Supplementary Information dynamically regulates group I metabotropic glutamate receptors. Jia Hua Hu, Linlin Yang, Paul J. Kammermeier, Chester G. Moore, Paul R. Brakeman, Jiancheng Tu, Shouyang Yu, Ronald S. Petralia, Zhe Li, Ping Wu Zhang, Joo Min Park, Xinzhong Dong, Bo Xiao, Paul F. Worley Contents Supplementary Figures 1 13
Figure S1 1 100 Human (1) -------------------------------------------------- -------------------------------------------------- Rat (1) -------------------------------------------------- -------------------------------------------------- Mouse (1) -------------------------------------------------- -------------------------------------------------- Chicken (1) -------------------------------------------------- -------------------------------------------------- Zebrafish (1) -------------------------------------------------- -------------------------------------------------- Drosophila (1) MLLPSGRSNSDVYPLQNVNTTTASASSVGGSKPLLLTRTSSPQLTTLQTLNGGGIGGGGGANAHTALRTNSSAAFAATLAQETAGSLPGTGTQATATTST 101 200 Human (1 ) ---------------------------------------------------------------------------------MDVFSFVKIAKLSSHRTKS Rat (1) -----------------------------------------------------------------------------------------MRSHSCHRTKS Mouse (1) -----------------------------------------------------------------------------------------MRSHSCHRTKS Chicken (1) -----------------------------------------------------------MEEAEMNPSEKLATSFKVIWFTCVGKGAIAMRRRTDHRNK S Zebrafish (1) ---------------------------------------------MITVCNWVINSSSSFFTRSSFPPQPFVPQCHCSPPPKLQIGRTFERRDGSRRDK S Drosophila (101) TASATTTTTAISTTTTAISTTATATATTAAASVNAVNTDFGAGALYGPILGPSHAGNAPIGNWGEIDRHLDAVQEKLKAGVHLAKDGRLYYCNHLTQ S Human Rat Mouse Chicken Zebrafish Drosophila 201 300 (20) SG-----------------WPPPSGTWGLSQVPPYGWEM TANRDGRDYFINHMTQAIPFDDPRLESCQIIPPAPRKVEMRRDPVLGFGFVAGSE KPVVVR (12) SG-----------------WPPPSGTWGLNQVPPYGWEMMTNRDGRDYFINHMTQAIPFDDPRFDSCQIIPPAPRKVEMRRDPVLGFGFVAGSE KPVVVR (12) SG-----------------WPPPSGTWGLNQVPPYGWEMMTNRDGRDYFINHMTQAIPFDDPRFDSCQIIPPAPRKVEMRRDPVLGFGFVAGSE KPVVVR (42) SG-----------------WPHPSGTWSLNQGTPYGWEMTANRDGRDYFINHMTQSATFEDPRIESCQITPPAPRKVEMRRDPVLGFGFVAGSE KPVVVR (56) FPRLYIVFTSTVLCLNGE CQTCVSNVAVMKEES KSCVICPHGKKKYRSIL HHLSQSSSLEEVRVDVEKFVPPAPRKVEMR RDPVLGFGFVAGSE KPVVVR (201) SG-----------------WLPACEDWSKHEELSYGWE RAIDSKGRSYYI NHLNKTTTYEAPECIRWDEHPPEPRLVQLQRSASLGFGFVAGSE RPVIVR Human Rat Mouse Chicken Zebrafish Drosophila 301 400 (103) SVTPGGPSEGKLIPGDQIVMINDEPVSAAPRERVIDLVRSCKESILLTVIQPYPSPKS-----AFISAAKKARLKSNPVKVRFSEEVIIN-----GQVSE (95) SVTPGGPSEGKLIPGDQIVMINDEPVSAAPRERVIDLVRSCKESILFTVI QPYPSPKS-----AFISAAKKARLKSNPVKVRFSEEVIIN-----GQVSE (95) SVTPGGPSEGKLIPGDQIVMINDEAVSAAPRERVIDLVRSCKESILLTVIQPYPSPKS-----AFISAAKKARLKSNPVKVRFSEEVIIN-----GQVSE (125) SVTPGGPSEGKLIPGDQIIMINDEPVSTAPRERVIDLVRSCKESILLTVVQPYPSPKS-----AFISAAKKARLKSNPVKVRFSEEVIIN-----GQVSE (156) SVTPGGPSEGKLIPGDEIVMINEEPVSSAPRERVIDLVRSCKESIILTVVQPYPSPKS-----AFISAAKKAKLKSNPVKVRFAEEVIIN-----GQVPE (284) FVTEGGPSIGKLQPGDQILAVNGEDVKDAPRDHVIQLVRACETQVSLLVC QPMAHCVLPGRKSTLLSAGKRAKLRTRPSRVRFAESVCVNGAPLFPPSAF Human Rat Mouse Chicken Zebrafish Drosophila 401 500 (193) TVKDNSLLFMPNVLKVYLENGQTKSFRFDCSTSIKDVILTLQEKLSIKGIEHFSLMLEQRTEGAGTKLLLLHEQETLTQVTQRPSSHKMRCLFRISFVPK (185) TVKDNSLLFMPNVLKVYLENGQTKSFRFDCSTSIKDVILTLQEKLSIKGIEHFSLMLEQRTEGAGTKLLLLHEQETLTQVTQRPSSHKMRCLFRISFVPK (185) TVKDNSLLFMPNVLKVYLENGQTKSFRFDCSTSIKDVILTLQEKLSIKGIEHFSLMLEQRIEGAGTKLLLLHEQETLTQVTQRPSSHKMRCLFRISFVPK (215) TVKDNSLLFMPNVLKVYLENGQTKSFRFDCSTSIKDVILTLQEKLSIK CIEHFSLMLEQRVEGSGTKLLLLHEQETLTQVTQRPSSHKMRCLFRISFVPK (246) SVKDNFLLFMPNVLKVFLENGQTKSFKFDCNTSIK--------------- -------------------------------------------------- (384) SLGDICVPPMANVLKVFLENGQTKSFKYDATTTVQDVVSSLLDKLCL CCGELFSL VLEHVKSLKRNKLTLLDPQESLARIAARPGAHKLRCLFRITFVPI Human Rat Mouse Chicken 501 600 (293) DPIDLLRRDPVAFEYLYVQSCNDVVQERFGPELKYDIALRLAALQMYIATVTT-KQTQKISLKYIEKEWGLETFLPSAVLQSMKEKNIKKALSHLVKANQ (285) DPIDLLRRDPVAFEYLYVQSCNDVVQERFGPELKYDIALRLAALQMYIATVTT-KQTQKISLKYIEKEWGLETFLPSAVLQSMKEKNIKKALSHLVKANQ (285) DPIDLLRRDPVAFEYLYVQSCNDVVQERFGPELK YDIALRLAALQMYIATVTT-KQTQKISLKYIEKEWGLETFLPSAVLQSMKEKNIKKALSHLVKANQ (315) DPIDLLRRDPVAFEYLYVQSCNDVVQERFGPELKYDIALRLAALQMYIATVTT-KQTQKISLKYIEKEWGLETFLPSAVLQSMKEKNIKKALSHLVKANQ Zebrafish (281) -------------------------------------------------- -----------------EWGLAQFLPPAVLSSMKEKNIKKALTHILKTNQ Drosophila (484) SAAELAQRDLNALDYLYMQCCNDVNQERFAPELQPELALRLAAL HMHQHALANNFAPAKLTVKLVER EFGLERFVPVSLFEGMKRKELRRLISHFLKVNA Human Rat Mouse Chicken Zebrafish Drosophila 601 D-domain 700 (392) NLVPPGKKLSALQAK VHYLKFLSDLRLYGGRVFKATLVQAEKRSEVTLLVGPRYGISHVINTKTNLVALLADFSHVNRIEMFSEEESLVRVELHVLDVKP (384) NLVPPGKKLSALQAK VHYLKFLSDLRLYGGRVFKATLVQAEKRSEVTLLVGPRYGISHVINTKTNLVALLADFSHVNRIEMFTEEESLVRVELHVLDVKP (384) NLVPPGKKLSALQAK VHYLKFLSDLRLYGGRVFKATLVQAEKRSEVTLLVGPRYGISHVINTKTNLVALLADFSHVNRIEMFTEEESLVRVELHVLDVKP (414) NLVPPGKKLSALQAK VHYLKFLSDLRLYGGRVFKATLVQGEKRSEVTLLVGPRYGISHVINTKTNLVALLADFSHVNRIEMFTEDESSVRVELHVLDVKP (314) NLVPPGKKLTALQAK VHYLKYLSDLRLYGGRVFKSTLVQ GEKHTEVTLLVGPRYGISHVINTKTNLVALLADFSHVNRIEM YTEDERNVRVELHVLDVKP (584) EMTGSSKVLTQLQAK LHYLDIIASLPSYGAKCFSTNQRE G---MERVLLVSPRFGLS QISSARNSVPQPISAIEDFTHVVVHREDDITCSVSIYMLGDRA
Human Rat Mouse Chicken Zebrafish Drosophila Human Rat Mouse Chicken Zebrafish Drosophila Human Rat Mouse Chicken Zebrafish Drosophila Human Rat Mouse Chicken Zebrafish Drosophila 701 800 (492) ITLLMESSDAMNLACLTAGYYRLLVDSRRSIFNMANKKNTATQETGPENK GKHNLLGPDWNCIPQMTTFIGEGEQEAQITYIDSKQKTVEITDSTM CPKE (484) ITLLMESSDAMNLACLTAGYYRLLVDSRRSIFNMANKKNAGTQDTG TENK GKHNLLGPDWNCMPQMTTFIGEGEQEAQITYIDSKQKAVEMTDSTLCPKE (484) ITLLMESSDAMNLACLTAGYYRLLVDSRRSIFNMANKKNAGTQDTG SENK GKHNLLGPDWNCMPQMTTFIGEGEQEAQITYIDSKQKTVEMTDSTLCPKE (514) ITLLMESSDAMNLACLTAGYYRLLVDSRRSIFNMANKKNAG NRETGIENR GKHNLLASE WNCVPKTTTFLADGDQETQMPFGDPKQKAIDVSESLLCQKE (414) ITLIMESSDAMNLACLTAGYYRLLVDSRRSIFNMANN---- SNTAGHDTR IKHNYQAID WNYG --T----CGGCNEPHPEYAVCGRRECDISPSEIHQAH (681) VKFIMEDRDACEFSLVLG GYYRLLTG---NVLNVLRERE PNEEDNAPAFL SQHTVLPAS WSYL LPFHNRAHSVNFLITPPYHPMPQQAVLSQPQPVQQQQ 801 900 (592) HRHLYIDNAYSSDGLNQQLSQPGEAPCEADYRSLAQRSLLTLSGPETLKKAQESPRGAKVSFIFGDFALDDGISPPTLGYETLLDEGPEMLEKQRNLYIG (584) HRHLYIDNTYSSDELSQPLTQPGDAPCEADYRSLAQRSLLTLSGPDTLKKAQESPRGAKVSFIFGDLALDDGMSPPTLGYERMLDENPEMLEKQRNLYIS (584) HRHLYIDNSYSSDELNQPLTQPGDAPCEADYRSLAQRSLLTLSGPDTLKKAQESPRGAKVSFIFGDLALDDGMSPPTIGYERMLEENPEMLEKQRNLYIS (614) HRHLYIENTYNSGGFDQHLTKQDPTDAEES-RNFNQHSLLSLSGLESSKKAQDSPRGAKVSFIFGDHNLDG-INPQTLGYERLLDESPEVLEKQRAIYI N (504) HMHGDEKAERQSRGSHMHAQPYFNIPKGKP-------------------- Q-ESPRSAKVSFIFGDPPLDS-VNPQNLGYHRLMEDIPEVLEEHR-YLY A (778) QLSYATDLDLHSVMATELLEEAAKDSGLSDSSGSNYCSESRTQLAVEA KN EQVLRRVQELQKLVQCSEQYLS EQEQELQHVPLQQLQQQQREQTQQQMGQ 901 1000 (692) SANDMKGLDLTPEAEGIQFVENSVYANIG--DVKSFQAAEGIEEPLLHDICYAENTDDAEDEDEVSCEEDLVVGE MNQP----------AILNLSGSSDD (684) SANDMKNLDLTPDTDSIQFVANSVYANIG--DVKNFEAPEGIEEPLLHDICYAENTDDAEDEDEVSCEEDLVVSEINQP----------AILDLSGSSDD (684) SANDMKNLDLTPDTDSIQFVANSVYANIG--DVKNFEAPEGIEEPLLHDICYAENTDDAEDEDEVSCEEDLVVGE MNQP----------AILDLSGSSDD (712) NANDIKGLELSPDAESIQFATNAVYATIN--DGKIFGAAEGIEEPLLHDICYAENTDDAEDEDEVSCEEDIMVGE INRP----------ALLSLSGSSDD (581) QQKDYKPLEASLEVDGFQYGTHMVYG-----DAKIFGTTEGIEEPLLHDICYAETTDDAEEDDDISCEEDMMM MMGEMK------DKAN SLLSLSESSDD (878) SVAMLEFDSDCDSLNSSKVSSTEESNHVSQPSLKHSDSLTLLAETISHELTGITQGLNAIPEPISASAPPTPA TPKRKPSLCSTS- SPRPQRKLNGFSQL 1001 Homer site 1100 (792) IIDLTSLPPPE--GDDNEDDFLLRSLNMAIAAPPPGFRDSSDEEDSQSQAASFPEDK---EKGSSLQNDEIPVSLIDAVPTSAEGKCEKGLD NAVVSTLG (772) IIDLTTLPPPE--GDDNEDDFLLRSLNMAIAAPPPGFRDSSDEEDTQSQATSFHENK---EQGSSLQNEEIPVSLIDAVPTSAEGKCEKGLD PTVVSTLE (772) IIDLTTLPPPE--GDDNEDDFLLRSLNMAIAAPPPGFRDSSDEEDTQSQATSFHEDK---EQGSSLQNEEIPVSLIDAVPTSAEGKCEKGLD PAVVSTLE (800) IIDLTSLPPPE--GDDNEDDFLLHTLNMAIAAPPPGFRDSSDEEDSQNQATQPRDDK---EQASNLGS DDIPVSLIDAVPTNTEGKCEKGLDDAVVSTLQ (670) IIDLTSLPPPPEGDYEEDSDDLLESLNLAIAA PPPGFRDSSDEDDHHDRKMSKN---------------DIPVSLIDSVPTQVAGGSEEVFN DAVVSTLQ (977) LSDLQALGTDFSQSESDSESVASPAQSPTTRR PQQLLQAG SKQQQQQQQH QQLAQRSSFGLHSPDGTH FGAETKDYNLREYLQQLKEISNVP DTDVAARQ Human Rat Mouse Chicken Zebrafish 1101 1200 (875) ALEALSVSEEQQTS DNSG---VAILRAYSPESSSDSGNETNSSEMTESSELATAQKQSENLSRMFLATHEGYHPLAEEQTEFPASKTPAGGLPPKSSHAL (867) ALEALSE--EQQKSENSG---VAILRAYSPESSSDSGNETNSSEMTEGSELAAAQKQSESLSRMFLATHEGYHPLAEEQTEFPTSKTPSVGLPPKSSHGL (867) ALEALSE--EQQKSENSG---VAILRAYSPESSSDSGNETNSSEMTEGSELAAAQKQSESLSRMFLATHEGYHPLAEEQTEFPTSKAPSVGLPPKSSHGL (895) ALEALAASEEQQTNDNSG---VAILRAYSPESSSDSGNETNSSEMTESSELAAAQKHSENTAR MFLTTSEGYQPLVEEQTEFPIAKSQAGGPGMKSSQPL (755) ALEALAASEDQSHPQSDNSSGVEISRSFSPESSSDSGNETNSSEMTESSELAAAQKLSENSLK MFVATAEGYQTLAEEETEFRLAPSLDDPQSS------ Drosophila (1077) LSEIYGFEVREDTFIETDTDLIDLRAIPPPQTPDELDSLSLLNAAPPKGFEGKAEELDSFLQQ MLVAPPTQKATPAKELTPEEIMSFIIPPPPNLEAQPE 1201 1300 Human (972) AAR---PATDLPPKVVPSKQLLHSDHMEMEPETMETKSVTDYFSKLHMGSVAYSCTSKRKSKLADGEGKAPPNGNTTGKKQQGTKTAEMEEEASGKFGTV Rat (962) AAR---PATDLPPKVVPSKQILHSDHMEMEPETMETKSVTDYFSKLHMGSVAYSCTSKRKSKLAEGEGKCPLSGNVPGKKQQGTKIAETEEDTKGKVGTV Mouse (962) AAR---PATDLPPKVVPSKQILHSDHMEMEPETMETKSVTDYFSKLHMGSVAYSCTSKRKSKLPEGEGKSPLSGNIPGKKQQGTKIAEIEEDTKGKAGTV Chicken (992) AGC---QAADLQSKVVPSKQILHSDNMEMEPETMETKSVTDYFSKLHMGS LVYSCTSKRKSKM TESEVKAPSDGNATVKKQQGTKKAETDEELKAKFGTI Zebrafish (849) -----------------AVAN LRSDHFEMEPETMETKSLTEYFSKMHRDV MRGMAQGKVEEQDS GMKSSAEAAARQLN--------IAESEDLVGMYSNF Drosophila (1177) Human (1069) Rat (1059) Mouse (1059) Chicken (1089) RPEECLYSNSVQAKREFFQAVANGNNSIQQNNHKDQAAHVIEYPTVERKS KFSCCPTSKKEEAA SLEEAAPLQPPPRTPAIEQTTPPPARPPKSAELLQR 1301 1400 SSRDSQHLSTFNLERTAFRKDSQRWYVATEGGMAEKSGLEAATGKTFPRASGLGAREAEGKEEGAPDGETSDGSGLGQGDRFLTDVTCASSAKDLDNPED SSRD NPHLSTFNLERTAFRKDSQRWYVASDGGVVEKSGVEAPAMKAFPRGPGLGNREAEGKEDGTMEGEADDASGLGQGERFLSDMACVASAKDLDNPED SSRD NPHLSTFNLERTAFRKDSQRWYVASDGGVVEKSGMEAPAMKVFPRGPGLGNREAEGKEDGTVEGGADDASVLGQG DRFLTDMACVASAKDLDNPED SARDSQ RLSTFNVERTAFR---QRWYGA DDGAADKPS-PETANGKTFPRVPVRGKTETDCKDEVDPEVDQDDTSGLSQG ENFLSDM TPVSSAKDLNDAED Zebrafish (924) NGRDSQ CSAPFDMESMSYAFQ---------------------GSNQRLHMILNNKSEEHIYSEVAGDSAPLHADRVAQ KVSLEDSIDLNASSPAKEQYLV Drosophila (1277) YSPKK QVRIVGHQPVVSVPLRELCPPQLPPRSSPTASSEPATPQTATPATSAVSAPKKPPLPPIANRPRPVNGTPTPV SNTSPPIPATTRLPNAHSNGGQ Human (1169) Rat (1159) Mouse (1159) Chicken (1185) 1401 1500 ADSSTCDHPSKLPEADESVARLCDYHLAKRMSSLQSEGHFSLQSSQGSSVDAGCGTGSSGSACATPVESPLCPSLGKHLIPDASGKGVNYIPSEERA PGL TDSPTCDHATKLPEAEDNVARLCDYHLAKRMSSLQSEGHFSLQSSQGSSVDTGCGPGSSSSACATPVESPLCPSMGKHLIPDASGKGGSYISPEERVAGH TDSPSCDHATKLSEAEDNVARLCDYHLAKRMSSLQSEGHFSLQSSQGSSVDTGCGPGSSSSACATPVESPLCPSMGKHMIPDASGKGGRYISPEERAPGH TDLSADDHPSKLPEAEQSVARLCEYHLAKRMSSLQSEGHFSLQSSQCSSVDAGCSTG--SSTCATPVESPLCTS DIKHVISDPSMKGIAYIPADERAAIL Zebrafish (1003) ERARSEHQQSKGASSEQEVTRLYEYHLSKRMSSLQSE GVHSLQSSQCSSI DAGCSTG--SSSCVTPMDSPLCTVD SVHLHTESSMKGLGYTNQNVDGTFL Drosophila (1377) PTLPKKPQQLHGEKLFIKNGHLIDGEALLAKTDVAMA GLLIKLDQVAAQC SAAQSAGGGTSIDEDKFQRARNELTEQTLALVTASKFLVASMSDMTLINL
Human (1269) Rat (1259) Mouse (1259) Chicken (1283) 1501 1550 PNHGATFKELHPQTE----------------------------------------------------------------GMCPRMTVPALHTAINTEPLF PNHGATFKELHPQTE----------------------------------------------------------------GMCPRMTVPALHTAINADPLF PNHGATFEELHPQTE----------------------------------------------------------------GMCPRMTVPALHTAINADPLF SNHGTTYKDLHQQPE---------------------------------------------------------------- AVCHRMTVPVTHSAINAEPLF Zebrafish (1101) RKHHGQAGAESGREGSQRLPKIRETTVVFFIATVYMLSSPCLVDICSDAADGDKRGFALTECPSSSSSRAQLVLCQARG DGTSRMLPCLLVHSASVH PLW Drosophila (1477) PDHLTSCLTAIRRITELAQDMTR----------------------------------------------------HTSAP LQTRNIVLKVHDVASSFREL 1601 1637 Human (1305) GTLRDGCHRLPKIKETTV------------------- Rat (1295) GTLRDGCHRLPKIKETTV------------------- Mouse (1295) GTLRDGCHRLPKIKETTV------------------- Chicken (1319) GTLREGCHRIPKIKETTV------------------- Zebrafish (1201) LPQGGGKGILNKTTMGSGSIHTFYGNEMAVIIQAVVR Drosophila (1525) VGVQIGPIGAGQLALQAECLANVLATLLRSLRVFSP- Supplementary Figure 1: The amino acid sequence alignment of from various creatures. Red residues shaded in yellow are identical in all the predicted proteins, dark blue residues shaded in light blue are identical in > 50% of the predicted proteins, similar residues are shown in black shaded in green. D domain and Homer binding site are indicated.
Figure S2 1 100 h (1) MDVFSFVKIAKLSSHRTKSSGWPPPSGTWGLSQVPPYGWEMTANRDGRDYFINHMTQAIPFDDPRLESCQIIPPAPRKVEMR RDPVLGFGFVAGSEKPVV hpreso2 (1) --------METLDSQRVQDRLLAAPGCSSPSGQQELFSSHVMQEES----------------ANDMECEQLPAEILRQVTV HRDPIYGFGFVAGSERPVV hpreso3 (1) -----MEELETSLFQTRKAHRIEQMVARWLRRSRDSSARAKVAAADG---------------PARNPTQTLIPVRHTVKIDKDTLLQDYGFHISESLPLT 101 200 h (101) VRSVTPGGPSEGKLIPGDQIVMINDEPVSAAPRERVIDLVRSCKESILLTVIQPYPSP-KSAFISAAKKARLKSNPVKVRFSEEVIINGQVSETVKDNSL hpreso2 (77) VRSVRPGGPSENKLLAGDQIVAINEEDVSEAPRERLIELIRSAKEFIVLTVLHTHQSP-KSAFISAAKKAKLRSNPVKVRFSEQVAVGETDA KMMKKEAL hpreso3 (81) VVAVTAGGSAHGKLFPGDQILQMNNEPAEDLSWERAVDILREAEDSLSITVVRCTSGVPKSSFLTEEKRARLKTNPVKVHFAEEVLISGHS----QGNSL 201 300 h (200) LFMPNVLKVYLENGQTKSFRFDCSTSIKDVILTLQEKLSIKGIEHFSLMLEQRTEGAGTKLLLLHEQETLTQVTQRPSSHKMRCLFRISFVPKDPIDLLR hpreso2 (176) LLIPNVLKVFLENGQIKSFTFDGRTTVKDVMLTLQDRLSLRFIEHFALVLEYAGPEQNHKFLLLQDKQPLAYVVQRTHYHGMKCLFRISFFPKDPVELLR hpreso3 (177) LCMPNVLKLYLENGQTKAFKFEANTTVKDIILTVKEKLSIRSIEYFALALEEQYSIS--RLHLLHEEELIQQVVEREESHDYRCLFRVCFVPKDPLDLLK 301 400 h (300) RDPVAFEYLYVQSCNDVVQERFGPELKYDIALRLAALQMYIATVTTKQTQKISLKYIEKEWGLETFLPSAVLQSMKEKNIKKALSHLVKANQNLVPPGKK hpreso2 (276) RDPAAFEYLYIQSRNDVIRERFGMDPKPEMLLGLAALHIYITVSATRPSQKISLKNVEKEWGLEPFLPPSLLQVIKEKNLRKSLSQQLKAHQTHPSCGTK hpreso3 (275) EDPVAFEYLYLQSCSDVLQERFAVEMKCSSALRLAALHIQERIYACAQPQKISLKYIEKDWGIENFISPTLLRNMKGKDIKKAISFHMKRNQNLLEPRQK 401 D-domain 500 h (400)--LSA LQAKVHYLKFLSDLRLYGGRVFKATLVQAEKRSEVTLLVGPRYGISHVINTKTNLVALLADFSHVNRIEMFSEEESLVRVELHVLDVKPITLLME hpreso2 (376)--GSA IQAKLQYLRILNELPTFTGVLFNTVGLD- EKQSATTLLVGPRHGISHVIDLKTNLTTVLSEFSKISKIQLFRENQGVARVETSIMDAKPLVLLME hpreso3 (375) QLISA AQLRLNYLQILGELKTYGGRIFNATLMLQDRESYIALLVGAKYGISQVINSKLNIMSTLAEFANISRVELTEESEKVSVVKVYLQDVKVLTLLLE 501 600 h (498) SSDAMNLACLTAGYYRLLVDSRRSIFNMANKKNTATQETGPENKGKHNLLGPDWN CIPQMTTFIGEGEQEAQITYIDSKQKTVEITDSTMCPKEHRHLYI hpreso2 (473) WPEATNFACLIAGYCRLLLDSRKMVFSRPASQPLPPPMIKADYMHSAHRPVTGGH LGKKESSYVG----------------------------------- hpreso3 (475) SNSAKDLACLIAGYYRLLVDPVTSIFLWPGNKQQAHRVSAEEGYESRACSDSEES SEVDCVLEPLSDRRLVKLAPCRSLIK------------------- 601 700 h (598) DNAYSSDGLNQQLSQPGEAPCEADYRSLAQRSLLTLSGPETLKKAQESPRGAKVS FIFGDFALDDGISPPTLGYETLLDEGPEMLEKQRNLYIGSANDMK hpreso2 (538) ----SVGTSPRKSSRCTPPPADSELVSFCYLHMREQ------RKEQESRTDVNENLIFFEETRPRTKSDPTS---------------------------- hpreso3 (556) EEQPPGNSPTPEVARRGPSTCGASSTTDSAESEASDSANTESRGYRTSGSSESMD ALEEDDLDTCSSSRSTFFHFGSPGLAESIDSDSQEERSGIETSGF 701 800 h (698) GLDLTPEAEGIQFVENSVYANIGDVKSFQAAEGIEEPLLHDICYAENTDDAEDEDEVSCEEDLVVGEMNQPAILN------------LS GSSDDIIDLTS hpreso2 (600) ------KSSGQGYEVVPDDFDAASLDHEPCASRARSYTLDNSLGAEALNFYCDSC KAKLQEQLGPRKGGKPGSS-----------------RDNIVDLMS hpreso3 (656) LCLLDLAQRANPQCQKTEFSESAALETFGWAPELSTVRLDPRLYEGSHADYYSLCSSVSPASYLSDSSESTASRQGGAPPAWGQQGEAQPSSMLEPLA 801 Homer site 900 h (786) LPPP-EGDDNEDDFLLRSLNMAIAAP--PPGFRDSSDEEDS-QSQAASFPEDKEKG------SSLQNDEIPVSLIDAVPTSAEGKCEKGLDNAVVSTLGA hpreso2 (677) LPPPGSEEEEEEEDETTSLLPAIAAP--PPGFRDNSSDEDDPKRRAVQSQEQGRHL------RGLLYDEIPVTLIDSVQTRTVRDHAQELDDALVSTLQA hpreso3 (756) LHPPLAFEDGSSDEEYYDAADKLTPPGPPSGPRDVSTAEPSATSLQNKASTSSPENSLPCGPDGRQPSRRGGVKKYAKTLRKRRSFLQTDDTSQVSFPLV 901 1000 h (876) LEALS VSEE---QQTSDNSGVAILRAYSPESSSDSGNETNSSEMTESSELATAQKQSENLSRMFLATHEGYHPLAEEQTEFPASKTPAGGLPPKSSHALA hpreso2 (768) LEALA ASEDGPHPPPPQTAGLIVLATITPESSLDSGHETNSSELTDMSEMMSAMKQHQNTTYFLAQHLNKDSLLARKDLPFRIQSCAAQAVLTAPYSLGR hpreso3 (856) PSASLESVDDVCYYDREPYLALGAPSPTVSSLQDMQGEPGLLETKALGLLAPLRETKSTNPASRVMEMEPETMETKSVIDSRVSSISAIRFRIDPNNKEN 1001 1100 h (973) ARPATDLPPKVVPSKQLLHSDHMEMEPETMETKSVTDYFSKLHMGSVAYSCTSKRKSKLADGEGKAPP-----------NGNTTGKKQQGTKTAEMEEEA hpreso2 (868) PDPNPSLQPIATGQSPGPPGARRKLPQSEGQVQGERTYSLAVHPALSPQLSEQKNLSLLSPVPEDKGPGHTRAGLEMSLRAATSSLSEEQVSELRDNLPK hpreso3 (956) SGVVPAASSSASTPHCSNPGSSGPDTAQARPSQ-ILPLSQDLDGIAPKEPTIEHGDSSFSLSSGDPNPDRACLAS----NPGLNNVSQGDTLELQLEPHV 1101 1200 h (1062) SGKFGTVSSRDSQHLSTFNLERTAFRKDSQRWYVATEGGMAE----KS--GLEAATG------KTFPRASGLGAREAEGKEEGAPDGETSDGSGLGQGDR hpreso2 (968) EVRLSPKLILDPKSSVTPAIISAALQQVVHNKSLVTAGGALGNPPSRGERRLEASMGRPEVSMMSSSASKNLKFKISPSAPETSWNSQHQLGAEVSSSPR hpreso3 (1052) QLEMGLESFCTNHIQETAPKYTEPLLSPRDEPRSDECGINPGEK-IASIPTKEEPQGQLSLERDREVTNKNGTNVFQEESRKDSGDSPGDVSNNVSQTLD
1201 1300 h (1150) FLTDVTCASSAKDLDNPEDADSSTCDHPSKLPEA--DESVARLCDYHLAKRMSSLQSEGHFSLQ--SS-QGSSVDAGCGTGSS------------G---- hpreso2 (1068) APTGSRADSLHLSQQEDSLPVQNFPPKSYLLRTS--RESVGKQATGEVAGKGGPVGGKPTLQKQGTISSQGEKAQLESTPKRSKLEETSLVPRATYPMAL hpreso3 (1152) ISSPAGKIVTSLSLDAPVTGTEQIPPHPPRDPQGQSREPPGQGCQAQEQKLFVELDLDPDFFLGKQTVSPAVPPEGIKAEAPNHVTGQDIAPRDSPEWVC 1301 1400 h (1229) --------SACATPVESPLCPSLGKHLIPDASGKGVNYIPSEE-----RAPGLPNHGATFKELHPQTEGMCPRMTVPALHTAINTEPLFGTLRDGCHRLP hpreso2 (1166) QS------PSCQSRSHSPSCQPHGHSPSSQSRGQSPSCQPRGQSPLRSQAASRQVSTMPSRKLETTLNGAHSTSEGPAKPKSSRGPFRLRNLFSATFPTR hpreso3 (1252) FNPEPSLPEPLPCPQEDPHLETSNHCLLSEGKSDSSSICLSAEKSFLCFAPESHPEVSASLRVATSLGFAGMNEMVAPRIGMDQCSCQFSYATCFRGPQP 1401 1500 h (1316) KIKETTV--------------------------------------------------------------------------------------------- hpreso2 (1260) QK KETDERQAQLQKVKQYELEFLEELLKPPSQGELPGTEYLQPPAPGRCSCQLRS SPVQQGPGMSREQRRSCDCKRICRGGRPQATQTPVPSLRGRERDR hpreso3 (1352) E----TEEEDR--DLEAHPMAPLTS---PPSAGSPVVLPWRPARAHSCTTAPLSR KSHIWPEYCSRALRQLKATPASTPEGFIQLMESLLELQDILETSW 1501 1600 h (1316) ---------------------------------------------------------------------------------------------------- hpreso2 (1360) VLPSQRQPEAGPGVSLSSPINVQRIRSTSLESRECRSDPESGVSCLTTCASGGEC LGAPNYRKLMRRYSISELDQGDRASLTSDVYPHPPLGMLPREAKE hpreso3 (1443) GVGNKHPPEKCTWHFTESRSRLCMGSQKLLSSCRHVIRMDQSPEEMQGAVRDTFQ HLVQLAGLCFQFTDCSRCSARHREAAGNLRDVVYTYHQFIEAAKS 1601 1700 h (1316) ---------------------------------------------------------------------------------------------------- hpreso2 (1460) VEASLPIA LGPKSRSLESPTLGDPSYVQVAPETKGPRQMAVFSLPEEVYRKPAELDEDSESSKCCSIRYCFYYRKCDMADDASDGKDELSYSIPMKILPG hpreso3 (1541) TCERGYHD LSVKLLARQCTALTAAVFCLTQKFRASTAL----------------- --------------------------------------------- 1701 1800 h (1316) ---------------------------------------------------------------------------------------------------- hpreso2 (1560) MKLDEQVVPVVSRTLQVLDAATCSSSSPEASRTQEIDLRVSTFEGSLAKINALRAHAYGLPDGFLAARLDTNELLTVLRQCVASPEARAPKPYVSQISEY hpreso3 (1541) ----- ------------------------------------------ ---------------------------------------------------- 1801 1900 h (1316)---------------------------------------------------------------------------------------------------- hpreso2 (1660) KLELALKFKELRASCRRVANVDKSPTHMLAAITGSFQVLSSLIETFVRLVFIVRSEAQRQELLAKVEEVVRNYTFLLRAAEESTARNLNQQQQQQQQQQQ hpreso3 (1541)----- ----------------------------------------------------------------------------------------------- 1901 1950 h (1316) -------------------------------------------------- hpreso2 (1760) QQQQQQQQQQQQQQQQVAAAAGAATEHPPGSPTSATVMSTFTHSLKTLIK hpreso3 (1541) -------------------------------------------------- Supplementary Figure 2: The amino acid sequence alignment of human Preso family. Red residues shaded in yellow are identical in all the predicted proteins, dark blue residues shaded in light blue are identical in > 50% of the predicted proteins, similar residues are shown in black shaded in green. D domain and Homer binding site are indicated.
Figure S3 Preso2 Preso3 Supplementary Figure 3: Preso family expression patterns by in situ hybridization. 1Kb C terminal coding region of each gene was used for antisense and sense probes that were labeled with [35S].
Figure S4 a Myc : Myc Preso2: Myc Preso3: Myc Homer1c: b Lysate IP: anti HA mglur5: Myc : IP: Homer1c anti HA Lysate anti Myc c Blot with anti myc d Lysate IP: anti Lysate IP: anti mglur2: Myc : mglur4: Myc : anti mglur2 anti mglur4 anti Myc anti Myc e mglur5 C term: S1126A S1126A λ Phosphatase: ps mglur 50 KD mglur5 50 KD
Supplementary Figure 4: Preso family: co IP assays with Homer and mglurs. a, and Preso2 co IP with Homer1c, but Preso3 does not. Myc Homer1 and Myc Presos were transfected into HEK293T cells. Detergent lysates of the cells were incubated with anti Homer1c antibody and then analyzed by Western blotting with anti Myc antibody. b, mglur5 co IPs with. Myc and HA mglur5 were transfected into HEK293T cells. Detergent lysates were incubated with anti antibody and blotted with anti Myc and anti HA antibody. c, mglur2 does not co IP with. Myc and mglur2 were transfected into HEK293T cells. Detergent lysates were incubated with anti antibody and precipitates analyzed by Western blotting with anti Myc and anti mglur2 antibody. d, mglur4 does not co IP with. Myc and mglur4 were transfected into HEK293T cells. Detergent lysates were incubated with anti antibody and precipitates analyzed by Western blotting with anti Myc and anti mglur4 antibody. e, Characterization of mglur5 phosphorylation antibody. The phosphorylation site specific anti mglur5 ps1126 antibody detects wild type mglur5 C terminus from transfected HEK293 cells, but not mglur5(s1126a) mutant. λ phosphatase treatment of the immunoblot abolishes reactivity of wild type mglur5 C terminus. A phosphorylation independent anti mglur5 C terminus antibody is used to detect expression of wild type and mutant mglur5 C terminus.
Figure S5 a protein WW PDZ RA FERM Homer Ligand PDZ Ligand PPPGFRD ETTV locus 90kb EX2 EX3 EX4 EX3 Targeting vector LoxP frt PGK-neo frt EX3 LoxP Nhe I NheI Short Arm Long Arm LoxP frt frt LoxP 90kb floxed EX2 PGK-neo EX3 EX4 allele - allele EX2 LoxP Cre-recombinase expression 90kb EX4 b Cortex Cerebellum Cortex Cerebellum / / / / / / / / / / / / 517 bp 353 bp 409 bp 245 bp c Actin / d e Cortex Hippocampus Striatum Cerebellum Actin / Homer1 Homer2 Homer3 GluN1 GluA1 W1 W2 K1 K2 W1 W2 K1 K2 W1 W2 K1 K2 W1 W2 K1 K2 GluA2/3 Actin
Supplementary Figure 5: PKAP1 knockout mice generation. a, Schematic representation of the gene targeting strategy. The relevant portion of the locus Exon 3 was inserted in the LoxP/PGK neo cassette to make a conditional knockout. CMV CRE mice were employed to generate / mice. b, RT PCR analysis after CMV CRE expression. Two independent primer sets in different exons were used. c, Western blot analysis shows absence of immunoreactivity in / mouse brain. d, Western blot analysis shows absence of immunoreactivity in / cortical neurons. e, Western blot analysis showing no significant difference of Homer1, Homer2, Homer3, NMDA receptor GluN1, GluA1 and GluA2/3 protein expression between 8 week old and / mouse brain. W: ; K: /.
Figure S6 a Duration of response (s) 100 80 60 40 20 0 Grm5 / / MPEP Grm5 / / MPEP b Duration of response (s) 400 350 300 250 200 150 100 50 Grm5 / / MPEP Grm5 / / MPEP * ** -20-10 0 10 20 30 40 50 60 Time after formalin injection (min) 0 0-10min 10-60min Supplementary Figure 6: Grm5 / mice show reduced response to formalin induced inflammatory pain. a, b, Formalin induce inflammatory pain in Grm5 / mice. a, The duration of behavioral responses to hind paw formalin injection in 5 min intervals in Grm5 / mice and their littermates with or without MPEP (30mg/kg, i.p.) pre injection. b, Results from a grouped into two phases. *P < 0.05, **P < 0.01, n = 7 11 for each group.
Figure S7 a / 20 mv 20 ms 300 na b 10 / 8 Spike number 6 4 2 0 0 200 400 600 800 1000 Injected current (na) Supplementary Figure 7: Excitability of DRG neurons is not different between and / mice. The small diameter DRG neurons were cultured and injected with depolarizing currents stepwise from 100 to 900 na with 100 na interval. Each injected current lasted 200 ms and was separated by an interval of 4 s. The excitability was evaluated by the number of action potentials. and / DRG neurons do not show a significant difference of excitability. Eight neurons from 3 mice and 10 neurons from 3 / littermates were recorded.
Figure S8 a b Duration of response (s) 180 160 140 120 100 80 60 Homer2 / Homer3 / 800 Homer2 / Homer3 / * * * * * 40 20 0-20 -10 0 10 20 30 40 50 60 Time after formalin injection (min) Duration of response (s) 700 600 500 400 300 200 100 0 0-10min ** 10-60min Duration of response (s) c 140 Grm5 120 R/R 100 80 60 40 20 0-20 -10 0 10 20 30 40 50 60 Time after formalin injection (min) * d Duration of response (s) 700 600 500 400 300 200 100 0 Grm5 R/R 0-10min ** 10-60min Duration of response (s) 120 100 e Homer1a / 80 60 40 20 0-20 -10 0 10 20 30 40 50 60 Time after formalin injection (min) Duration of response (s) f 400 * 350 Homer1a / 300 250 200 150 100 50 0 0-10min 10-60min
Supplementary Figure 8: Formalin induced inflammatory pain responses in Homer2 / Homer3 / mice, Grm5 R/R mice and Homer1a / mice. a, b, Formalin induce inflammatory pain is increased in Homer2 / Homer3 / mice. a, The duration of behavioral responses to hind paw formalin injection in 5 min intervals in Homer2 / Homer3 / mice and their controls. b, Results from a, grouped into two phases. *P < 0.05, **P < 0.01, n = 12 and 8 for and Homer2 / Homer3 / mice. c, d, Formalin induced inflammatory pain is increased in Grm5 R/R mice. c, The duration of behavioral responses to hind paw formalin injection in 5 min intervals in Grm5 R/R mice and their littermates. d, Results from c grouped into two phases. *P < 0.05, **P < 0.01, n = 8 for each group. e, f, Formalin induce inflammatory pain is reduced in Homer1a / mice. e, The duration of behavioral responses to hind paw formalin injection in 5 min intervals in Homer1a / mice and their littermates. f, Results from e, grouped into two phases. *P < 0.05, n = 9 for each group.
Figure S9 b c Lysate PI IP HA : F806R HA Homer1c: G89N W24A Homer3 Homer1c mglur1 IP 3 R IP: anti- Shank Homer1c Lysate PSD95 IP: anti Supplementary Figure 9: Full length western blot for Figure 1. Samples were loaded on a 4 12% gel and blotted for the antibodies indicated in the figure. Dotted lines indicated that the bands were used for inclusion in Figure 1.
Figure S10 Myc mglur5: F8 06 R Δ FL HA : Δ W W b PD Z Δ FE RM F8 06 R a Myc FERM: HA mglur5: F1128R mglur5 / co IP mglur5 / co IP mglur5 / Lysate mglur5 / Lysate / IP FERM / IP FERM / Lysate Myc : W T 10 2 1~ 0 92 0 Myc : 1~ W T 10 2 1~ 0 92 0 1~ W T HA mglur5: HA mglur5: FR e c IP: anti-myc IP: anti YV F YV / Lysate mglur5 / co IP mglur5 / Lysate mglur5 / co IP mglur5 / Lysate / IP / Lysate / IP / Lysate IP: anti Myc IP: anti Myc
Supplementary Figure 10: Full length western blot for Figure 2. Samples were loaded on a 4 12% gel and blotted for the antibodies indicated in the figure. Dotted lines indicated that the bands were used for inclusion in Figure 2.
Figure S11 a b Homer1c: mglur5: : Homer1c IP Lysate Lysate Homer1c IP Homer1c: mglur5: mglur5tsaa: FR: Lysate Homer1c IP Lysate mglur5 mglur5 ps mglur Homer1c Homer1c Supplementary Figure 11: Full length western blot for Figure 3. Samples were loaded on a 4 12% gel and blotted for the antibodies indicated in the figure. mglur5 dimers were used for mglur5 band quantification. Dotted lines indicated that the bands were used for inclusion in Figure 3.
Figure S12 a b c HA mglur5: HA CDK5/P35: HA MEK DD: mglur5 ps mglur mglur5 ps mglur Control UO126 Purvalanol A HA : HA CDK5: d CDK5 IP: anti Lysate HA : HA ERK1: MEK DD P35 CDK5 ERK1 e HA mglur5: HA : HA ERK1: mglur5 dimer mglur5 monomer ERK1 f Lysate PI IP CDK5 IP: anti Lysate g Lysate PI IP ERK1/2 IP: anti mglur5 Lysate IP: anti CDK5 IP: anti ERK Supplementary Figure 12: Full length western blot for Figure 4. Samples were loaded on a 4 12% gel and blotted for the antibodies indicated in the figure. mglur5 dimers were used for mglur5 band quantification. Dotted lines indicated that the bands were used for inclusion in Figure 4.
Figure S13 a Cx Sc c / / / / Ctl DH Ctl DH Ctl BD Ctl BD mglur5 / co IP mglur5 / co IP mglur5 / Lysate ps mglur ps mglur mglur5 / Lysate Actin / Lysate Homer / IP Homer / IP b / Lysate / ERK1/2 / co IP mglur5 / IP d mglur5 / Total mglur5 / Surface Actin / Total / / Ctl D5 Ctl D5 Ctl D30 Ctl D30 ERK1/2 / Lysate Actin / Surface mglur5 / Lysate
Supplementary Figure 13: Full length western blot for Figure 5. Samples were loaded on a 4 12% gel and blotted for the antibodies indicated in the figure. mglur5 dimers were used for mglur5 band quantification. Dotted lines indicated that the bands were used for inclusion in Figure 5.