; 0.03; = YVF Lysate cortex and spinal cord com cord spinal and cortex + + = 5; 5; = mGluR5 −/−

+ + + CMV-cre

FR Preso1 Control P nature nature neurons and were not not were and neurons Homer1c ) and cultured cortical cortical cultured and )

± mGluR5 = 0.04; 0.04; = Fig. 5 Fig. + + 3.3% of WT in cortex, IP

n TSAA = 5; + + + ±

36% of untreated, mGluR1 mice. mice. a Preso1 NEUR Lysate n ). Additionally, Additionally, ). Fig. 5 Fig. TSAA + + P Preso1 = 8; 8; = = 0.04; 79.1 mGluR2 + + + OSCI Preso1 −/− b ). Preso1 Preso1 ). P −/− ± ). 98 kDa 38 kDa 188 kDa = 0.03; 0.03; = 33.8% 33.8% brain, brain, mice mice EN −/− C ± E - - ­

© 2012 Nature America, Inc. All rights reserved. behavioral pain response that is followed by a second, Formalinsustained injectionresponse into the mouse hind paw in results increased is response Pain in an initial, transient Homer3 sistent with absence the ofobserved an ofeffect does not function by regulating mGluR5 surface expression. This is± con decreases of surface mGluR5 (65.4 WT, culture was not different between WT and in neurons of surface the on expression mGluR5 surface. cell the on group I mGluRs and modify signaling by changing expression of mGluR in in both homologous and heterologous receptor signaling pathways. for dynamic regulation of mGluR5 phosphorylation and HomerP binding Preso1 ± treated in 22.9% P not increases of mGluR5 phosphorylation and Homer binding in butWT ERK TrkBBDNF,increase of activates to which pathway. homologousreceptorfeedback effect Wethe examined also a Homer in binding enhanced mediate receptorto the phosphorylate is consistent with mGluR5 activation of CDK5 and ERK P P Preso1 47 (binding: stimulation DHPG after changed in shown are figure this for blots western Full-length intervals. confidence 95% bars, ( and anti-ERK1. ( cells, and lysate. HEK293T anti-HA into with detergent blotted and cotransfected were anti-mGluR5 Preso1 with HA-tagged and precipitated mGluR5 were lysates HA-tagged mGluR5. with ERK1 of immunoprecipitation increases ( Quantitation ( pSer-mGluR. or mGluR5 with blotted and immunoprecipitated was ** Lysates (below): cells. HEK293T into Quantitation 4 anti-HA. and cotransfected were active) anti–pSer-mGluR anti-mGluR5, (constitutively with DD HA-MEK blotted or were HA-CDK5/HA-p35 and mGluR5 HA-tagged phosphorylation. 4 Figure nature nature c d HA-MEK DD: HA-CDK5/p35: HA-mGluR5: a HA-ERK1: HA-Preso1: = 0.04; 67.3 10.1% in in 10.1% 15.1% of untreated, untreated, of 15.1% = 0.09; 0.09; = = 0.3; phosphorylation: 71.7 71.7 phosphorylation: 0.3; = = 0.001; 65.3 65.3 0.001; = ) CDK5 immunoprecipitates with Preso1 from HEK293T cells. ( cells. HEK293T from Preso1 with immunoprecipitates CDK5 ) µ pSer-mGluR Tamalin M) or CDK5 (purvalanol A, 20 20 A, (purvalanol CDK5 or M) Preso1 Preso1 ERK1 MEK DD n mGluR5 IP: anti-Preso1 = 6; 6; = −/− −/− CDK5 −/−

p35 NEUR Preso1 binds proline-directed kinases and enhances kinase-mGluR5 binding. ( binding. kinase-mGluR5 enhances and kinases proline-directed binds Preso1 , , , , Fig. 5 Fig. 2 −/− n n + + genotype on mGluR5 surface expression Preso1 7 P = 4; 4; = = 5; 5; = , calcineurin inhibitor protein (CAIN) = 0.14; 0.14; = + + neurons (binding: 157.7 157.7 (binding: neurons ± + + OSCI 14.8% in treated c ± + ). These observations suggest that Preso1 is required is Preso1 that suggest observations These ). 18% in untreated in 18% P g Preso1 −/− P + + Lysate ) Preso1 immunoprecipitates with ERK1/2 from mouse brain detergent lysate. PI, preimmune; IP, immunoprecipitation. Error Error IP, immunoprecipitation. preimmune; PI, lysate. detergent brain mouse from ERK1/2 with immunoprecipitates Preso1 ) = 0.0002; 79.7 79.7 0.0002; = = 0.007; 54 54 0.007; = + + EN , , Fig. 5 Fig. n C n = 3; 3; = 28 kDa 38 kDa 49 kDa 188 kDa 188 kDa E −/− = 5; 5; =

38 kDa 98 kDa d advance online publication online advance , , ). Furthermore, DHPG induced identical identical induced Furthermore,DHPG ). P n = 0.04; 0.04; = = 4; 4; = P ± = 0.00002; 66.8 66.8 0.00002; = Preso1 ± µ 22.4% in untreated in 22.4%

Preso1 pSer-mGluR5 M) for 30 min reduced basal phosphorylation of mGluR5 in cortical neurons at 14 days days 14 at neurons cortical in mGluR5 of phosphorylation basal reduced min 30 for M) 12.9% in treated treated in 12.9% ± ±

P (% of control) 8.3% in treated treated in 8.3% 16.7% in WT, Preso1 1,000 1,200 1,400 = 0.77; phosphorylation: 182.1 182.1 phosphorylation: 0.77; = Fig. 5 Fig. −/− 200 400 600 800 mGluR5 monomer e ±

Control 0 14.5% in treated WT, treated in 14.5% , −/− mGluR5 dimer n CDK5/p35 Preso1 −/− = 5; d HA-ERK1: HA-Preso1: HA-mGluR5: mice ** ), suggesting that Preso1that suggesting ), , ,

MEK DD ** n Preso1 P Homer1 = 4; 4; = ERK1 2 ± ± −/− = 0.76; 6 2 , and found similar similar found and , 8 7.7% in untreated untreated in 7.7% n 9.5% in untreated in 9.5% and Norbin IP: anti-mGluR5 3 = 4; (115.6 Preso1 0

Preso1 Preso1 . P

+ + + + = 0.02; 69.8 69.8 0.02; = 2 −/− P 5 Fig. 5 , , which then + + = 0.02; 65.2 Homer2 b pSer-mGluR −/− ± −/−

−/−

+ 21.6% of , , mGluR5 c , , , , 2 ). This n d n n 9 n ) ERK1 immunoprecipitates with Preso1 from HEK293T cells. ( cells. HEK293T from Preso1 with immunoprecipitates ERK1 ) bind + + + + = 3; 3; = = 4; 4; = = 4; 4; = = 5; 5; = Lysate −/− + + ± -

+ Control P Preso1 phase: 68.7 ing the second phase to the same degree in kilogram body weight, intraperitoneally) decreased pain responses dur in s n littermates (first phase: 94.6 during the second phase, but not the first phase, Preso1compared with their WT mGluR5 in formalin-induced inflammatory pain. ( mice inducedinflammatory pain response mice, WT inbut not in formalin- the reduced substantially antagonist, specific mGluR5 an Fig. 6a ( littermates WT their in responses with compared inthesecond phase, but not inthefirst phase, after formalin injection examinedwe agonistsantagonistsandcal have been implicated in inflammatory pain by means of pharmacologi pain that is associated with tissue inflammation and is termed inflammatory nrae in increased tory pain response, we confirmed that inflammatory pain behavior was bindingto mGluR5 is important to limit the intensity of the inflamma excitabilities( rootganglion neurons from andWT tribute to formalin-induced pain Preso1 increasedinflammatoryresponseindicatetheinpaindata that These = 8; = 0.002; 214.7 UO126 38 kDa 98 kDa 188 kDa We next asked whether formalin-induced inflammatory pain is altered in Preso1 3 Purvalanol A 1 a P −/− . This model is widely used in pain research, and group I mGluRs −/− −/− , , upeetr Fg 6a Fig. Supplementary b b 188 kDa 188 kDa = 0.72; second phase: 347.6 ). Furthermore, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), , below): * below): , mice. , is mediated by mGluR5. Because ongoing sensory inputs con Supplementary Figure 12 Figure Supplementary n f −/− ± ) Preso1 immunoprecipitates with CDK5 from mouse brain brain mouse from CDK5 with immunoprecipitates Preso1 ) = 6; P a 11.7 s in treated WT, < 0.01, 0.01, < Homer2 , ) CDK5/p35 or MEK DD expression increases mGluR5 mGluR5 increases expression DD MEK or CDK5/p35 ) SupplementaryFig. 7 Grm5 n Preso1 ± P = 8; = Preso1 f 47.3 s in treated CDK5 = 0.56; second phase: 207 pSer-mGluR5

P (% of control) −/− < 0.05, ** 0.05, < 100 120 Lysate −/− P 20 40 60 80 −/− IP: anti-CDK5 n 0 mice and confirmed decreased pain responsespaindecreased confirmed and mice = 0.002;=

= 3 each group. ( group. each 3 = Control mice exhibited greater behavioral pain responses Homer3

PI PurvalanolUO126 ** ± 3 24.2 s in WT, . To further validate the role of mGluR5,ofTorole validatethe . further IP * P 3 , Fig.6a b −/− 2 < 0.01, 0.01, < Preso1

28 kDa 98 kDa A , we measured the excitability of dorsal . hs rsls ofr a oe for role a confirm results These ). n . = 6; ie ad n n Gu5 mutant mGluR5 an in and mice, ± ). To). assess thenotion that Homer 52.8 s in WT, Preso1 , c −/− HA-Preso1: HA-CDK5: P b b ). Notably,). per mgMPEP(30 = 0.13; 76.8 n Preso1 ) Inhibitors of MEK (UO126, (UO126, MEK of Inhibitors ) Preso1 , = 4 for each group. group. each for 4 = CDK5 ± n n −/− 33.5 s in treated WT, = 9; 88 = 6; IP: anti-Preso1 in vitro in mice and found similar ERK1/2 g Preso1 −/− P + + = 0.0002; n and WT mice (first t r a Lysate ± = 9; 576.3 + + IP: anti-ERK ± 25 s in (DIV). mGluR5 mGluR5 (DIV). e Supplementary 24.7 s in treated ) Preso1 Preso1 ) Lysate + + PI C I IP Fig. 6a Preso1 Grm5

± 38 kDa 98 kDa 28 kDa 98 kDa e l 113.2 n = 6; −/− , −/− b s ).  - - - - ,

© 2012 Nature America, Inc. All rights reserved. spinal cord spinal of pain pathway level that [Ca a reach critical that suggest and P WT, treated in 5.2 P WT, treated and with MPEP reduced the number of c-Fos–positive neurons in the WT Preso1 n P 19.6 1–2: (lamina in the neurons positive WT and formed c-Fos immunostaining in lumbar level L4–L5 spinal[Ca cord cellular from increase that circuit pain the in activated cord by formalin injection into the hind paw, and it identifies neurons binding with mGluR5. mechanismentwitha whereby deletion Homer1aof increases Homer Homer1 decreased in mice with selective deletion of the was behavior pain inflammatory that found ( receptor( the knock-inmouse in which Homer cannot bind in shown are figure this for blots western Full-length intervals. confidence 95% bars, Error min. 30 DHPG D30, 5 min; DHPG D5, group. each * (right): Quantitation blotting. western and biotinylation surface for (100 DHPG with treated were Cultures neurons. and WT between different not is mGluR5 of expression total and ** * (right): Quantitation control. Ctl, blot. western and immunoprecipitation ml ng 20 100 (DH; DHPG with treated were Cultures neurons. cortical 14 DIV not but WT in coimmunoprecipitation Homer–mGluR5 and phosphorylation serine mGluR5 TrkB increases receptor BDNF or ( anti-mGluR5. or ERK anti- with blotted were immunoprecipitates and panels) (bottom lysates the and anti-mGluR5, with immunoprecipitated were and WT from lysates Forebrain in decreased is mGluR5 with ( group. each * antibodies. indicated with blotted and pan-anti-Homer with immunoprecipitated or anti–pSer-mGluR and WT from lysates (Sc) cord spinal and (Cx) Cortex mice. of cord spinal and cortex in decreased are coimmunoprecipitation Homer–mGluR5 and ( binding. Homer and phosphorylation mGluR5 of increase dependent 5 Figure t r a  mechanism. mGluR5-dependent a through induction, SupplementaryFig. 8a

= 18; 18; = = 0.03; = 0.000009; lamina 3–4: 3.7 3.7 3–4: lamina 0.000009; = = 0.007; lamina 3–4: 6.1 6.1 3–4: lamina = 0.007; P c-Fos expression is induced in neurons of the lumbar dorsal spinal spinal in of neurons is dorsal lumbar the induced expression c-Fos < 0.01, < 0.01, ± Preso1 0.8 in treated treated in 0.8 µ M) for 5 min or 30 min and processed processed and min 30 or 5 min for M) −/−

gene ( C I P −1 Preso1 Preso1 is required for activity- for required is Preso1 Supplementary Figure 13 Figure Supplementary Fig. Fig. 6c = 0.03; lamina 5–6: 5.8 5.8 5–6: lamina 0.03; = , , ) for 30 min and lysed for pan-Homer pan-Homer for lysed and min 30 ) for −/− n n P = 18; 18; = b e l Preso1 n = 3–8 for each group. ( group. each for = 3–8 < 0.05, ** < 0.05, ) ERK1/2 immunoprecipitation immunoprecipitation ) ERK1/2 spinal cord to the same degree (lamina 1–2: 14.9 Grm5 = 18; 18; = Homer1a −/− n , Preso1 d = 18; = 18; s Preso1 ). ). These findings corroborate behavioral pain assays mice after formalin injection and found more c-Fos ± P −/− R/R Preso1 2.7 in WT, in 2.7 a = 0.0001; 0.0001; = P P µ ) mGluR phosphorylation phosphorylation ) mGluR c mice were blotted with with blotted were mice M) or BDNF (BD; (BD; BDNF or M) < 0.05, < 0.05, = 0.03; 13.6 13.6 0.03; = or mGluR5FRmiceor ) Activation of mGluR5 mGluR5 of ) Activation −/− P −/− P Preso1 – = 0.62; 7.8 7.8 = 0.62; −/− DIV 14 cortical cortical 14 DIV < 0.01, < 0.01, d mice) −/− ). Furthermore,). we ± increases the number of neurons in the the in neurons of number the increases Preso1 P 1.0 in WT,in 1.0 Preso1 , , < 0.05, < 0.05, n n −/− ± Fig. 6c Fig. = 3–6 for for = 3–6 n = 18; = 18; 0.7 in treated WT, treated in 0.7 3 . = 25; 26.6 26.6 = 25; 0 spinal cord than in WT spinal cord spinal in WT cord than spinal n −/− ( ± ± = 5–8 for for = 5–8 d Supplementary Fig. 8e −/− Preso1 ± 2.22 in treated treated in 2.22 Preso1 1.1 in WT, in 1.1 mice. mice. ) Surface ) Surface 1.2 in treated treated in 1.2 P

mice mice

, d = 0.009; lamina 5–6: 6.2 6.2 5–6: lamina = 0.009; n

). Furthermore, preinjection preinjection Furthermore, ). = 25; 8.7 8.7 = 25; −/− −/− 3 3 ±

)

4.1 in in 4.1 Homer1a n = 25; 11.1 11.1 25; = a mGluR5/Surface d mGluR5/Lysat c mGluR5/Lysat Preso1/Lysat mGluR5/co-I ± mGluR5/co-I n Preso1 Preso1 Preso1 mGluR5/Tota pSer-mGluR 2.1 in in 2.1 Actin/Lysat Actin/Surface = 18; 18; = pS-mGluR Actin/Total Homer/IP Homer/IP 2+ isoform of the 2+ ] 3 , −/− −/− −/− f 4 ] ] for c-Fos P Preso1 ), consist . We per . P e e P e e = 0.001; 0.001; = , , , , , , ± l ± n n n 2.4 in in 2.4 Ct WT Ct 2.8 in = 18; 18; = = 18; = 18; = 22; WT ± WT l l Cx

Preso1 DH 1.2 1.2 D5 −/− - - Preso1 , Ct Preso1 Ct

–/– D l D l WT consistent thanmore a be selective to agonist found for was mGluR5 cocktail (DHPG), This as is mGluR. consistent stimulate to used were (100 NBQX and blocker, receptor assays. Glutamate (100 neurons from WT and in enhanced of cord spinal in Preso1 expression c-Fos and pain behavioral increased mGluR5 to similar pattern expression an with cord ( spinal dorsal the in expressed is Preso1 Enhanced mGluR-mediated Ca in s 2.2 2.4 s in Preso1 in in 6.9 5 1: d (day through persisting and CFA injection after d 3 beginning responses pain thermal increased s, 3.3 identical initial pain response (WT: 13.6 and showed they an pain responses, thermal in basal littermates their (CFA) chronic pain model. B H 5 Sc –/– –/–

We also tested pain responses in the complete Freund’s adjuvant adjuvant Freund’s complete the in responses pain tested Wealso Preso1 Preso1 Preso1 Ct Ctl WT WT l D30 n −/− C D −/− 188 kDa –/– 188 kDa 188 kDa 188 kDa Preso1 49 kDa = 9; 9; = Preso1

Preso1 Preso −/− Ctl −/− mice, we predicted that group I mGluR function would be be would function mGluR I group that predicted we mice, , , tl advance online publication online advance n BD D30 , , , = 9; 9; = n 1 n Preso1 P –/– –/– = 9; −/− = 9; −/− = 0.96; 0.96; = 49 kDa 188 kDa 188 kDa 188 kDa 49 kDa 188 kDa 49 kDa 188 kDa 49 kDa , , , , n

P pSer-mGluR Homer–mGluR5 binding n P P = 9; = 0.02; day 4: 11.1 11.1 4: day 0.02; = −/− = 9; 9; = = 0.26; day 3: 11.7 (% of WT) (% of WT) = 0.83; day 2: 10.2 100 120 100 120 20 40 60 80 20 40 60 80 dorsal spinal cord neurons. We neurons. cord these spinal cultured dorsal 0 0 Preso1 µ Fig. 6 Fig. P M) combined with APV (100 WT P

= 0.03; day 5: 13.6 Surface/total mGluR5 Co-IP/lysate Co-IP/lysate mGluR5 = 0.03; 0.03; = Preso1 ** Cx mGluR5 (% of ctl) (% of ctl) Cx

(% of ctl) * 120 160 240 280 200 120 160 120 160 200 240 280 40 80 −/− 40 80 40 80 0 e 0 0 Preso1 ). However, However, ). mice and performed calcium imaging WT −/− 2+ WT WT ** Fig. 6 Fig. * Sc Sc * µ * –/ response in Ct D5 mice were not different from WT ** M), an AMPA receptor blocker, receptor AMPA an M), – * Preso1 Preso1 l Preso1 ± ** ** ± ± 3.6 s in WT, 2.1 s WT,in 2.1 ± e 5.0 s in WT, ± b mGluR5/Lysate ). 2.2 s, ERK1/2/Lysate 2.9 s in WT, in s 2.9 –/

ERK1/2/co-I –/ –/ – Preso1 – – ± nature nature mGluR5/IP 4.2 s in WT, Supplementary Fig. Supplementary 3 n

5 Surface/total mGluR5 pS-mGluR (% of ctl) pS-mGluR (% of ctl) = 9; 240 280 200 120 160 . On the basis of the the of basis the On . 120 160 200 240 280 40 80 40 80 Preso1 (% of ctl) −/− 120 160 0 0 40 80 P n 0 = 9; 7.5 n Preso1 mice exhibited exhibited mice NEUR WT µ n WT WT = 9; 6.6 = 6.6 9; ** M), an NMDA = 9; 7.3 * WT Preso1 * n ** * −/− = 9; 2.6 2.6 9; = Preso1 n ** ** OSCI Preso1 −/− = 9; 9.9 neurons D30 Ct Preso1 ± 188 kD 188 kD 38 kDa 38 kD –/ l 3.3 s in : 13.6 – DH Ct * ± BD Ct ± –/ EN 2.6 s 2.6 –/ l 5.5 s – l – –/ a

a a – C 3 ± ± ± E )

© 2012 Nature America, Inc. All rights reserved. of of ( neurons. 10 (100 NBQX and Ca peak (right) population 7 Figure Preso1 3.28 (WT: neurons WT in response Preso1 depolarization with the that observation mGluR1 signaling is enhanced by preceding nature nature purvalanol A conditions, respectively. * respectively. A conditions, purvalanol For n Ca of traces shows panel Left min. 10 in as stimulated Ca a delayed showing neurons of percentage the increase inhibitors a a = 390 neurons in 8 experiments, 420 neurons in 9 experiments and 330 neurons in 10 experiments for vehicle, UO126 and purvalanol A, respectively. purvalanol and UO126 vehicle, for in 10 experiments neurons 330 and in 9 experiments neurons 420 in 8 experiments, = neurons 390 c WT /MPEP Preso1 Preso1 Con 340 nm/380 nm ratio WT Preso1 Duration of response (s) 0.5 1.0 1.5 2.0 2.5 µ Preso1 –2 10 12 14 0 M) blocked the Ca the blocked M) 20 40 60 80 0 0 0 0 0 −/− −/− –/ –/ –10

0 – – /MPE NEUR −/− Enhanced group I mGluR-mediated calcium response in the spinal cord neurons of of neurons cord spinal the in response calcium I mGluR-mediated group Enhanced Stimulate : 4.47 neurons until that washout persisted ( b Time afterformalininjection(min) −/− ) ) A neurons expressing GFP or GFP-Preso1. Same assay as in in as assay Same GFP-Preso1. or GFP expressing neurons 1 0 40 Preso1 WT /MPEP Preso1 WT neurons, neurons, Preso1 P Preso1 OSCI Time (s 3 ± µ 6 a 2 0 Ip 0.44 fold increase over basal Ca 80 . . Stimulation induced a rise of [Ca , and after 2 min, kinase inhibitors UO126 (4 (4 UO126 inhibitors kinase 2 min, after , and M). Pretreatment with group I mGluR antagonists Bay 36-7620 (mGluR1 antagonist, 20 20 antagonist, (mGluR1 36-7620 Bay antagonists I mGluR group with Pretreatment M). –/– –/– Wash out −/− /MPEP 3 0 EN ) transgene reduces Ca reduces transgene 120 2+ n neurons was significantly greater than that in in that than greater significantly was neurons C = 360 neurons in 8 experiments, 270 neurons in 7 experiments and 190 neurons in 6 experiments for vehicle, UO126 and and UO126 vehicle, for 6 experiments in neurons 190 and 7 experiments in neurons 270 8 experiments, in neurons = 360 response. Calcium concentration was measured by Fura-2 340 nm/380 nm ratio. ratio. nm nm/380 340 Fura-2 by measured was concentration Calcium response. 4 0 E 2+ ± Preso 160 WT Control

0.33 fold increase over basal Ca basal over increase fold 0.33 responses of WT and and WT of responses * advance online publication online advance 5 0 1 –/ * 6 0 – * d b e Number of c-Fos–positive cells per 0 2+ 340 nm/380 nm ratio P 2.0 2.5 0.5 1.0 1.5 Response latency (s) section after formalin injection responses from WT neurons, with example of delayed rise of [Ca of rise delayed of example with neurons, WT from responses < 0.05, ** < 0.05, 10 12 14 16 18 10 15 20 25 30 35 40 0 0 2 4 6 8 0 5 Basa b 2+ Duration of response (s) 0 80 10 20 30 40 50 60 70 response in DIV 2 DIV in response Lamina l ** Stimulate 0 0 Day 0 0 0 0 0 0 0 * 1–2 ** 40

Preso1 1 2+ 0–10 min Fig. Fig. 7

P Day Time (s Preso1 WT /MPEP Preso1 WT , < 0.01. Error bars, 95% confidence intervals. confidence 95% bars, Error < 0.01. 2+ n

80 2

= 74; Day ] ] in both WT and Wash out Lamina * −/− ** a 3–4 –/– –/– * ) 3 120 ). ). The calcium ** Preso1 WT /MPE Preso1 WT DIV 14 dorsal spinal cord neurons stimulated with glutamate (100 (100 glutamate with stimulated neurons cord spinal dorsal 14 DIV Day /MPEP P

4 * = 0.00003; GFP-Preso1 GFP 2+ 160

µ Day 10–60 min Preso1 WT Preso1 –/– –/– , , M) or purvalanol A (5 A (5 purvalanol or M) ** ** Lamina * **

n 5 2+ / MPEP P 5–6 = 90; 90; = ** * increase in WT but not not but WT in increase –/– a −/− ; ; n dorsal spinal cord neurons. Representative traces and population responses responses population and traces Representative neurons. cord spinal dorsal = 23 for GFP neurons, neurons, GFP for = 23 340 nm/380 nm ratio c initiation and used for calcium imaging after 2 intod. dorsalGFP-Preso1–expressing spinal cord neurons from a response in ( (20 7 Fig. n and WT littermate of paw hind into adjuvant Freund’s complete of injection 5 d after for and before monitored was in increased is adjuvant Freund’s * group. each in mice ( ipsilateral. Ip, 500 bars, Scale right. at magnified are regions Boxed formalin. after min 90 cord spinal of L4–L5 in expression c-Fos * ( injection. before MPEP and WT littermate comparing intervals 5-min in injection formalin paw hind to responses behavioral of duration in increased is in adjuvant 6 Figure than that in WT (WT: 11.5 11.5 (WT: WT in that than Ca with neurons of age again increased until washout in some neurons ( basal Ca Ca basal over expressingcomparedneurons2.38GFP (GFP:to neurons showed a markedly reduced mGluR-mediated calcium response decreased over an interval of 2–9 min in most neurons, but [Ca but neurons, most in min 2–9 of interval an over decreased Ca We that the inhibitors. kinase observed by is altered proline-directed the Ca mGluR-dependent whether ( responses mGluR sustained inhibits and site, binding Homer the at phosphorylation mGluR increase to responses in 1.0 2.0 3.0 4.0 Fig. Fig. 7 P = 9 for each group. Error bars, 95% confidence intervals. confidence 95% bars, Error group. each = 9 for Preso1 0 We next asked whether the enhanced mGluR-mediated calcium calcium mGluR-mediated enhanced the whether asked next We Our model predicts that Preso1 facilitates proline-directed kinases kinases proline-directed that facilitates Preso1 predicts Our model < 0.05, ** < 0.05, 2+ µ Stimulate response to sustained treatment with glutamate, APV and NBQX M), an mGluR1 antagonist, blocked the sustained calcium response 0 a a ). Pretreatment with MPEP (10 (10 MPEP with Pretreatment ).

), confirming that ), itconfirming is on dependent group I mGluR. transgene. GFP or GFP-Preso1 constructs were electroporated 2+ Kinase inhibitor 100 µ Increased pain response to formalin or complete Freund’s Freund’s complete or formalin to response pain Increased M) or vehicle (control) were added, followed by washout at washout by followed added, were (control) vehicle or M) , Preso1 Preso1 n 200 Without Ca With Ca = 19; Preso1 P Preso1 2+ Preso1 < 0.01, < 0.01, Preso1 d ) Statistical data from from data ) Statistical 300 , Time (s) n −/− −/− P Preso1 2+ = 23; GFP-Preso1: 0.46 GFP-Preso1: 23; = n −/− 400 = 5 × 10 mice. ( mice. rise spinal cord neurons would be rescued by expressing = 19 for GFP-Preso1 neurons. ( neurons. GFP-Preso1 for = 19 P −/− −/− −/− 2+ < 0.05, ** < 0.05, neurons can be reversed by Preso1. rise n mice. ( mice. b mice and is dependent on mGluR5. ( mGluR5. on dependent is and mice dorsal spinal cord neurons. Neurons were were Neurons neurons. cord spinal dorsal 500 µ = 6–9 for each group. ( group. each for = 6–9 ) Results from from ) Results 2+ m at left, 200 200 left, m at 2+ −/− Wash out rise was significantly higher in in higher significantly was rise ] that reverses on washout. For WT neurons, neurons, WT For washout. on reverses ] that a µ 600 mice. ( mice. n , −9 M) and MPEP (mGluR5 antagonist, antagonist, (mGluR5 MPEP and M) = 90 for WT neurons; neurons; WT for = 90 ± b a ) Formalin-induced inflammatory pain pain inflammatory ) Formalin-induced ; 4.3%, 4.3%, 700 ) Representative traces (left) and and (left) traces ) Representative Fig. 7 Preso1 P < 0.01. ( < 0.01. c 2+ Preso1 ) c-Fos immunohistochemistry of immunohistochemistry ) c-Fos

2+ Preso1 c b n Neurons with Ca rise (%) in response spinal cord neurons . . −/− a ), indicating that increased Ca = 390; 390; = µ Fig. Fig. 10 20 30 40 50 grouped into two phases. phases. two into grouped n 0 m at right. Con, contralateral; contralateral; Con, right. m at = 18–25 sections from 3–5 3–5 from sections = 18–25 mice. Latency to withdrawal withdrawal to Latency mice. µ Preso1 −/− e −/− M) and with Bay36-7620 Bay36-7620 with and M) 4 ) Pain response to complete complete to response ) Pain ± mice on the day of culture ). Accordingly, we asked asked we Accordingly, ). mice with or without without or with mice ** 0.35 fold increase over increase fold 0.35 c µ Preso1 WT ** , M), APV (100 (100 APV M), d Fig. 7 −/− ) Formalin-induced ) Formalin-induced n ± c * mice. * mice. t r a = 74 for for = 74 ) MEK or CDK5 CDK5 or ) MEK 0.36 fold increase fold 0.36 −/− c ). ). The percent : 22.3 22.3 : Preso1 P a C I ) The ) The < 0.05, < 0.05, Purvalanol UO126 Control Preso1 Preso1 µ

± M) M) –/ e l 7.4%, 7.4%,

–

−/− 2+ −/− 2+

s A



- ]

© 2012 Nature America, Inc. All rights reserved. Pain induced by inflammation also causes central sensitization and and sensitization central causes also inflammation by induced Pain independent mGluR5 signaling may contribute to inflammatory pain. contribute to stress effects in fear conditioning AMPA of receptors scaling homeostatic receptor becomes agonistsignaling independentmGluR if I Homer group does notthat bind anddemonstrated crosslink have the studies Previous n spinal cord neurons showing this delayed response (UO126: 27.6 P [Ca of rise delayed a exhibiting neurons of percentage the of increase threefold to two- a in resulted UO126 of Addition death. cell (4 to due not was the that response indicated delayed microscopy Nomarski by assessed viability cell n t r a  hypersensitivity activation of the mGluR receptor. Inhibition of mGluR signaling signaling mGluR of Inhibition receptor. mGluR the of activation in either of state mGluR or steady expression conditions surface after level the influence not do binding Homer dynamic and Preso1 First, and kinases receptor coupled canonical the mechanism from of GPCR distinct desensitization is mediatedmechanism byThis G mGluR. protein– and to binding Homer or dynamic, highly Homer expression that modify models genetic in several phenotypes is mechanism findings support a consistent model Preso1– across biochemical kinase–Homer and The behavioral mGluR. directed of regulation negative dependent, activity- that mediate kinases for protein proline-directed anchoring an as role its supports and complex, signaling mGluR I group the of part as an essential functions that Preso1 The study indicates present DISCUSSION [Ca of increases mGluR-dependent blocking in action acute their ways, path are inhibitors to likely signaling multiple alter kinase Although inhibitors did kinase not contrast, change of the percentage ity is also elevated during inflammation and regulates pain signaling phosphorylation of a potassium channel due to excitability of as a neuronal mediator implicated and been has tor kinase activation by receptors such as TrkB proline-directed increased to due response receptor heterologous a as or glutamate, by activation mGluR5 increased to due regulation mGluR5 may occur normally as part of a homologous receptor down of inhibition Preso1-dependent CFA the model. in pain chronic and model formalin the in pain inflammatory underlies that activation Tamalin including surface, cell (CAIN) protein inhibitor the calcineurin on mGluR of expression by changing signaling modify and I group mGluR bind that proteins ways remains unknown. The Preso1 mechanism is distinct from other kinases receptor protein–coupled G to response in desensitize reportedly mGluRs I the of intrinsic specificity G protein–coupled receptor Groupkinases. cificity to broad-substrate proline-directed kinases and thereby mimic spe confer can that mGluR for protein anchoring an of importance the highlights also it but crosstalk, receptor of possibility the affords This pathways. signaling many by activated are rather the but for receptor, specific not are response this mediate that kinases proline- directed the Second, membrane. plasma the from removal of than rather binding Homer of consequence direct a as result to appears

= 270; = 360; 360; = = 0.0004; purvalanol A: 23.8 purvalanol = 0.0004; Preso1 appears to limit the amplitude and duration of mGluR5 mGluR5 of duration and amplitude the limit to appears Preso1 3 µ 2+ 9 M), or purvalanol A (5 (5 A purvalanol or M), . Phosphorylated ERK is upregulated during inflammatory pain pain inflammatory during upregulated is ERK Phosphorylated . ] is consistent with our model of Preso1 function. Preso1 of model our with consistent is ] C I 1 P P 4 = 0.32; purvalanol A: 25.0 . This mechanism underlies the effect of Homer1a in driving = 0.03; 0.03; = e l 4 3 s 3 8 , and recent studies suggest a special role for the the for role special a suggest studies recent and , Fig. 7 Fig. , but the natural relevance of the suggested path suggested the of relevance natural the but , c ). The reversal by washout and subsequent subsequent and washout by reversal The ). µ ± β M) 2 min after onset of stimulation stimulation of onset after min 2 M) 5.6%, 5.6%, -arrestin pathway -arrestin 2+ ] (UO126: 28.3 28.3 (UO126: ] ± 5.3%, 2 n 8 4 and Norbin and 0 = 330; 3 . CDK5 expression and activ 0 and has been suggested to to suggested been has and 2 6 n or the bradykinin recep = 190; 4 2 . Accordingly, agonist- P = 0.005; 3 7 ± P 2 in several ways. ways. several in 9 5.7%, 5.7%, = 0.60; . Fig. Fig. 7 Preso1 n proline- ­ Fig. = 420; 420; = ± 6.6%, c ). ). By

7 −/− c 2 4 7 1 ). ------. ,

eton, including the Rac-activation domain, and has been reported to reported and has been domain, Rac-activation the including eton, anchoring an the cytoskel with that can domains interact includes Preso1 protein, as functions Preso1 that notion the with Consistent receptors protein–coupled G including substrates, enzymes potential near signaling multiple recruiting by complexes transduction isms. This is action to similar that of AKAPs, which coordinate signal organ higher for protein anchoring kinase proline-directed a of ple protein anchoring kinase specific. output be could mGluRs I group crosslinking mGluR1 coupling to intracellular IP example, For outputs. all inhibit uniformly not does mGluR5 to mGluRs ing I group here, monitored signal in several output mGluRs pathways (see introduction), I and Homer bind group of responses the inhibited uniformly binding Homer and Preso1 Although ogy. pharmacol distinctive this to contributes Preso1 that possible is It ligands) (biased outputs signaling alter differentially to agents cal syndrome retardation mental Alzheimer’s disease X fragile including diseases function. of mGluR regulation through sensitization amygdala in in recordings. B.X. generated Preso1 antibodies, designed and performed experiments support on the behavioral experiments and dorsal root ganglion neuron electrophysiological experiments. X.D. provided intellectual input and technical Supplementary Figure 7 the experiments in did initial characterizations. S.Y. generated the Preso1 antibody. R.S.P. performed Preso1identified as a Homer binding protein. J.T. and B.X. cloned Preso1 and Supplementary Figure 4 Figure Figure 4 L.Y. performed and analyzed experiments in and J.-H.H. designed, performed and analyzed experiments included in and Other Communication Disorders Intramural Research Program (R.S.P.). Program of China (20009CB941400; B.X.); and the National Institute on Deafness (NS050274 (P.F.W.); NS054791 and GM087369 (X.D.)); National 973 Basic Research Health (MH084020) and National Institute of Neurological Disorders and Stroke National Institute on Drug Abuse (DA010309), National Institute of Mental This work was supported by US National Institutes of Health grants from the with behavioral experiments and Y.-X. Wang for help with the immunogold. We thank J. Roder of University of Toronto for Note: Supplementary information is available in the the pape the of in version available are references associated any and Methods M signaling. GPCR of understanding comprehensive a for important system nervous central the of outside expressed are Presos also for signaling. GPCR role their in examined regulating been not have mRNAs they but system, Preso3 nervous the in and expressed broadly are Preso2 mGluR. I group to changes binding dynamic Homer of coordinate uniquely Presos kinases, that proline-directed predicts for which consensus a also is that site binding However, a Homer contain group I only mGluRs signaling. in GPCR that suggesting 7tm/), Preso family members may have a broader role binding sites and D-domains http://www.gpcr.org/(GPCR databases; with interact AU Acknowledgment ethods Figure The The yeast protein STE5 provides a for precedent a proline-directed cognitive of therapies for targets important are mGluRs I Group T Supplementary Supplementary Figures 1 H O 3 . . C.G.M. generated and the identified phospho-mGluR antibody in . P.J.K. performed and analyzed electrophysiology experiments in R R 1 and provided intellectual input and technical support for CONT 4 advance online publication online advance 4 . Hence, it is possible that Preso1 also contributes to central 4 β 7 . Accordingly, the effect of Preso1 on the signaling of of signaling the on Preso1 of effect the Accordingly, . -Pix RIBU Figure 1 r 5 6 . T , , and are for notable the of ability pharmacologi . Many GPCRs encode predicted FERM domain FERM . predicted Many encode GPCRs . . P.-W.Z. generated . P.R.B. performed the yeast-two hybrid screen that s I 1 ON . . Z.L. performed and analyzed experiments in – 4 S 6 6 8 , but Preso1 appears to be the first exam first the be to appears Preso1 but , and 8 , , and wrote a first draft of the manuscript. 3 receptors is increased by Homer Grm5 Figures Figures 1 Grm5 R/R online version of the pape

−/− mice. J.M.P. performed nature nature mice, J. Worley for help and

2 1 and 8 , and they will be be will they and , NEUR Supplementary Supplementary Figures Figures 1 Preso1 OSCI 4 −/− online online r 5 . EN – and and

7

C 4 4 9 6 E ------. .

© 2012 Nature America, Inc. All rights reserved. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.html. http://www.nature.com/ at online available is information permissions and Reprints Published online at The authors declare no competing interests.financial wrote the versionfinal of the manuscript. mouse generation. P.F.W. the overall project, supervised experiments designed and nature nature COM

rad, L.R. Orlando, Huang, G.N. Sharrocks, A.D., Yang, S.H. & Galanis, A. Docking domains and substrate-specificity of organizers family: 4.1 protein the of genetics The P.J. Bryant, & K.B. Hoover, B. Xiao, An, N., Blumer, J.B., Bernard, M.L. & Lanier, S.M. The PDZ and band 4.1 containing H. Zhong, H.W.Lee, CDK5. of decade A Tsai,L.H. & R. Dhavan, F.Ango, Brakeman, P.R. GABA inhibit receptors glutamate metabotropic I Group M.A. Diana, & M. Galante, 3-kinase-Akt-mammalian phosphoinositide the of Activation E. Klann, & L. Hou, kinases protein mitogen-activated of Regulation L. Mao, & Wang,E.E. Fibuch, J.Q., S. Park, Tu, J.C. proteins Homer S.R. Ikeda, P.F. Worley, & J.C., Tu, B., Xiao, P.J.,Kammermeier, of visions new transporters: monoamine and Glutamate S.G. Amara, & Torres,G.E. Perisynaptic P. Somogyi, & R. Shigemoto, J.D., Roberts, physiology, Z., Nusser, receptors: R., Lujan, glutamate Metabotropic P.J. Conn, & C.M. Niswender, Bhave, G., Karim, F., Carlton, S.M. & Gereau, R.W.t. Peripheral group I metabotropic C. Chiamulera, Lüscher, C. & Huber, K.M. Group 1 mGluR-dependent synaptic long-term depression: 557–569 (2009). 557–569 metabotropic glutamate receptors by cyclin-dependent kinase 5. proteins. Homer scaffolding kinases. MAP for determination cytoskeleton. and membrane the 21 proteins. synaptic homer-related, of complexes multivalent with receptors G-proteins.with interaction its and 3 G-proteinsignalingactivatorof subcellularoflocation regulatestheFrmpd1 protein (2009). 363–374 (2008). morphogenesis. spine dendritic regulates that (2001). Homer. protein intracellular the by receptors. Neurosci. J. production. endocannabinoid through synapses cell interneuron-Purkinje at release glutamate metabotropic for required is depression. pathway long-term receptor-dependent signaling rapamycin of target receptors. glutamate by (2008). mGluR-LTD.for essential Arc/Arg3.1 of translation dynamic the receptors. IP3 with receptors glutamate channels. calcium potassium M-type N-type and to receptors glutamate metabotropic I group of coupling regulate function. and form hippocampus.rat the in spinesdendritic location of metabotropic glutamate receptors mGluR1 and mGluR5 on dendrites and disease. pharmacology,and (2001). mice. in nociception modulate receptors glutamate mice. mutant null mGluR5 in absent disease. and (2010). circuitry for implications and mechanisms P , 707–716 (1998). 707–716 , ET NEUR I et al. NG et al. et et al. et et al. et et al. et Nature t al. et FI

et al. 24 Homer binds a novel proline-rich motif and links group 1 metabotropic Homer regulates the association of group 1 metabotropic glutamate metabotropic 1 group of association the regulates Homer OSCI Agonist-independent activation of metabotropic glutamate receptors glutamate metabotropic of activation Agonist-independent Elongation factor 2 and fragile X mental retardation protein control protein retardation mental X fragile and 2 factor Elongation N t al. et , 4865–4874 (2004). 4865–4874 , Preso, a novel PSD-95-interacting FERM and PDZ domain protein domain PDZ and FERM PSD-95-interacting novel a Preso, et al. t al. et http://www.nature.com/doifinder/10.1038/nn.310 A uclua dnmc o tp I PA n neurons. in PKA II type of dynamics Subcellular

NFAT binding and regulation of T cell activation by the cytoplasmic 386 NC Curr. Opin. Neurobiol. Opin. Curr. EN Homer: a protein that selectively binds metabotropic glutamate hshrlto o te oe-idn dmi o gop I group of domain homer-binding the of Phosphorylation enocn ad oooo siuat fet o ccie are cocaine of effects stimulant locomotor and Reinforcing , 284–288 (1997). 284–288 , IA C J. Neurochem. J. L E I

Annu. Rev. Pharmacol. Toxicol.Pharmacol.Rev. Annu. NTE Science advance online publication online advance Trends Biochem. Sci. TrendsBiochem. R J. Neurosci. J. Curr. Opin. Cell Biol. Cell Opin. Curr. E Nature

S 319 J. Biol. Chem. Biol. J. T Nat. Neurosci. Nat.

S 100 Neuron Eur. J. Neurosci. J.Eur. , 476–481 (2008). 476–481 ,

J. Neurosci. J. 17 411 , 1–11 (2007). 1–11 , Nat. Rev. Mol. Cell Biol. Cell Mol. Rev. Nat. , 304–312 (2007). 304–312 ,

20 , 962–965 (2001). 962–965 ,

21 . Neurosci. J. , 7238–7245 (2000). 7238–7245 , , 717–726 (1998). 717–726 ,

283 4 a. Neurosci. Nat.

25 , 873–874 (2001). 873–874 , 24 12 , 24718–24728 (2008).24718–24728 , , 448–453 (2000). 448–453 ,

, 6352–6361 (2004). 6352–6361 , , 229–234 (2000). 229–234 , 8

Neuron 50 , 1488–1500 (1996).1488–1500 ,

, 295–322 (2010). 295–322 , 28 J. Neurochem. Neuron 14546–14556 ,

3 65 .

4

2 Neuron

445–459 ,

417–423 , , 749–759 , 59 , 70–83 , Neuron

110

62 , ,

35. 34. 33. 32. 31. 30. 29. 28. 27. 26. 25. 24. 36. 49. 48. 47. 46. 45. 44. 43. 42. 41. 40. 39. 38. 37.

Alvarez, F.J., Villalba, R.M., Carr, P.A., Grandes, P. & Somohano, P.M.Differential Somohano, P.& P.A.,Carr,Grandes, R.M., F.J.,Villalba, Alvarez, Greenberg,MembranedepolarizationM.E.& McFadden, calciumG.M.,Sheng, and D.K. Cozzoli, Puig, S. & Sorkin, L.S. Formalin-evoked activity in identified primary afferent fibers: Hunskaar, S. & Hole, K. The formalin test in mice: dissociation between inflammatory J.H. Hu, H. Wang, L.T. Ferreira, by activated pathways J. Kitano, signaling Intracellular M.E. Greenberg, & R.A. Segal, Daly,Gallagher,Bear,S.M., M.F.C.A., signal-regulated Extracellular Huber,K.M. & of coupling the alters RGS2 of Expression S.R. Ikeda, & P.J. Kammermeier, Kim, S.J. Wong, W. & Scott, J.D. AKAP signalling complexes: focal points in space and time. and space in points focal complexes: signalling AKAP Wong,W.J.D. Scott, & protein multiple tethers Ste5 E.A. Elion, & D.M. Lyons, B., K.Y.,Satterberg, Choi, J.P.Yuan, mental X fragile J.P. Rocher, of theory mGluR the of implications Therapeutic M.F. Bear, pain of generator a sensitization: plasticity Synaptic R.W.t. Gereau, & G. Bhave, Central G.C., Bird, W.,V., Li, Neugebauer, C.J. Woolf, & A. Latremoliere, Tronson,N.C. Cdk5 of Roles Molecular A.B. Kulkarni, & T.K. Pareek, A., Futatsugi, E., Utreras, Hu, H.J., Alter, B.J., Carrasquillo, Y., Qiu, C.S. & Gereau, the R.W. Metabotropic of glutamate interactions Bradykinin-regulated T. Michel, & S. Haldar, S.G., Bernier, receptor glutamate metabotropic of Regulation S.S. Ferguson, & G.K. Dhami, M.G. Caron, & R.J. Lefkowitz, L.M., Bohn, R.T., Premont, R.R., Gainetdinov, itiuin f eaorpc ltmt rcpos a 1, n 5 n h rt spinal rat the in 5 cord. and 1b, 1a, receptors glutamate metabotropic of distribution (1990). 571–582 inducec-fos transcription via phosphorylation oftranscription factor CREB. (2009). alcoholism. for implications functional signaling: activity. phase-2 suppresses lidocaine systemic pain. non-inflammatory and Homer1a. signaling. 5 receptor (2009). 28986–28994 signaling. and endocytosis receptor glutamate metabotropic cytohesins. factor exchange nucleotide guanine the and receptors glutamate metabotropic 1 group of formation factors. neurotrophic CA1. (2004). area hippocampal in depression long-term receptor-dependentglutamate metabotropic for required is activation kinase protein channels. Ca2+ N-type and K+ M-type Neuron to 1a receptor glutamate metabotropic Nat. Rev. Mol. Cell Biol. Cell Mol. Rev. Nat. (1994). 499–512 in mating for required cascade kinase MAP the in kinases receptors. IP3 by TRPC1 Chem. agents. therapeutic novel of series a towards approach chemistry retardation. metabotropic of roles differential pain: 5. arthritic and 1 of receptors glutamate model a in amygdala the in plasticity. neural central by hypersensitivity fear. on effects stress neurons. horn Signaling. Pain in dorsal cord spinal in (2007). 13181–13191 signaling Kv4.2 extracellularmodulatesnociceptivesignal-regulatedplasticityvia 5receptor kinase- Chem. Biol. J. mitogen-activated protein kinase pathway with the endothelial nitric-oxide synthase. endocytosis. (2006). and desensitization signaling, Neurosci. functions. neuronal and receptors protein-coupled G of Desensitization cells. Purkinje cerebellar in signaling 1 receptor glutamate metabotropic of potentiation short-term J. Comp. Neurol. Comp. J.

11 22 et al. et

et al. , 680–695 (2011). 680–695 , 27 et al. et Neuron , 819–829 (1999). 819–829 , et al. et t al. et Genes Brain Behav. Brain Genes , 107–144 (2004). 107–144 , t al. et et al. et et al. et Homeostatic scaling requires group I mGluR activation mediated by mediated activation mGluR I group requires scaling Homeostatic

Transient upregulation of postsynaptic IP3-gated Ca release underlies J. Neurosci. J. t al. et 275 Tamalin, a PDZ domain-containing protein, links a protein complex protein a links protein, Tamalin,domain-containing PDZ a Homer binds TRPC family channels and is required for gating of gating for required is and channels family TRPC binds Homer obn s n noeos euao o mtbtoi glutamate metabotropic of regulator endogenous an is Norbin

68 Gu5 eaie lotrc ouaos vriw a medicinal a overview: modulators allosteric negative mGluR5 Drug Discov. Today Ther. Strateg. TodayTher.Discov. Drug Binge drinking upregulates accumbens mGluR5-Homer2–PI3K accumbens upregulates drinking Binge Metabotropic glutamate receptor 5/Homer interactions underlie interactions 5/Homer receptor glutamate Metabotropic , 30707–30715 (2000). 30707–30715 , acnui ihbtr rti (AN atnae Gop I Group attenuates (CAIN) protein inhibitor Calcineurin , 1128–1142 (2010). 1128–1142 , Annu. Rev. Neurosci. Rev. Annu. Science Biol. Psychiatry Biol.

422

5 Cell , 959–970 (2004). 959–970 ,

, 464–487 (2000). 464–487 , 28 J. Neurosci. J. Pain

326 J. Neurosci. J. 114 , 4350–4355 (2008). 4350–4355 ,

4

30 , 393–398 (2005). 393–398 , , 1554–1557 (2009). 1554–1557 , , 777–789 (2003). 777–789 , , 103–114 (1987). 103–114 ,

68

22 , 1007–1015 (2010). 1007–1015 ,

23 19 , 1280–1289 (2002). 1280–1289 , J. Pain J. , 52–63 (2003). 52–63 , , 463–489 (1996). 463–489 , hrao. Ther. Pharmacol. Pain

. Neurosci. J. . Neurosci. J. 10

6 64 , 105–111 (2009). 105–111 , , 895–926 (2009). 895–926 , , 345–355 (1996). 345–355 , t r a S. cerevisiae S. . il Chem. Biol. J. . Neurosci. J.

24 29 111 Curr. Top. Med. Top.Curr. 4859–4864 , 8655–8668 , C I 260–271 , Annu. Rev. Annu. . Neuron Cell e l

284

78 27

s 4  , , , ,

© 2012 Nature America, Inc. All rights reserved. each well of a 12-well plate (Corning) with cover slips coated with poly- rons were prepared from embryonic day 15 pups. 5 × 10 onic day 18 pups were prepared as reported previously N specifications. Cells were harvested 2 d after transfection. manufacture’sto 6 Fugene withTransfections performed FBS. were 10% with C Committee of Johns Hopkins University School of Medicine for mice and rats. mentswereprotocolsperformedunder approved CareAnimalandUsebythe experi experiments. All the forall were used mice 250experiments, totaland were used in all the experiments. Only adult male mice were used for behavioral Homer2 termate controls and blind to mouse genotype. lit on performed were experiments the all and mice, C57/Bl6J onto erations cycle. light:dark h h:12 12 22 atmaintained was water.colonyroomand chow The rodent mice in size or behavior by casual observation. ated as previously reported Homer, and were described previously mutant(mGluR5FR)mGluR5areplace genewithmicethenotbind that does and CMV-cre Preso1 injected into blastocysts, and chimeras were mated to C57BL/6 mice to produce blottingfor homologous recombination. Correctly targeted cellESclones were with selected Southernby confirmed and were clonesPCR, werepicked,byandscreenedG-418, cells ES cells. ES into electroporated and linearized was mouse neo/Exonback3 into Bsu36I and BstBI sites of the13-kb BAC fragment of the tor.The vec loxP/PGK-neo a intosubcloned and BstBI and Bsu36I by released was 3 fragmentcontaining exon of3 M 1:4,000, Myc-HRP (Santa Cruz, clone 9E10) at 1:3,000. 1:10,000, c-Fos (Calbiochem, PC38) at 1:4,000, HA-HRP (Roche, clone 3F10) at atAC-74) clone (Sigma-Aldrich, actin 1:1,000, at sc-173) Cruz, (Santa CDK5 1:1,000, at 9102) Signaling, (Cell ERK 1:5,000, at (JH2793) Homer3 1:5,000, at (JH2623) Homer2 1:5,000, at (JH2621) immunoprecipitation,Homer1for at 1:500, NR1 (Millipore, 05-432) at 1:1,000, pan-Homer (Santa Cruz, sc-17842) (Neuromab, clone K28/43) at 1:5,000, GluA1 (JH1710) at School1:1000, of GluA2Medicine)) (JH1707) at 1:1,000, Shank (Neuromab, clone N23B/49)(BD, at610965) at1:2, 1:10,000, PSD95IP and their dilutions are as follows: mGluR5 (Upstate, 06-451) at 1:10,000,other All antibodies were previously mGluR1or described were acquired commercially, protocolapproved UseandCareAnimalby Committee Sichuanof University. rabbitSichuanUniversitya atgenerated under was whichantigen, 330-aa the againstantibody Preso1 the exceptCovance, generatedby were antibodies bit (ELVALTPPpSPFRD)peptide antigenas (1:3,000forwestern blot). rab These mGluR5 rat usingrabbits female two in generatedwas antibodyphospho-Ser was used for western blot (1:2,000) and immunostaining (1:100). Group for I immunoelectronmGluR microscopy (1:100), and the antibody from 330-aa antigen terminal 20 aa or 330 aa as antigens. The antibody from 20-aa antigen was used Antibodies. with Myc or HA tags. All constructs were verified by sequencing. vectors pEGFPC1 (Clontech), pGEX 4T2 (Pharmacia), and pRK5 (Genentech), mutant will be supplied upon request. PCR products were cloned into expressionMutagenesis Kit (Stratagene) or by megaprimer method. The sequence for each deletions and point mutations were made either using QuikChange Site-DirectedE ONLINE METHODS nature nature Growthmediumconsisted (Invitrogen)ofMEM supplemented horse5% with xpression constructs. ell culture and transfection. euronal culture and electroporation. ouse models. ouse Micewere group housedplasticin mouse cages accesswithfreeto standard Grm5 Preso1 floxed heterozygotes.floxed These Preso1 −/− −/− Preso1 mice to producetomice NEUR −/− Homer3 ( Preso1 antibodies were generated in rabbits using the rat Preso1 C mGluR5 mice. gene ( gene targeting construct was generated by inserting the loxP/PGK- the inserting bygenerated was construct targeting

OSCI Preso1 −/− Supplementary Fig. 7 SupplementaryFig. mice and their age-matched, same-background WT mice −/− EN All the expression constructs were made by PCR. Internal −/− mc wr otie fo Jh Roder John from obtained were mice ) C mice were generated as follows. A 13-kb NheI BACNheI 13-kb A follows.generatedas were mice Preso1 2 E Preso1 3 2 R (gift from Alan Sharp (Johns Hopkins University , and are healthy, fertile and not different from WT HEK293T cells were cultured in DMEM medium Preso1 +/ −/− − Preso1 mice, which weremice,which crossedgenerate to WT mice were backcrossed at least five gen five least at backcrossed were mice 3 was subcloned into thepBSvector. Exon 3 . Neuronal cortical cultures from embry Homer2 floxed heterozygotestomatedfloxed were ). The resultingconstructtargeting The ). Grm5 −/− Homer3 −/− 3 0 . Dorsal spinal cord neu 5 mice, neurons were added to −/− mice were gener Grm5 ± 5 2 °C with a with °C 2 0 R/R . . Grm5 l mice or -lysine. R/R ------

centrifugedat 13,200 r.p.m. (16,100 twice). Cultures were harvested in RIPA min 5buffer for and once (briefly and7.4) (pH sonicated.glycine mM containing100 PBS++ with Homogenates were for 30 min at 4 °C. Unreacted biotin was quenched by washing cells three times incubatedcontainingthenPBS++ with sulfo-NHS-SS-biotinmg/ml 1 (Pierce) ice, washed twice with ice-cold PBS++ (PBS, 1 mM CaCl nylation assay as previously reported Surface biotinylation assay. SDS loading buffer and analyzed by gel electrophoresis and western blotting. tationbuffer containing 1% Triton X-100. The protein samples were eluted with an additional 1 h. The protein beads were A–washed or proteinthree timesG–Sepharose with slurryimmunoprecipi (Amersham-Pharmacia Biotech)with was0.5–2 added for ples were sonicated. After centrifugation, the supernatant containing(300 Complete EDTA-Free protease inhibitors 1 (Roche)mM Nawas added and sam ers of immunoprecipitation buffer (PBS, pH 7.4, with 5 mM EDTA,for the5 mMcoimmunoprecipitation EGTA, assay as previous reported C (Amaxa) after neuron dissociation. Calcium imaging was performed after 2 d. For the rescue experiment, electroporation was performedconditionedgrowth medium.neuronsDIV 14 were for calciumused imaging. using a Nucleofector kit 100 U/ml streptomycin (Invitrogen). Neurons were fed twice per week with glia- serum (Hyclone), 2% B27, 1% glutamine (Invitrogen), 100 U/ml penicillin, and Na The intracellular pipette solution (ICS) contained (in mM): KCl 135, Mg-ATP 3, adjusted to 7.39 using NaOH and osmolarity adjusted to 310 tionmOsm (in withmM): NaCl sucrose.140, KCl 4, CaCl chamber with medium (the extracellular solution: ECS) of theDRGneurons werecultured coveronandwere d,transferredslips for following1 into a composi cation of expressing cells. (0.02 cDNA protein fluorescent green enhanced with coinjected were neurons All ng/ 100 at injected was Preso1 Invitrogen); (pCDNA3.1+; ng 100–130 at injected were constructs mGluR5 and mGluR1 All day. following the performed were experiments clamp Patch °C. 37 at overnight cultured and dissociated, mechanically and enzymatically decapitation, and from adult male Wistar rats (175–225 g, total 14 rats) following CO rons was measured as previously reported E Openlab (Improvision) and analyzed with Igor Pro. Hamamatsuaand wheel,filter camera. Images CCD withOrca were collected 2000-U inverted microscope equipped with a fluorescence arc lamp, excitation was performed at 340 and 380 nm in 2 mM Ca ture in aCSF buffer with 1 Fura-2/AM for 45 min. Dorsal spinal cord neurons were loadedC at room tempera were mounted and photographed with a Nikon microscope. (Vector)kit ABC anusing manufacturer’s accordingthe to instructions.Slices processed were sections times, six PBS with washing After °C. 4 at overnight antibody was diluted in blocking solution (1:4,000) and incubated with sections tions were then blocked for 2 h at room temperature in blocking solution. c-Fos and incubated with 0.3% H Immunohistochemistry. against the Preso1 C-terminal 20 aa. postfixation immunogold labeling as described Immunoelectron microscopy. RIPA buffer three times (5 min each time). All procedures were done at 4 °C. was rotated with streptavidin beads (Pierce) for 2 h. Precipitatessupernatant waswere savedwashed as withthe total protein. The remaining 85% of the homogenate lectrophysiology. alcium imaging. oimmunoprecipitation assays. Whole-cell current-clamp recordings were made using cultured DRG neurons. 2 -ATP 0.5, CaCl 0.5, -ATP µ g g / 3 VO µ µ l; l; pEGFPN1; BD Biosciences-Clontech, Palo Alto, CA) for identifi g of the appropriate antibody for 3 h at 4 °C. Then 50 4 , 10 mM sodium pyrophosphate, 50 mM NaF, and 1% Triton X-100) HEK293T cells were loaded at 37 °C in aCSF buffer with 1 Group I mGluR–coupled calcium current in the SCG neu 2 1.1, EGTA 2, HEPES 10, glucose 5, with pH adjusted to adjusted pH with 5, glucose 10, HEPES 2, EGTA 1.1, Mouse spinal cord L4–5 sections were washed in PBS µ M Fura-2/AM for 30 min. Ratiometric Ca 2 DIV 14 cortical neurons were used for surface bioti O 2 in PBS for 30 min. After washing with PBS, sec Immunoelectron microscopy was performed by Mouse brain tissues or HEK293T cells were used g 2 3 ) for) 20minat °C.4Fifteen percent of the 2, MgCl 0 . Briefly, cortical neurons were cooled on 7 . . Briefly, both ganglia were removed 2 2, HEPES 10, glucose 5, with pH 2+ 5 1 solution with a Nikon Eclipse , using rabbit Preso1 antibody 3 2 0 , 0.5 mM MgCl . Briefly, 500 microlit doi:10.1038\nn.3103 µ µ l where indicated. where l l) was then mixed µ l of 1:1 protein 2 euthanasia 2+ imaging 2 ) and µ µ l M −1 ------

© 2012 Nature America, Inc. All rights reserved. ouin n aie Temlpi sniiiy a asse b rcrig paw recording by assessed was sensitivity Thermal-pain saline. in solution plantarregion of one hindpaw of eachmouse wasinjected with6 injection. formalin before min mg/kg 20 (30 subcutaneously MPEP administered studies, was each Tocris) Tween-80; 10% during in pharmacologic For recorded min. was 60 paw for hind interval 5-min injected the biting or licking spent (10 described as formalin, assays. Behavioral experiments were performed at room temperature (~25 °C). 700B amplifier and the pCLAMP 9.2 software package (Axon Instruments). All clamp recordings, action potential measurements were performed with an Axon 7.36 using KOH and osmolarity adjusted to 300 mOsm with sucrose. In current doi:10.1038\nn.3103 CFA-induced pain response was performed as described µ l, 5% in saline) into the dorsal side of one hind paw. The total time time total The paw. hind one of side dorsal the into saline) in 5% l, Inflammatory pain responsiveness was assessed using using assessed was responsiveness pain Inflammatory 5 2 . Mice were injected subcutaneously with formalin formalin with subcutaneously injected were Mice . 5 3 . Briefly, the intra µ l 50% CFA50%l -

as means the behavior data, which were analyzed by one-way ANOVA. Values are presented 53. 52. 51. 50. Statistical analysis. test) before CFA injection and 1–5 d after injection. withdrawal latency on exposure to a defined radiant-heat stimulus (Hargreaves

i, Q. Liu, A.Y.Kim, R.S. Petralia, Y.M. Lu, chloroquine-induced pruritus. chloroquine-induced TRPV1. of subunit synapses. silent (1999). for 31–36 basis molecular a suggests development LTP. CA3 normal but (LTP) potentiation Neurosci. long-term J. CA1 reduced and learning ± 95% confidence interval. t al. et t al. et et al. et

17 Sensory neuron-specific GPCR Mrgprs are itch receptors mediating itch receptors are Mrgprs GPCR neuron-specific Sensory ie akn mtbtoi guaae eetr so impaired show 5 receptor glutamate metabotropic lacking Mice Pirt, a phosphoinositide-binding protein, functions as a regulatory a as functions protein, phosphoinositide-binding a Pirt, , 5196–5205 (1997). 5196–5205 , t al. et All data were analyzed by two-tailed Student’s Cell eetv aqiiin f MA eetr oe postnatal over receptors AMPA of acquisition Selective

133 , 475–485 (2008). 475–485 , Cell

139 , 1353–1365 (2009). 1353–1365 , nature nature NEUR a. Neurosci. Nat. t OSCI -test except EN

C 2 E ,