I-Cdk5 I-Phosphorylates i-Dopamine I-D2 I-Receiver kunye ne-Attenuate Signal Downing (2013)

Citation: Jeong J, Park YU, Kim DK, Lee S, Kwak Y, et al. (2013) I-Cdk5 Phosphorylates I-Dopamine D2 i-Receptor kunye ne-Attenuates i-Downstream Signaling. PLoS ENYE 8(12): e84482. doi:10.1371/journal.pone.0084482

umhleli: James Porter, kwiYunivesithi yaseNorth Dakota, eUnited States yaseMelika

I funyenwe: Ngamana i-20, 2013; Zamkelwa: NgoNovemba 14, 2013; Yashicilelwe: Disemba 31, 2013

Copyright: © 2013 Jeong et al. Eli linqaku lofikelelo oluvulelekileyo lisasazwe phantsi kwemiqathango ye Ilayisenisi yeCreation Commons Licribution, evumela ukusetyenziswa, ukuhanjiswa, nokuvelisa okungahambisani naluphi na uhlobo, ukuba unobumba bokuqala kunye nomthombo baxelwe.

Inkxaso: Lo msebenzi uxhaswe ngezibonelelo (NRF-2012R1A2A2A01012923 kunye ne-NRF-2012R1A4A1028200) evela kurhulumente waseKorea (MSIP) kwaye iphinde ixhaswe phantsi kwenkqubo yentsebenziswano yamazwe ngamazwe elawulwa yi-NRF yaseKorea (2012K2A1A2033117) Research Institute (2031K415A2004A2006) Inkqubo yoPhando oluSisiseko ye-MSIP (XNUMX-XNUMX). I-SKP yaba ngumamkeli we-XNUMX kunye ne-XNUMX ye-National Alliance for Research on Schizophrenia and Depression (NARSAD) amaBhaso aPhandi oLutsha. Abaxhasi bemali babengenayo indima ekuyilweni kokufunda, ukuqokelela idatha kunye nohlalutyo, isigqibo sokupapasha, okanye ukulungiswa kombhalo wesandla.

Injongo yokunyanzela: Ababhali baye bavakalisa ukuba akukho mfuno ekhuphisanayo.

intshayelelo

Ukubonakaliswa kweDopamine kubandakanyeka kwimisebenzi eyahlukeneyo yobuchopho kubandakanya ulungelelwaniso lwemoto, ulawulo lweemvakalelo kunye neendlela zokuvuza [1]. Elona candelo liphambili lokubonakaliswa kwe-dopamine kwi-vertebrates yenziwa yi-striatal medium spiny neurons (MSNs) ebonisa ngokukhethekayo i-subset ye-dopamine receptors kwaye ifumane igalelo le-dopaminergic ikakhulu kwindawo ye-ventral tegmental (VTA) kunye ne-substantia nigra (SN).) [2]. I-Dopamine receptors zi-G protein-coupled receptors (GPCR) ezinemimandla esixhenxe ye-transmembrane kwaye iqulethe ii-subtypes ezimbini, I-D1-efana ne-D2-efana ne-receptors, edibanisa izenzo ezihambelanayo kwi-dopamine signing [1]. Umzekelo, i-dopamine D1-like receptors (D1, D5) ivule i-adenylyl cyclase nge-G._Iimpawu kunye nokwandisa inqanaba le-intracellular ye-cAMP, kodwa i-dopamine D2-like receptors (D2, D3, D4) inhibit adenylyl cyclase nge-G._αi kunye nokunciphisa inqanaba le-intracellular le-camp [1], [3].

Phakathi kwe-dopamine receptors, i-D2 receptor (DRD2) ibandakanyeka kwi-pathophysiology yeengxaki ezininzi zengqondo ezibandakanya i-schizophrenia kunye nokulutha kweziyobisi. [4], kangangokuba amachiza amaninzi okulwa nee-antipsychotic ajolise ngokuyinxenye kwi-DRD2. Kukwayaziwa ukuba umsebenzi we-DRD2 uhambelana kakuhle neziphumo zokuziphatha zeziyobisi zokusetyenziswa kakubi kwimodeli yezilwanyana [5]. I-Antidepressants kunye nokusebenza kakuhle kwe-mood stabilizer kuye kwadityaniswa nokuguqulwa kwi-cell surface expression ye-DRD2 okanye i-intracellular signing ephantsi edityaniswe yi-PKA, ERK kunye ne-GSK3. [1], [4], [6]. Ngaphandle kwezi ndima zibalulekileyo ze-DRD2 kwingqondo, iindlela zokulawula ezineenkcukacha ezinikezela ukungafani kunye nobunzima kwiipropathi ze-DRD2 aziqondwa ngokupheleleyo.

Ukuguqulwa kobungqina bobungqina bubonisa ukuba ukuguqulwa kweenguqu ezininzi emva kokuguqulelwa kubandakanyeka ekulungisweni kakuhle komsebenzi we-DRD2. I-glycosylation ebanzi ye-DRD2 yatyhilwa kwizifundo zokuqala zokulebula ifoto-affinity [7], kunye nokwakhiwa kwebhondi ye-disulfide ngaphakathi kwe-DRD2 nayo ichongiwe njengento ebalulekileyo yokuguqulwa kwe-ligand. [8]. Ngaphezu koko, iindawo ze-phosphorylation ze-DRD2 zaqala zachongwa ngu in vitro i-assay kunye ne-radioisotopes, ukubonelela ngeendlela zeendlela ezahlukeneyo zokulawula ezilawulwa yi-kinases eyahlukeneyo [9]. Enyanisweni, iprotein kinase C (PKC) ilawula i-DRD2-mediated mobilization ye-intracellular calcium kwaye imodareyitha ukusebenzisana kwe-DRD2 kunye neeprotheni ze-cytoskeletal. [10]. I-Phosphorylation yi-GPCR kinase 2 (GRK2) ilawula iipateni ze-agonist-induced rensitization ze-DRD2 [11].

I-Cyclin-dependent kinase 5 (Cdk5) yi-proline-directed serine/threonine kinase enomsebenzi okhethekileyo ngenxa yokubonakaliswa okuthe ngqo kwengqondo kwezinto zayo ezibalulekileyo, p35 kunye nep39 [12]. I-Cdk5 ibandakanyeka kwiinkqubo ezahlukeneyo ze-neuronal ezibandakanya ukufuduka kwe-neuronal kunye nesikhokelo se-axon, kunye ne-Cdk5 kunye ne-p35 yeempuku ezingenanto zibonisa iziphene ekubekweni kwecortical. [13]. Kutshanje, kuye kwaboniswa ukuba i-phosphorylation ye-WAVE1 kunye ne-ephexin nge-Cdk5 ilawula i-dendritic spine morphogenesis. [14]. Ngaphaya koko, i-Cdk5 ikwalawula amanqanaba embonakalo yomphezulu we-NMDA receptor, NR2B, kunye ne-NR2A-mediated NMDA currents. [15], [16]. Kuyaphawuleka ukuba iziqwenga ezininzi zobungqina zibonisa ubudlelwane obusondeleyo phakathi kwe-Cdk5 kunye nenkqubo ye-dopamine. I-Cdk5 phosphorylates tyrosine hydroxylase (TH), ilawula uzinzo lwayo, kwaye ngaloo ndlela igcina i-homeostasis ye-dopaminergic [17]. Kwi-postsynaptic neurons, xa intsalela ye-T75 ye-dopamine kunye ne-cyclic-AMP ilawulwa yi-phosphoprotein-32kD (DARPP-32) i-phosphorylated yi-Cdk5, inokuthintela umsebenzi we-PKA kwaye ngaloo ndlela ichasene ne-dopamine DRD1-mediated PKA signing. [18]. Okubangela umdla kukuba, xa i-cocaine, i-agonist engathanga ngqo ye-dopamine receptors, ilawulwa ngokungaguqukiyo kwiimpuku, i-mRNA kunye namanqanaba eprotheyini ye-Cdk5 yokwanda kwe-spiny neurons. [19]. Ngokudibeneyo, i-Cdk5 ibonakala ibandakanyeka kwi-synaptic adaptation eyenziwe ngamachiza. Kolu phononongo, sibonisa intsebenziswano esebenzayo ye-DRD2 kunye ne-Cdk5 eyandisa ngakumbi indima ye-Cdk5 kumqondiso we-dopamine.

Impahla nenkqubo

IAndibodies

Iiserum ezichasayo zomvundla zaphakanyiswa ngokuchasene neepeptides eziqukethe i-phospho-serine 321 (pS321) ye-loop yesithathu ye-intracellular ye-DRD2 (D2i3). I-Phospho-peptide, i-CNPDpSPAKPEK (PEPTRON), yayisetyenziselwa ukwenza ikholamu ye-peptide-conjugated for affinity purification (20401, PIERCE). I-anti-pS321 ye-antibody yenziwe yatyetyiswa yinkqubo yokucoca i-affinity elandela umyalelo womenzi. I-phospho-antibody ehlambulukileyo igcinwe kwi-PBS kunye ne-0.1% ye-sodium azide kunye ne-0.1% ye-gelatin. I-anti-mouse anti-Cdk5 antibody (sc-249) kunye ne-anti-rabbit i-anti-p35 antibody (sc-820) yayisetyenziselwa ukuchithwa kweNtshona kunye ne-immunocytochemistry ye-Cdk5 / p35. I-anti-mouse i-anti-GFP antibody (sc-9996) yayisetyenziselwa ukugonywa kunye nokuchithwa kweNtshona ye-DRD2-GFP. I-anti-Rabbit anti-FLAG antibody (sc-807), i-anti-Rabbit anti-HA (sc-805), i-anti-mouse ye-GST antibody (sc-138), kunye ne-anti-mouse anti-GAPDH (sc- 32293) zathengwa kwiSanta Cruz Biotechnologies.

izilwanyana

Impuku ye-p35 yokunkqonkqoza yayisisipho sobubele esivela kuGqr. Katsuhiko Mikoshiba kwi-RIKEN Brain Science Institute e-Japan kwaye isetyenziselwa inkcubeko ye-neuron yokuqala. Iiseti zokuqala ze-genotyping zaziyi-5′- GGTCTCCTCTTCTGTCAAGAAG, 5′-GCTCTGCTAGACACATACTGTAC kunye ne-5′- TCCATCT GCACGAGACTAGT njengoko kuchaziwe ngaphambili [20]. Iigundane ze-ICR kunye neempuku ze-Sprague Dawley zazisetyenziselwa ukulungiswa kwe-lysate yengqondo. Zonke iinkqubo zezilwanyana zivunyiwe yiPohang University of Science and Technology Institutional Animal Care and Use Committee.

IPlasmid Yakha

Umntu we-DRD2 ende ye-isoform kwi-EGFP-N1 ye-plasmid vector kunye ne-loop yesithathu ye-intracellular ye-DRD2 (i-212-373 i-amino acid iintsalela ezibandakanya i-29 eyongezelelweyo i-amino acid eyongezelelweyo kwi-DRD2 ende isoform) kwi-pFLAG-CMV-2 ye-plasmid vector yasetyenziswa. I-Cdk5 yomntu yafakwa kwi-pCMV-HA ye-plasmid vector kwaye i-p35 yabantu yafakwa kwi-pcDNA 3.1 ye-plasmid vector. I-Cdk5 yomntu ifakwe phantsi komgqugquzeli we-cytomegalovirus (CMV) kunye ne-p35 yomntu kwi-pcDNA 3.1 vector ukwenza i-dual expression construct (Cdk5 / p35) ye-immunocytochemistry, i-receptor internalization assay, [³⁵S]-GTP_γUvavanyo olubophayo lwe-S, uvavanyo lokubopha i-radioligand kunye ne-cAMP ye-enzyme immunoassay.

I-In vitro Kinase Assay

IP-iqhagamshelwe in vitro i-kinase assay yenziwa ngolu hlobo lulandelayo. Ingqondo enye yempuku yalaliswa kwi-3 mL erythrocytes lysis buffer (ELB) (50 mM Tris (pH 8.0), 250 mM NaCl, 5 mM EDTA, 0.1% NP-40) ngemivumbo engama-20 ye-Dounce homogenizer ukufumana i-lysates yobuchopho ehomogenized . I-lysates ifakwe kwi-ice ye-30 min, i-sonicated, kwaye i-centrifuged kwi-16,000 × g nge-10 min. I-supernatants i-immunoprecipitated kunye ne-anti-rabbit anti-p35 antibody ukufumana i-Cdk5 / p35 esebenzayo. Iprotheni ye-Cdk5/p35 entsonkothileyo kunye necociweyo ye-GST yodibaniso yaxutywa ne-adenosine 5′-triposphate, [γ-³²P] (NEG-502H, PerkinElmer) kwaye ifakwe kwi-kinase buffer (30 mM HEPES (pH 7.2), 10 mM MgCl₂, 0.2 mM DTT) kwi-1 h kwiqondo lokushisa [18], [21]. ICdk5/p25 ecocekileyo (14–516, Millipore) nayo yasetyenziselwa in vitro i-kinase assay njengoko kuchaziwe ngasentla. I-2 × isampuli yokulayisha i-buffer yongezwa kumxube wokusabela kwaye ibilisiwe kwi-100 ° C. Iisampulu zaye zafakwa kwi-SDS-PAGE kwaye ijeli eyomileyo yavavanywa yi-autoradiography.

I-Liquid Chromatography (LC)-Mass Spectrometry (MS)/MS Analysis

Iprotheni ye-GST-D2i3 edibeneyo yahlalutywa yi-LC-MS/MS ilandela i-IP-iqhagamshelwe in vitro i-kinase assay. Senze ukuchongwa kwe-peptide yedatha ye-LC-MS/MS sisebenzisa i-X!!Tandem (uguqulelo lukaDec-01-2008). Ifayile nganye ye-RAW yedatha yaguqulelwa kuqala kwi-mzXML kusetyenziswa umbhobho we-trans-proteomic (TPP; inguqulo 4.3). Izikena ze-MS/MS kwii-mzXML eziguquliweyo zaye zaphantsi kophando ngokuchasene nesiseko sedatha yeprotheyini ye-UniProt (ikhutshwe ngo-2010_07) kubandakanywa ulandelelwano lwe-GST-D2i3 kusetyenziswa i-X!!Tandem. Ukunyamezela kwakubekwe kwi-3 Da ye-ion ye-precursor kunye ne-2 ye-Da ye-ion fragment. Kwasetyenziswa i-enzyme ethile ye-trypsin. Izinketho zokuguqulwa eziguquguqukayo zisetyenziselwa i-carbamidomethylation ye-cysteine ​​(57.021 Da), i-oxidation ye-methionine (15.995 Da), i-hydrolysis ye-asparagine (0.987 Da) kunye ne-phosphorylation ye-serine (79.966 Da).

I-Immunoprecipitation

I-Immunoprecipitation yenziwa kwi-cell lysates kwi-ELB lysis buffer. I-anti-GFP ye-antibody yongezwa kwii-lysates kwaye ifakwe kwi-3 h kwi-4 ° C. Ii-Immunocomplexes zacocwa kusetyenziswa iprotein-A agarose. I-precipitates ifakwe kwi-SDS isampula yokulayisha i-buffer ye-30 min kwi-37 ° C, kwaye ixhomekeke kwi-SDS-PAGE kunye ne-Western blots.

GST Tsala-phantsi Assay

I-10 µg ye-GST ecocekileyo kunye ne-GST-D2i3 zafakwa kwi-lysate yobuchopho bempuku i-1.5 h kwi-4°C. I-30 µL ye-glutathione (GSH)-conjugated amaso e-Sepharose 4B (17-0756-01, GE Healthcare) elungelelaniswe ne-lysis buffer yongezwa kwaye yafakelwa i-1 h eyongezelelweyo. Ubuhlalu baqokelelwa nge-centrifugation kwi-2,000 ×g kwaye ihlanjwe nge-lysis buffer amaxesha ama-4 [22], [23]. Amanzi aye ahlalutywa yi-Western blotting usebenzisa i-anti-Cdk5 kunye ne-anti-p35 antibodies.

Immunocytochemistry

Iiseli ze-HEK ze-293 ezitshintshiweyo kunye ne-neurons ye-striatal ekhuliswe kwii-coverlips zahlanjwa kanye nge-phosphate buffered saline (PBS) kwaye zilungiswe ngokuntywiliselwa kwi-4% paraformaldehyde / PBS ebandayo ye-30 min. I-antibody ephambili yahlanjululwa kwisisombululo sokuthintela (i-2% ye-serum yehashe kunye ne-1% ye-Triton X-100 kwi-PBS). I-Alexafluor-647-conjugated anti-mouse antibody (i-A20990, i-Invitrogen) kunye ne-Alexafluor-568-conjugated anti-rabbit antibody (A11011, i-Invitrogen) isetyenziswe njenge-antibodies yesibini. I-Hoechst yayisetyenziselwa ukwenza i-nucleus staining. Imifanekiso ifunyenwe nge-confocal microscopy (Olympus, FluoView-1000).

Uvavanyo lwangaphakathi lwe-Receptor

I-24 h emva kokudluliselwa, iiseli zaphathwa nge-1 µM quinpirole (Q102, Sigma) kwi-30 min kunye ne-90 min kwi-37 ° C. Iiseli zaphinda zamiswa kwi-2 mL ye-PBS ebandayo kunye ne-200 µL ye-aliquots yasetyenziswa kwi-reaction nganye. Unyango lweziyobisi lwenziwa kwindawo yokushisa kwe-3 h kwezi zinto zilandelayo; 3 nM [³H]-spiperone (NET-565, PerkinElmer), 3 µM sulpiride (895, TOCRIS), 10 µM haloperidol (H1512, Sigma). I-Hydrophobic [³H]-i-spiperone yayisetyenziselwa ukuleyibhela ii-receptors ezibonakalisiweyo zizonke kwaye i-hydrophilic sulpiride yayisetyenziselwa ukubuyisela i-membranous receptor-bound [³H]-imiqondiso ye-spiperone. Izibonakaliso ze-receptor ezihambelana ne-Membrane zibalwe ngokukhupha ixabiso le-intracellular receptor ukusuka kwixabiso elipheleleyo elibonakalisiweyo. Iiseli zahluzwa kwisihluzo se-GF/B (Millipore) kwaye zahlanjwa amaxesha e-3 ngesithinteli sokuhlamba (50 mM Tris-HCl (pH 7.4), 100 mM NaCl). Izihluzo zomiswa kwaye i-radioactivity eseleyo yalinganiswa kusetyenziswa ikhawunta ye-liquid scintillation [24].

ULungiselelo lweMembrane yeSeli

Iiseli ezidibeneyo kwii-100 mm zenkcubeko-izitya emva kokudluliselwa zihlanjwe nge-PBS ebandayo ye-ice kwaye zivunwe kwi-1 mL HME buffer (25 mM HEPES (pH 7.5), 2 mM MgCl₂, 1 mM EDTA). I-lysates ye-Homogenized yayiyi-centrifuged kunye ne-500 × g ye-15 min kunye ne-supernatants emva koko i-centrifuged kunye ne-36,000 × g ye-30 min. Iipellets ziphinde zaxhonywa kwi-HME buffer zisetyenziselwe ukuvavanya.

[³⁵S]-GTP_γS Ukubophelela uvavanyo

Amaqhezu e-membrane yeseli afakwe ngaphambili kunye ne-1 µM quinpirole (Q102, Sigma) kwi-assay buffer (25 mM HEPES (pH 7.5), 1.5 mM MgCl₂, 100 mM NaCl, 1 mM EDTA kunye ne-0.01 mM GDP) kwi-10 min. [³⁵S]-GTP_γI-S (NET-030H, i-PerkinElmer) yongezwa kwi-concentration yokugqibela ye-3 nM kwi-30 µL kwaye iqhutyelwe ngakumbi kwi-90 min. I-170 µL yesithinteli sokubanda komkhenkce (10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM MgCl₂, kunye ne-0.1 mM GTP) yongezwa ukumisa ukuphendula. Iimbumba zahluzwa kwisihluzi se-GF/B (i-Millipore) kwaye zahlanjwa amaxesha e-3 nge-buffer yokuhlamba (50 mM Tris-HCl (pH 7.4), 100 mM NaCl). Izihluzi zomiswa kwaye i-radioactivity yalinganiswa kusetyenziswa ikhawunta ye-scintillation [25], [26].

Radioligand Binding Assay

Iinwebu zeseli ezilungisiweyo zifukanywe nge-0.01 nM [³H] -spiperone (NET-565, PerkinElmer) kunye nokunyuka koxinzelelo lwe-quinpirole (Q102, Sigma) kwi-30 min kwi-assay buffer (25 mM HEPES (pH 7.5), 1.5 mM MgCl₂, 100 mM NaCl, 1 mM EDTA). Iimbumba zahluzwa kwisihluzi se-GF/B (i-Millipore) kwaye zahlanjwa amaxesha e-3 nge-buffer yokuhlamba (50 mM Tris-HCl (pH 7.4), 100 mM NaCl). Ukusabela kuye kwapheliswa kukuhluzwa okukhawulezayo ngokusebenzisa izihluzi ze-GF/C. Intsalela ye-radioactivity yalinganiswa kusetyenziswa ikhawunta ye-liquid scintillation [27]-[29].

I-CAMP Enzyme Immunoassay

Iiseli ze-HEK ze-293 ezitshintshiweyo zenziwa kwangaphambili nge-10 µM rolipram (R6520, Sigma) kwi-1 h, kwaye emva koko iphathwa nge-0.1 µM ye-forskolin (F6886, Sigma) kunye nokunyuka kwe-quinpirole (Q102, Sigma) kwi-30 min. I-neuron ephuculweyo ye-striatal esisiseko yaphathwa nge-10 µM rolipram ngeyure enye, emva koko i-1 µM i-dopamine ye-1 h. [22]. Iilysates zeseli zalungiswa nge-0.1 M HCl kunye namanqanaba e-cAMP afunyenwe ngekhithi ye-cAMP enzyme immunoassay kit (Sapphire Bioscience) kulandela umyalelo womenzi.

Primary Cultured Striatal Neuron

Indawo ye-striatal yahlukaniswa kwi-mouse embryonic brain (E15). I-tissue edibeneyo yahlulwe kwimidiya encinci ebalulekileyo (MEM) (11095, i-Invitrogen) equkethe i-0.25% ye-trypsin (T4549-100, i-Sigma) kunye ne-0.1% ye-DNase I kwi-6 min kwi-37 ° C. Iiseli zaphinda zamiswa kwi-plating media (MEM nge-0.01 M HEPES (pH 7.4) kunye ne-10% (vol/vol) ye-horse serum (16050-122, GIBCO)). IiNeurons zakhuliswa iintsuku ezisi-7 in vitro (DIV 7) kwi-MEM kunye ne-B27 supplement (17504-044, i-Invitrogen) ngaphambi kokuba isetyenziswe kwi-cAMP enzyme immunoassays.

iziphumo

I-Cdk5 Phosphorylates iSerine 321 kwi-Third Intracellular Loop ye-DRD2 in vitro

Ukuchonga i-Cdk5 substrates zenoveli, senze uphando olucwangcisiweyo sisebenzisa (S/T)PX(K/H/R) njengolandelelwano lwemvumelwano yokuqondwa kweCdk5. [30] kwaye ichonge i-DRD2 njengenxalenye yomgqatswa. Ukulandelelana kwemvumelwano, kuquka i-serine 321, ifumaneka kwi-loop yesithathu ye-intracellular ye-DRD2 (D2i3) apho iindlela ezahlukeneyo zokulawula ziye zabandakanyeka. [3], [10], [11]. Ulandelelwano lugcinwa ngokuguquguqukayo kwi-DRD2 kwii-vertebrates, oku kuthetha ukubaluleka kokusebenza kwentsalela (Umzobo 1A).

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Umzobo 1. I-Cdk5 phosphorylates serine 321 kwi-loop yesithathu ye-intracellular ye-DRD2 i-In vitro.

(A) Ulungelelwaniso lolandelelwano lwe-amino acid ebonisa imimandla egciniweyo ye-DRD2 evela kwiindidi ezahlukeneyo (ezinomthunzi). Indawo enokubakho yeCdk5 phosphorylation iboniswa inkwenkwezi. (B) I-IP-iqhagamshelwe in vitro i-kinase assay ene-recombinant GST-D2i3 kunye ne-GST-D2i3 iiprotheyini eziguqukayo. I-Cdk5/p35 entsonkothileyo etyetyisiweyo kwisicatshulwa sobuchopho bempuku nge-anti-p35 immunoprecipitation yayisetyenziselwa ukuphendula kwe-kinase. I-autodiograph yeeproteni ze-phosphorylated ibonisiwe kunye ne-Coomassie ekhazimlayo eluhlaza okwesibhakabhaka enemibala yejeli efanayo. I-arrowhead ibonisa isignali ye-radioactive ehambelana ne-GST-D2i3s kunye nentloko yotolo evulekileyo ibonisa iimpawu ze-radioactive ezivela kwi-p35. (C) I-spectrum ye-MS / MS yeqhekeza le-peptide ye-phosphorylated ye-D2i3. Iipateni zokuqhekeka kwethiyori ziboniswe ngezantsi kwe-spectrum. Phakathi kwazo zonke ii-ion eziqhekezayo, i-y- kunye ne-b-ion efunyenweyo ibonakaliswe kwi-spectrum. Iy₆ y₇ iioni zibonisa ngamandla iphosphorylation ye serine 321. (D) I-In vitro i-kinase assay kunye ne-Cdk5 / GST-p25 ecocekileyo ecocekileyo esebenzisa i-GST-D2i3 kunye ne-GST-D2i3 iiprotheni eziguqukayo. Iiprotheyini ze-phosphorylated zaboniswa kwi-autoradiograph, kunye ne-Coomassie eqaqambileyo yebala eliluhlaza. I-arrowhead ibonisa isignali ye-radioactive ehambelana ne-GST-D2i3 kunye notolo oluvulekileyo lubonisa iimpawu ze-radioactive ezivela kwi-GST-p25.

I-doi: 10.1371 / journal.pone.0084482.g001

Ukuvavanya umthamo we-Cdk5 ukuya kwi-phosphorylate D2i3, senze i-IP-linked in vitro i-kinase assays usebenzisa i-Cdk5 / p35 esebenzayo ephuculweyo esuka kwi-mouse brain lysate nge-p35 immunoprecipitation kunye ne-recombinant ecocekileyo ye-GST-D2i3 (iintsalela ze-amino acid 212-373) iiprotheni njenge-substrates. Siye sabona iimpawu ze-phosphorylation kwiiprotheni ezihlambulukileyo ze-GST-D2i3 kunye ne-GST-D2i3 S297A, kodwa umqondiso wancipha kakhulu usebenzisa i-GST-D2i3 S321A.Umzobo 1B). Ukuqinisekisa ngakumbi i-phosphorylation ye-serine 321 kwi-GST-D2i3, senze uhlalutyo lwe-LC-MS/MS lweesampulu ezivela kwi-IP-exhunyiwe. in vitro i-kinase assays usebenzisa i-LTQ XL mass spectrometry. Ngokuqhubekayo, i-phospho-peptides ehambelana nobunzima be-phospho-serine 321 peptides yafunyanwa (Umzobo 1C). Ukuqwalasela ukuba ukufunyanwa kwedatha exhomekeke kwidatha ngexesha lokuhlalutya kwe-LC-MS / MS kudla ukufumanisa iiprotheni ezininzi kwisampulu. [31], le datha ibonisa ukuba i-serine 321 i-residu yindawo evelele ye-phosphorylation ye-Cdk5 kummandla we-D2i3. Ukungqina i-phosphorylation ngqo ye-serine 321 kwi-GST-D2i3 nge-Cdk5, in vitro i-kinase assay usebenzisa i-Cdk5 / GST-p25 ecocekileyo kunye ne-recombinant ecocekileyo ye-GST-D2i3 iiprotheni zenziwa. Sichonge isibonakaliso esibalulekileyo se-phosphorylation kwi-GST-D2i3 eyayingekho kwi-GST-D2i3 S321A (Umzobo 1D). Xa zidibene, ezi ziphumo zibonisa ukuba intsalela ye-D2i3 S321 yithagethi ekhethiweyo ye-Cdk5-mediated phosphorylation.

I-Cdk5 Phosphorylates iSerine 321 kwi-Third Intracellular Loop ye-DRD2 kwiiSeli

Ukuchonga i-phosphorylation ye-serine 321, siphakamise i-antibody ethile kwi-phospho-serine 321 (pS321). Iisampulu ezivela kwi-IP edityanisiweyo in vitro i-kinase assay yahlalutywa yi-Western blotting usebenzisa i-anti-pS321 antibody. IiBlots zibonise ibhanti eyahlukileyo kwi-kinase reaction eyayixhomekeke kwi-GST-D2i3 (Umzobo 2A). Ukuqinisekisa i-phosphorylation enokwenzeka ye-serine 321 kwi-DRD2 nge-Cdk5 kwiiseli, i-anti-GFP immunoprecipitates esuka kwi-HEK 293 iiseli ezibonisa i-DRD2-GFP kunye ne-DRD2 S321A-GFP kunye okanye ngaphandle kwe-HA-Cdk5 kunye ne-p35 zahlalutywa yi-Western blotting usebenzisa i-GFP kunye ne-GFP. anti-pS321 amajoni omzimba. Iimpawu zebhendi eziqatyiweyo ze-anti-GFP ezaziwa ngokuba ngenxa ye-glycosylation egqithisileyo ye-DRD2 zibonwa kuphela xa kukho i-DRD2-GFP, kunye ne-anti-pS321 i-antibody ifumene iimpawu ezifanayo ze-DRD2 kuphela nge-Cdk5 / p35 i-co expression.Umzobo 2B) [7]. Ukuqinisekisa ngakumbi i-phosphorylation ye-serine 321 nge-Cdk5, i-D2i3 (FLAG-D2i3) kunye nefom ye-mutant ye-D2i3 (FLAG-D2i3 S321A) yenziwe. I-FLAG-D2i3 kunye ne-FLAG-D2i3 S321A echazwe kunye okanye ngaphandle kwe-HA-Cdk5 kunye ne-p35 kwiiseli ze-HEK ze-293 zahlaziywa nge-SDS-gel mobility shift assay. Utshintsho olubalulekileyo lwe-Cdk5 oluxhomekeke kwi-Flag-D2i3, kodwa hayi kwi-FLAG-D2i3 S321A (Umzobo 2C). Siphinde savavanya inqanaba le-phosphorylation ye-DRD2 kwi-Ser321 phezu kokuvuselela i-agonist. Iiseli ze-HEK ze-293 ezibonisa i-DRD2-GFP kunye ne-Cdk5 / p35 eziyinkimbinkimbi zaye zakhuthazwa yi-quinpirole, kunye ne-anti-GFP immunoprecipitates ukusuka kwi-cell lysates yahlalutywa yi-Western blotting usebenzisa i-anti-GFP kunye ne-anti-pS321 antibodies. Sifumene ukuba i-Cdk5-mediated phosphorylation ye-DRD2 kwi-Ser321 ayizange ichaphazeleke yi-agonist stimulation, ebonakala ihluke kwi-GRK- kunye ne-PKC-mediated phosphorylations.Umzobo 2D) [32], [33]. Ngokudibeneyo, ezi ziphumo zibonisa ukuba i-Cdk5 inokwenza i-phosphorylate i-serine 321 intsalela ye-DRD2 kwindawo yeselula.

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Umzobo 2. I-Cdk5 phosphorylates serine 321 kwi-loop yesithathu ye-intracellular ye-DRD2 kwiiseli.

I-Cdk5-mediated phosphorylation ye-serine 321 yahlalutywa kusetyenziswa i-anti-pS321 antibody. (A) Iisampulu ezivela kwi-IP-edibeneyo in vitro i-kinase assay isebenzisa iiprotheni ze-GST-D2i3 zahlalutywa yi-Western blotting (WB) kunye ne-antibodies ebonakalisiweyo. Iintloko zotolo zibonisa i-GST-D2i3s. (B) I-DRD2-GFP kunye ne-DRD2 S321A-GFP ibonakaliswe okanye ngaphandle kwe-HA-Cdk5 kunye ne-p35 kwiiseli ze-HEK 293. I-Anti-GFP immunoprecipitates yahlalutywa yi-Western blotting usebenzisa i-anti-GFP kunye ne-anti-pS321 antibodies. I-bracket ibonisa iimpawu ze-DRD2 kunye nentloko yetolo evulekileyo ibonisa iimpawu ezingabonakaliyo ezivela kwi-anti-GFP immunoprecipitates. '% igalelo' yi-% umthamo we-lysate iyonke ye-IP reaction. Iimpawu ze-Cdk5 ezibuthathaka zibonakaliswe ngeenkwenkwezi. (C) Uvavanyo lokutshintsha kwe-gel yokuhambahamba. Iiseli ze-HEK ze-293 ezidluliselwe njengoko kubonisiwe zahlalutywa ngokucinywa kweNtshona. (D) Iiseli ze-HEK ze-293 ezitshintshiweyo zaphathwa nge-quinpirole kunye ne-anti-GFP immunoprecipitates zahlalutywa yi-Western blotting kunye ne-anti-GFP kunye ne-anti-pS321 antibodies. Intloko yetolo evulekileyo ibonisa imiqondiso engachazwanga evela kwi-anti-GFP immunoprecipitates.

I-doi: 10.1371 / journal.pone.0084482.g002

I-Cdk5/p35 Complex kunye ne-DRD2 ziNxulumene noMzimba

Siphande intsebenziswano enokubakho phakathi kwe-Cdk5/p35 kunye ne-DRD2 kuba uninzi lwee-substrates ze-Cdk5 zaziwa ngokunxulunyaniswa ngokwasemzimbeni ne-Cdk5/p35. [23], [34], [35]. Okokuqala, uvavanyo lokutsalwa phantsi kwe-GST lwenziwa. Iprotein ecocekileyo ye-GST-D2i3 yafakwa kwi-lysate yobuchopho bempuku kunye ne-GST yokutsalwa phantsi kwemvula yahlalutyelwe ukubhula kweNtshona. Njengoko kubonisiwe kwi Umzobo 3A, i-Cdk5 engapheliyo kunye ne-p35 ichongiwe kwi-precipitates yokudonsa, ebonisa ukusebenzisana komzimba phakathi kwe-DRD2 kunye ne-Cdk5 / p35 eyinkimbinkimbi (Umzobo 3A). Ngaphezu koko, i-HA-Cdk5 kunye ne-p35 zifunyenwe kwi-anti-GFP immunoprecipitates ukusuka kwi-HEK 293 cell lysates echaza i-DRD2-GFP kunye ne-Cdk5 / p35 (Umzobo 3B). Ukongezelela, senze uhlalutyo lwe-immunocytochemical kwaye sabona ukuba i-DRD2-GFP, i-HA-Cdk5 kunye ne-p35 ibonisa izibonakaliso ezibalulekileyo ze-co-localization kwindawo ye-membranous yeeseli ze-HEK 293.Umzobo 3C, iipaneli eziphezulu). Siphinde siphande i-co-localization ye-DRD2 kunye ne-Cdk5 / p35 kwimeko ye-neuronal. Ngokungaguqukiyo, i-DRD2-GFP ikwabonise ukubambisana okubalulekileyo kwendawo kunye ne-Cdk5 engapheliyo kunye ne-p35 kwi-neuron ekhulisiweyo ye-striatal (DIV7), ukuxhasa ngakumbi amakhonkco asebenzayo phakathi kwe-DRD2 kunye ne-Cdk5 / p35 (Umzobo 3C, iipaneli ezisezantsi). Iziphumo zibonisa ukuba i-DRD2 kunye ne-Cdk5 / p35 inokwenza i-complex kwaye ngoko, ixhase ingcamango yokuba i-DRD2 i-substrate ye-physiological ye-Cdk5.

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Umzobo 3. I-Cdk5/p35 inokwenza i-complex kunye ne-DRD2.

(A) Uvavanyo lwe-GST lokutsalwa phantsi kusetyenziswa iprotheyini ecocekileyo ye-GST-D2i3 enesicatshulwa sobuchopho begundane. Ukutsalwa kwemvula kwe-GST kuphantsi kohlalutyo lokucinywa kwaseNtshona. 'I-Bead' ibonisa ukutsalwa kwemvula ngaphandle kweeproteni ze-GST. (B) I-Immunoprecipitation ye-DRD2 kunye ne-Cdk5 / p35 eyinkimbinkimbi. I-Anti-GFP IP evela kwii-lysates ezivela kwiiseli ezitshintshileyo ziphantsi kohlalutyo lwe-Western blotting. I-bracket ibonisa iimpawu ze-DRD2 kunye nentloko yetolo evulekileyo ibonisa iimpawu ezingabonakaliyo ezivela kwi-anti-GFP immunoprecipitates. Ibhlothi evezwe ngokugqithisileyo yamagalelo ikwabonisiwe ngasekunene. (C) Uhlalutyo lwe-Immunocytochemical ye-DRD2 kunye ne-Cdk5 / p35. Iiseli ze-HEK ze-293 ezibonisa i-DRD2-GFP kunye ne-Cdk5 / p35 zihlanjululwe nge-anti-Cdk5 kunye ne-anti-p35 antibodies (iipaneli eziphezulu). I-DRD2-GFP ibonakaliswe yodwa kwi-neurons ye-striatal ekhulisiwe kwaye ifakwe i-anti-Cdk5 kunye ne-anti-p35 antibodies (iiphaneli eziphantsi). I-Hoechst yayisetyenziselwa ukwenza i-nucleus staining. Ibar yesikali yi-5 µm. Yonke imifanekiso ifunyenwe ngokusebenzisa i-microscopy ye-confocal (i-Olympus, i-FluoView-1000).

I-doi: 10.1371 / journal.pone.0084482.g003

I-Cdk5-mediated Phosphorylation ye-DRD2 Ithomalalisa uMsebenzi we-Receptor

Kuye kwaxelwa ukuba i-phosphorylation imodareyitha iipropati ezibalulekileyo ze-GPCR ezifana ne-G protein coupling, i-receptor internalization, i-intracellular localization, kunye nokudibanisa neeprotheni ze-modulator. [9], [11], [24]. AI-gonist-induced receptor internalization yinkqubo ebalulekileyo yokulawula ukuhanjiswa komqondiso. Siphande ukumodareyithwa kwe-Cdk5-mediated ye-DRD2 yangaphakathi. Iiseli ze-HEK ze-293 ezibonisa i-DRD2-GFP kunye ne-DRD2 S321A-GFP kunye okanye ngaphandle kwe-Cdk5 / p35 zifakwe kwi-1 µM quinpirole ukuze zenze i-agonist-ivuselelwe i-DRD2 ngaphakathi (Umzobo 4A). [³H] -izibonakaliso ze-spiperone ze-DRD2-GFP ezibonisa iiseli zancitshiswa kakhulu kwi-30 min yonyango ye-quinpirole kwaye yafunyanwa kwi-90 min. Okubangela umdla kukuba, [³I-H] -izibonakaliso ze-spiperone ze-DRD2-GFP kunye ne-Cdk5 / p35 ezibonisa iiseli nazo zancitshiswa kwi-30 min yonyango lwe-quinpirole kodwa ayizange ibuyiselwe kwi-90 min.Umzobo 4A, icandelo lesibini). Kwelinye icala, [³H] -izibonakaliso ze-spiperone ze-DRD2 S321A-GFP ezivakalisa iiseli zancitshiswa kwi-30 min kwaye zafunyanwa kwi-90 min, kungakhathaliseki ukuba i-co-expression kunye ne-Cdk5 / p35. Uphononongo lwangaphambili lubonise ukuba i-DRD2 yangaphakathi iphinda ibuyele kwi-plasma membrane ekuvuselelweni kwexesha elide kwe-agonist. [11]. Thus kubonakala ukuba i-phosphorylation ye-Cdk5-mediated ye-DRD2 ibandakanyeka kwiinkqubo zokuvuselela emva kwe-agonist-induced DRD2 internalization.

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Umzobo 4. I-phosphorylation ye-Cdk5-mediated ibeka imbonakalo ye-DRD2 ebusweni kunye nomqondiso osezantsi.

(A) imbonakalo yomphezulu DRD2 ilinganiswa nge [³H]-i-spiperone ebophelelayo uvavanyo. Iiseli ze-HEK293 ezitshintshiweyo zavuselelwa nge-1 µM quinpirole kwixesha elibonisiweyo kunye nokuvuna, kulandelwa yi-3 nM [³H]-unyango lwe-spiperone lwe-3 h. I-Radioactivity yalinganiswa kwaye imiqondiso yomphezulu yabalwa. Imivalo yemposiso imele intsingiselo ± SE (n = 8; *p<0.05, **p<0.01; Indlela enye ANOVA kunye novavanyo lwe-Dunnett post hoc: thelekisa zonke iikholamu vs. ikholamu yolawulo). (B) [³⁵S]-GTP_γUvavanyo olubophelelayo. Iinwebu zeseli zalungiswa ukusuka kwiiseli ezosulelwe njengoko kubonisiwe. Amalungiselelo eMembrane afukanywa nge-1 µM quinpirole elandelwa yi-3 nM [³⁵S]-GTP_γS kwi-90 min. Imivalo yemposiso imele intsingiselo ± SE (n = 8; *p<0.05, **p<0.01, ***p<0.001; Indlela enye ANOVA kunye novavanyo lwe-Bonferroni post hoc: thelekisa zonke izibini zekholamu). (C) I-Quinpirole-ekhuphisanayo [³H]-i-spiperone ebophelelayo uvavanyo. Amalungiselelo e-Membrane avela kwiiseli ezosulelekileyo afakwe kwi-0.01 nM [³H] -i-spiperone kunye nokunyuka koxinzelelo lwe-quinpirole kwi-30 min. Ukuhlehla okungahambelaniyo kufunyenwe yiGraphPad. Imivalo yemposiso ibonisa intsingiselo ± SE (n = 3). (D) i-cAMP i-enzyme immunoassays kwiiseli ze-HEK ze-293 ezitshintshiweyo. Iiseli ezitshintshiweyo zenziwa kwangaphambili nge-10 µM rolipram ye-1 h, kwaye emva koko ziphathwa ngokubambisana ne-0.1 µM ye-forskolin kunye nokunyuka kwe-quinpirole kwi-30 min. Ukuhlehla okungahambelaniyo kufunyenwe yiGraphPad. Imivalo yemposiso imele intsingiselo ± SE (n = 4; **p<0.01; enemisila emibini t- iimvavanyo). (E) I-neurons ye-striatal ekhuliswe kuhlobo lwasendle kunye ne-p35 knockout embryos (DIV 7) ziphathwe nge-10 µM rolipram ye-1 h ilandelwa yi-1 µM dopamine ye-1 h. Iibar zemposiso zibonisa ± SE (n = 4; **p<0.01; ezinemisila emibini t- iimvavanyo).

I-doi: 10.1371 / journal.pone.0084482.g004

Siphinde savavanya utshintsho olunokwenzeka lwe-agonist-evuselelwe iprotheyini ye-G kwi-DRD2 ehambelana ne-Cdk5-mediated phosphorylation isebenzisa [³⁵S]-GTP_γUvavanyo olubophelelayo [25], [26]. I-DRD2-GFP kunye ne-DRD2 S321A-GFP kunye okanye ngaphandle kweCdk5 / p35 zichazwe kwiiseli ze-HEK 293. Iimembranes zalungiswa kwaye zavuselelwa nge-1 µM quinpirole kunye nokuvunyelwa ngakumbi [³⁵S]-GTP_γS ukubandakanywa. I-DRD2-GFP kunye ne-Cdk5 / p35 echaza i-membrane yeseli ibonise ukukhubazeka kakhulu [³⁵S]-GTP_γUkubophelela kuka-S xa kuthelekiswa nazo zonke ezinye iimbumba zeeseli (Umzobo 4B). Ezi ziphumo zibonisa ukuba i-Cdk5-mediated phosphorylation down-regulates agonist-stimulated G protein binding at DRD2.

Ukongeza, quinpirole-ekhuphisanayo [³H] -i-spiperone i-assays yokubopha i-spiperone yenziwa ukuphanda naluphi na utshintsho olunokwenzeka kwi-agonist-affinity kwi-DRD2 nge-Cdk5-mediated phosphorylation. Ukubophelela kukhuphiswano lwe [³H] -spiperone phezu kwonyango lokunyuka kwe-quinpirole ukuya kulungiselelo lwe-membrane ukusuka kwi-transfected yalinganiswa. Ukukhuphisana ngokubophelela kwequinpirole kunye [³H]-spiperone e-DRD2-GFP kunye ne-DRD2 S321A-GFP yenza i-logK efanayo_i ixabiso (-9.789 ye-DRD2-GFP; -9.691 ye-DRD2 S321A-GFP), ebonisa ukuba ukudibanisa kwe-ligand kwi-DRD2 ayichatshazelwa kakhulu yi-Cdk5-mediated phosphorylation kwi-DRD2 (Umzobo 4C).

I-Cdk5-mediated Phosphorylation Phantsi-ilawula i-DRD2-cAMP yeNdlela yoMqondiso

Emva koko, siphande ukuba ukuguqulwa kwe-DRD2 nge-Cdk5 kuchaphazela iindlela zokubonisa ezisezantsi. Sibeke iliso kwi-DRD2-mediated inhibition ye-forskolin-ivuselelwe ukuveliswa kwe-cAMP nge-adenylyl cyclase kwiiseli ezibonisa i-DRD2-GFP kunye ne-DRD2 S321A-GFP usebenzisa i-cAMP enzyme immunoassay. I-DRD2-ebonisa iiseli ezibonisa ukunciphisa amanqanaba e-cAMP ekuphenduleni i-quinpirole ngendlela exhomekeke kwidosi. Okumangalisayo kukuba, i-co-expression ye-Cdk5/p35 yanciphisa kakhulu inhibition enkulu yokwakheka kwe-cAMP (Isazobe 4D, indawo yolawulo yasekhohlo). Ngakolunye uhlangothi, kwi-DRD2 S321A-GFP evakalisa iiseli, iifom ze-cAMP zaye zavinjelwa ngokufanelekileyo ekuphenduleni unyango lwe-quinpirole kungakhathaliseki ukuba kubonakaliswe kwi-Cdk5 / p35.Umzobo 4D, iphaneli yasekunene). Ezi ziphumo zibonisa ukuba i-Cdk5-mediated phosphorylation ye-DRD2 inciphisa umsebenzi wokuthintela we-DRD2 kwindlela yokubonisa i-cAMP esezantsi. Ukuqinisekisa ngakumbi isenzeko kwisimo esifanelekileyo ngokwasemzimbeni, siye sasebenzisa ii-neuron ezikhuliswe ngokusisiseko ukusuka kwiimbumba ezinqongopheleyo ezinqongopheleyo kwi-p35, i-activator ye-Cdk5 ebalulekileyo. I-neuron ye-striatal ephuculweyo esisiseko yaphathwa nge-1 µM dopamine kwaye yahlalutywa yi-cAMP enzyme immunoassay. I-Neurons ezisuka kwi-p35 yokunkqonkqoza iimpuku zibonise amanqanaba ancitshisiweyo e-cAMP xa kuthelekiswa nohlobo lwasendle lwe-neurons xa luvuselelwe nge-dopamine (Umzobo 4E). Taken kunye, sagqiba ekubeni i-phosphorylation ye-Cdk5-mediated ye-DRD2 iphumela ekunciphiseni kwethoni yokuthintela kwindlela ye-cAMP eyenziwa yi-DRD2.

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Sichonge i-DRD2 njengenxalenye yenoveli ye-Cdk5. I-phosphorylation ibonakala iphantsi-ilawula ukubonakaliswa kwe-DRD2 ebusweni ngokuchaphazela i-DRD2 emva kokungena ngaphakathi kwe-receptor ngaloo ndlela inciphisa i-DRD2 G._i-ukudibanisa kunye nendlela esezantsi ye-cAMP. Njengoko intsalela ye-phosphorylation ye-S321 ikhona kwi-DRD2 ende kunye ne-isoforms emfutshane, indlela ecetywayo kolu phononongo inokuba yindlela yokulawula ngokubanzi kwi-DRD2-mediated signing..

I-DRD2 kwi-spiny neurons ephakathi ayithathwanga kuphela njengeyona nto iphambili ye-dopamine receptor subtype kodwa iye yaqatshelwa ngokuchaphazeleka kwayo kutshintsho olukhoyo ekuphenduleni uvuselelo lokusingqongileyo. I-Agonist-induced desensitization kunye ne-rensitization ye-DRD2 ifundwe ngokubanzi [11], [24]. Ngokukodwa, inani lophononongo lubonise ukuba iziphumo zokuvezwa okungapheliyo kwengqondo, okufana ne-cocaine kunye ne-amphetamine, ephakamisa inqanaba le-dopamine engaphezulu kwi-striatal synapse, ihamba notshintsho oluguquguqukayo lwe-DRD2 postsynaptically. [36]. Ewe, abasebenzisi be-cocaine abangapheliyo bayaziwa ukuba bawanciphise amanqanaba e-DRD2 kwindawo yokubethelwa, kwaye ukufumaneka kwe-DRD2 kwi-nucleus accumbens (NAcc) ibonisa unxibelelwano olubi kunye nokufuna kwechiza kunye nokuziphatha okomeleza kwiimpuku kunye neeprimates. [37]-[39]. Ezi ziphumo zibonisa ukuba ukusebenza kwe-DRD2 kuchaphazeleka kakhulu kulawulo oluguquguqukayo okanye lwembuyekezo ekuphenduleni kwizivuseleli ezahlukahlukeneyo ezibandakanya ukuba sesichengeni seziyobisi ezingapheliyo. Iziphumo zethu zibonisa ukuba i-S321 i-residu kwi-loop yesithathu ye-intracellular ye-DRD2 ingaba yi-phosphorylated yi-Cdk5, ephumela ekunciphiseni kwimpembelelo yokuvimbela i-DRD2 kwindlela ye-cAMP. Olu nxibelelwano luphakamisa indlela yokulawula inoveli ehambelana ne-Cdk5 kwi-dopaminoceptive neurons enokuthi inxulunyaniswe nobume obuguquguqukayo bokufumaneka komphezulu we-DRD2.

Kufuneka kuqatshelwe ukuba i-Cdk5 yaziwa njengeyona nto iphambili ekulamleni utshintsho oluguquguqukayo lwemekobume ye-neuronal. Ngokomzekelo, ukuguqulwa kwesakhiwo kunye nokusebenza kwee-dendritic spines kwi-neurons yesekethe ye-limbic yenye yemiphumo yokuvezwa ngokuphindaphindiweyo kwengqondo. [40]. Olu tshintsho lukhatshwa lutshintsho olwahlukeneyo lwemolekyuli kubandakanya ukufakwa kweprotein ebophayo ye-cAMP (CREB) kunye ne-ΔFosB, izinto ezikhutshelweyo ezibonisa ummiselo ongapheliyo ekuphenduleni ulawulo lwe-cocaine olungapheliyo. [41], [42]. Okubalulekileyo, i-Cdk5 yithagethi ye-ΔFosB [19], kunye nezinto ezininzi ezibalulekileyo ezibandakanyekayo kwi-dendritic spine dynamics, njenge-PSD-95, i-p21-activated kinase (PAK), i-β-catenin, kunye ne-spinophilin, zichazwe njenge-Cdk5 substrates. [43]-[46]. Ngokungaguquguqukiyo, ukuguqulwa kofuzo okanye kwezekhemesti yomsebenzi we-Cdk5 kubangela utshintsho lwe-dendritic spine morphology kunye neempendulo zokuziphatha kwi-cocaine, okuthetha iindima ezibalulekileyo ze-Cdk5 kutshintsho lwemolekyuli kunye ne-morphological ye-mesolimbic dopamine circuits. [47], [48]. Iziphumo zethu zibonisa ukuba i-DRD2 yinoveli ekujoliswe kuyo ye-Cdk5 ibonelela ngolwazi olongezelelekileyo kutshintsho oluguquguqukayo lwenkqubo ye-dopamine ekuphenduleni ukuvezwa kweziyobisi ezingapheliyo ngenxa ye-ΔFosB-mediated up-regulation ye-Cdk5 inokubangela ukonyuka kwetonic kwiphosphorylation yeDRD2. . Ngaphezu koko, i-DRD2 yaziwa ngokuchaphazela iinkqubo ezininzi zeselula, kubandakanya ukulawulwa kwe-cAMP kunye neendlela ze-MAP kinase kunye neziganeko ezisezantsi zokukhuphela. [42], [49]. Ke ngoko, okufunyenweyo kolu phononongo akunakubonisa kuphela umgaqo othe ngqo we-DRD2 nge-Cdk5 kodwa ikwabonelela ngombono wenoveli kwiimpendulo eziguqukayo zenkqubo ye-dopamine ekuvezweni kweziyobisi ezingapheliyo.

Umbhali Wemivuzo

Kuyilwe kwaye kuyilwa imifuniselo: JJ YUP DH SKP. Yenza imifuniselo: JJ YUP DKK YK. Ihlalutywe idatha: JJ YUP DKK SL YK SAL HL YSG DH SKP. Ama-reagents anikezelweyo / izixhobo / izixhobo zokuhlalutya: YHS. Ubhale iphepha: JJ SKP.