Temporal Quantitative Proteomics of mGluR-induced Protein Translation and Phosphorylation in Neurons

Abstract

At neuronal synapses, activation of group I metabotropic glutamate receptors (mGluR1/5) triggers a form of long-term depression (mGluR-LTD) that relies on new protein synthesis and the internalization of AMPA-type glutamate receptors. Dysregulation of these processes has been implicated in the development of mental disorders such as autism spectrum disorders and therefore merit a better understanding on a molecular level. Here, to study mGluR-induced signaling pathways, we integrated quantitative phosphoproteomics with the analyses of newly synthesized proteins via bio-orthogonal amino acids (azidohomoalanine) in a pulsed labeling strategy in cultured hippocampal neurons stimulated with DHPG, a specific agonist for group I mGluRs. We identified several kinases with important roles in DHPG-induced mGluR activation, which we confirmed using small molecule kinase inhibitors. Furthermore, changes in the AMPA receptor endocytosis pathway in both protein synthesis and protein phosphorylation were identified, whereby Intersectin-1 was validated as a novel player in this pathway. This study revealed several new insights into the molecular pathways downstream of group I mGluR activation in hippocampal neurons, and provides a rich resource for further analyses. At neuronal synapses, activation of group I metabotropic glutamate receptors (mGluR1/5) triggers a form of long-term depression (mGluR-LTD) that relies on new protein synthesis and the internalization of AMPA-type glutamate receptors. Dysregulation of these processes has been implicated in the development of mental disorders such as autism spectrum disorders and therefore merit a better understanding on a molecular level. Here, to study mGluR-induced signaling pathways, we integrated quantitative phosphoproteomics with the analyses of newly synthesized proteins via bio-orthogonal amino acids (azidohomoalanine) in a pulsed labeling strategy in cultured hippocampal neurons stimulated with DHPG, a specific agonist for group I mGluRs. We identified several kinases with important roles in DHPG-induced mGluR activation, which we confirmed using small molecule kinase inhibitors. Furthermore, changes in the AMPA receptor endocytosis pathway in both protein synthesis and protein phosphorylation were identified, whereby Intersectin-1 was validated as a novel player in this pathway. This study revealed several new insights into the molecular pathways downstream of group I mGluR activation in hippocampal neurons, and provides a rich resource for further analyses. Activation of metabotropic glutamate receptors (mGluRs) initiates a broad array of signaling pathways that collectively modulate the efficiency of neuronal communication. mGluR-dependent signaling has been linked to cognitive functions such as attention, learning and memory, and disrupted mGluR signaling has been implicated in neurological disorders such as Fragile X Syndrome, mental retardation, schizophrenia, addiction and autism spectrum disorders (1Bhakar A.L. Dolen G. Bear M.F. The pathophysiology of fragile X (and what it teaches us about synapses).Annu. Rev. Neurosci. 2012; 35: 417-443Crossref PubMed Scopus (272) Google Scholar, 2Nicoletti F. Bockaert J. Collingridge G.L. Conn P.J. Ferraguti F. Schoepp D.D. Wroblewski J.T. Pin J.P. Metabotropic glutamate receptors: from the workbench to the bedside.Neuropharmacology. 2011; 60: 1017-1041Crossref PubMed Scopus (452) Google Scholar, 3Niswender C.M. Conn P.J. Metabotropic glutamate receptors: physiology, pharmacology, and disease.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 295-322Crossref PubMed Scopus (1159) Google Scholar). Group I mGluRs (mGluR1 and mGluR5), generally localized at the postsynaptic membrane, significantly contribute to synaptic function by modulating synaptic excitability, and inducing of synaptic Collingridge G.L. of in the synaptic activation of glutamate metabotropic PubMed Scopus Google Scholar, of synaptic depression by metabotropic glutamate PubMed Scopus Google Scholar, Collingridge G.L. molecular by metabotropic glutamate receptors of long-term PubMed Scopus Google Scholar, group I mGluR activation by 2010; PubMed Scopus Google Scholar, of the metabotropic glutamate receptor in long-term depression in 2011; PubMed Scopus Google Scholar). the form of by group I mGluRs the long-term depression of synaptic to as Group mGluR-dependent synaptic long-term and for and 2010; PubMed Scopus Google Scholar). to of the of relies on the and synthesis of new proteins in Bear M.F. for protein synthesis in hippocampal mGluR-dependent long-term PubMed Scopus Google of protein synthesis Collingridge G.L. of protein kinase and protein metabotropic glutamate long-term PubMed Scopus Google Scholar, in synaptic of hippocampal metabotropic glutamate long-term Neurosci. PubMed Scopus Google Scholar). the signaling pathways downstream of mGluR that and the proteins that newly synthesized in to mGluR activation, important to better mGluR-dependent mGluRs to which to form and in triggers the of from in in the and activation of protein kinase C.M. Conn P.J. Metabotropic glutamate receptors: physiology, pharmacology, and disease.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 295-322Crossref PubMed Scopus (1159) Google Scholar). from these pathways, mGluR has been to a of downstream kinase to metabotropic glutamate long-term depression and synaptic in the hippocampal J. Neurosci. PubMed Scopus Google kinase kinase F. J. of kinase and kinase by metabotropic glutamate PubMed Scopus Google and of the F. kinase pathways of long-term at Neurosci. PubMed Scopus Google Scholar, Bear M.F. protein kinase activation for metabotropic glutamate long-term depression in hippocampal Neurosci. PubMed Scopus Google Scholar, J. Group I metabotropic glutamate receptor long-term depression via protein kinase by and activation of mGluRs and protein kinase in the Neurosci. PubMed Google and Activation of the of signaling pathway for metabotropic glutamate long-term Neurosci. PubMed Scopus Google Scholar, for metabotropic glutamate long-term depression and Neurosci. PubMed Scopus Google Scholar, J. kinase to neuronal Neurosci. PubMed Scopus Google signaling of these pathways for the of these on the of such as and Activation of the of signaling pathway for metabotropic glutamate long-term Neurosci. PubMed Scopus Google Scholar, F. of by signaling pathways metabotropic glutamate long-term Neurosci. PubMed Scopus Google Scholar). of new protein synthesis that for the long-term in receptors the depression of synaptic Bear M.F. for protein synthesis in hippocampal mGluR-dependent long-term PubMed Scopus Google Scholar, in synaptic Fragile PubMed Scopus Google Scholar). The of these to the of and the synthesis of proteins for the internalization of and synaptic in the Neurosci. PubMed Scopus Google Scholar, by of receptor and of PubMed Scopus Google Scholar, the postsynaptic synaptic AMPA PubMed Scopus Google Scholar, The of synaptic AMPA receptor Rev. PubMed Scopus Google Scholar, G. of the of protein in metabotropic glutamate endocytosis of AMPA receptors in Neurosci. PubMed Scopus Google Scholar, synthesis of the mental protein mGluR-dependent with the 2011; PubMed Scopus Google Scholar, of mGluR-dependent in endocytosis PubMed Scopus Google Scholar, C.M. Collingridge G.L. P.J. The AMPA receptor endocytosis metabotropic glutamate receptor Neurosci. PubMed Scopus Google Scholar). it what molecular processes in to of the been identified, the of molecular that by mGluR activation understanding of Here, to the phosphorylation by mGluR activation in hippocampal neurons, we a phosphoproteomics using The of us to the of DHPG-induced mGluR to newly synthesized proteins in to mGluR activation, we labeling strategy J. of newly synthesized proteins for quantitative 2012; PubMed Scopus Google in with labeling for of on the phosphorylation we identified several kinases important in the of which we confirmed using specific kinase inhibitors. Furthermore, we a broad spectrum of protein synthesis and phosphorylation in the AMPA receptor endocytosis pathway. We several novel insights into the signaling mGluR activation and and validated Intersectin-1 to important in were in with the for the of by the of The were by the of were from as in J. of AMPA PubMed Scopus Google Scholar). neurons were on and at a of neurons were in with and at were at with using and were hippocampal neurons were stimulated with for stimulated This was in from were The were in and were for at in at in was with using as a At neurons were stimulated with for were with and in with and were at with the by for of was with a protein of protein of were at for and for at and for in the at The proteins were the with for at which the were to a of and was The were to a of the were using and in were using in using the The were with in and with were in and the The to the were with and the were with into to of were in and at The were with to were and on was for in and the was as in in in in in in and for was to The was in At a of at were from to a of to were for was at of the to a of was at a of was with of and a of were with J. and protein PubMed Scopus Google and were with the the protein of with was as a and protein and phosphorylation of and were as was as and to were was at at the protein and level. The was to for the and for the was and was The were further using J. The for of PubMed Scopus Google G. a PubMed Scopus Google Scholar, a new to PubMed Scopus Google M.F. using 2011; Google Scholar, to the of protein phosphorylation from PubMed Scopus Google and F. J. G. J. G. for the PubMed Scopus Google Scholar). hippocampal neurons were stimulated with and with and were was to neurons, to the of proteins that in to neurons were stimulated with in in the of to for the were to the and these were in a The as a and of of of and was in with the were and as specific and proteins that a in the in at of the were for further hippocampal neurons were in with and and with and in stimulated with for at were into with and at the of the and with into with were at with the by for of was with a protein synthesized proteins were from of protein using the protein for of proteins the with small protein was to protein for and was by were to and was to were with and was were for at and for in the at The were to the and with with and were to a new and proteins were with for at and at were and the was was using the were in and at labeling was to the using the were in and in of the were to the and at for which the was with were using the in and at further were in and in with a to X were on in and on was for in and was using a from to was to The was in At a of at were from to a of to were for at a of to a of was at a of with a of was with a of were using and for protein and by of was as a The protein of the protein of with was for with a of The was and was to was as and were for was and were on to in were using a at the protein and level. were to and which for the of in the The of the was with and using was that for the and were using a and at a was for the in a of were using as and the and The were using a using were which were as the The was using of activation PubMed Scopus Google Scholar, J. pathway in PubMed Scopus Google Scholar). The was on a and a the from the were as to which for were such as that phosphorylation on specific were to such as and was using with the as for and for of and and using of were on a to and were in the were on a with with and using in at by a with at The was at and at in the The was at and the at were with and a of were using of the was on the the and the important were and of the and the was as of the the and the the the of the The was as for the The was by the in and in the by G. of with the kinase neurons were with for of were stimulated with for to and were the hippocampal neurons were stimulated with for and to the neurons were with in for neurons were with in for at with and with were with a with of a of at and were for and was using the function in and of study the downstream of group I mGluR activation, hippocampal were stimulated with a specific agonist of group I mGluRs. We confirmed that this a in as has been Group mGluR-dependent synaptic long-term and for and 2010; PubMed Scopus Google Scholar, of AMPA receptor and synaptic 2012; PubMed Scopus Google Scholar). proteins that synthesized in to mGluR activation by DHPG, we a J. of newly synthesized proteins for quantitative 2012; PubMed Scopus Google Scholar, J. of newly synthesized proteins in using amino PubMed Scopus Google Scholar). hippocampal neurons were stimulated with in the of the bio-orthogonal to newly synthesized these in which a in to newly synthesized we to we in the of to for the we a in which neurons were with to the of proteins that in to the of protein neurons were and at to a proteins were via by a of and and using specific and proteins that a in the in at of the were for further a using F. J. G. J. G. for the PubMed Scopus Google confirmed of postsynaptic as as a postsynaptic and the newly synthesized proteins proteins in such as and in synaptic Fragile PubMed Scopus Google which the of this to protein DHPG-induced mGluR activation DHPG-induced activation of group I mGluR internalization of Group mGluR-dependent synaptic long-term and for and 2010; PubMed Scopus Google Scholar, Bear M.F. for protein synthesis in hippocampal mGluR-dependent long-term PubMed Scopus Google Scholar, G. of the of protein in metabotropic glutamate endocytosis of AMPA receptors in Neurosci. PubMed Scopus Google Scholar, of mGluR-dependent in endocytosis PubMed Scopus Google Scholar, C.M. Collingridge G.L. P.J. The AMPA receptor endocytosis metabotropic glutamate receptor Neurosci. PubMed Scopus Google Scholar). we newly synthesized proteins with functions to receptor endocytosis and the protein the and the neuronal protein which a of G. F. J. with of in the Neurosci. PubMed Scopus Google with this we identified of the of a protein for the of which was with the that of the the of Collingridge G.L. metabotropic glutamate receptor long-term depression in and on protein kinase PubMed Scopus Google and for to the changes in that with J. metabotropic glutamate long-term depression and Neurosci. 2011; PubMed Scopus Google we several proteins with roles in and and as as from these we of proteins and as as of both and the from these that postsynaptic mGluR activation has the in the to we identified and the of several protein and such as several and which as the protein proteins in a of and processes and been to in the study of and postsynaptic and for synaptic 2012; PubMed Scopus Google Scholar). the of proteins were identified as of the proteins in neurons of synaptic proteins by of protein PubMed Scopus Google Scholar). a of the of proteins and which processes we a using the J. integrated the of PubMed Scopus Google Scholar). of these in the of using the in J. F. G. function on the 2010; PubMed Scopus Google Scholar). This proteins as identified and from a of and these we of proteins with functions in processes mGluR signaling the with proteins of of these as as we several protein with in processes to protein The proteins in the of and several and and as as proteins and The with a in proteins and and as as both from the and and and This that mGluR group I activation initiates the of new to the of of protein proteins in protein several proteins and as as several of the and which molecular in protein J. of newly proteins in the of molecular Rev. PubMed Scopus Google Scholar). protein and the of what as a of protein which and new protein in the from the of protein synthesis to at a to as in and and to protein a protein and has been to for and of PubMed Scopus Google Scholar, that neuronal PubMed Scopus Google Scholar). of the in by of protein and of PubMed Scopus Google that a protein and of proteins by the for the of with we of proteins in protein in several of the and we identified protein with a in proteins in the of both several which been in to function in the of proteins and of 2010; PubMed Scopus Google Scholar). to several proteins and to the of phosphorylation in the phosphoproteomics of protein phosphoproteomics in signaling PubMed Scopus Google Scholar, J. The for phosphorylation by PubMed Scopus Google Scholar, F. J. of and for phosphoproteomics 2011; PubMed Scopus Google of phosphorylation in we that using on a and of several of with of protein we that from a neuronal of we phosphorylation the and for to hippocampal PubMed Scopus Google Scholar). This us to study phosphorylation on in hippocampal neurons, the for of The of was hippocampal neurons for with The in This phosphoproteomics in the of with a of which in at in at and were for a and a of the identified from both the and neurons with The of on and in with phosphoproteomics in signaling PubMed Scopus Google Scholar, and for to hippocampal PubMed Scopus Google Scholar, J. for from 2010; PubMed Scopus Google The of the was and the of the that a of signaling pathways by DHPG, with the that signaling pathways, a the from the Furthermore, of function with a in a for of postsynaptic receptor and and of the postsynaptic a was in a of that were significantly in to of the proteins from which these revealed J.T. a for and of quantitative 2011; PubMed Scopus Google Scholar). The proteins in by in phosphorylation and in with the such as and a with by at we in processes in such as protein kinase and kinase proteins in and and phosphorylation by at the and a in phosphorylation proteins for proteins with and small and generally protein we the phosphorylation from these to a of and processes such as protein kinase and kinase and processes such as and of signaling pathways by kinases and processes in Bear M.F. protein kinase activation for metabotropic glutamate long-term depression in hippocampal Neurosci. PubMed Scopus Google Scholar, Activation of the of signaling pathway for metabotropic glutamate long-term Neurosci. PubMed Scopus Google Scholar, G.L. depression in the Rev. Neurosci. 2010; PubMed Scopus Google Scholar, C.M. Metabotropic glutamate long-term molecular Rev. PubMed Scopus Google Scholar, C.M. Collingridge G.L. AMPA receptor metabotropic glutamate long-term Neurosci. PubMed Scopus Google Scholar). of several kinases important of these signaling kinase to important in protein kinase We to in at This in with in phosphorylation of and this was to by via the mGluR pathway of in via phosphorylation of Google Scholar). to we and activation, of at the of the protein kinase J. of by kinase by PubMed Scopus Google Scholar). phosphorylation has been to in protein of and of protein in hippocampal and fragile X mental protein the of for PubMed Scopus Google Scholar). to protein several proteins in protein by phosphorylation these the of the and of the of the several and and several proteins of the at the phosphorylation level. the protein important of at the of the G. J.P. synaptic and protein kinases in cultured PubMed Scopus Google was significantly at of the has been to phosphorylation of both and of synaptic protein by protein kinase and kinase the of and PubMed Scopus Google Scholar). for the of and which in of synaptic protein by protein kinase and kinase the of and PubMed Scopus Google Scholar). that of at in to changes the activation changes in phosphorylation were in several proteins We in phosphorylation in synaptic proteins such as and The was to mGluR at a that has been identified in was to by in J. J. synaptic phosphorylation and the of the postsynaptic PubMed Scopus Google Scholar). proteins kinase at phosphorylation as as proteins and identified to to significantly in these the protein which in of AMPA receptors in synaptic Rev. Neurosci. PubMed Scopus Google Scholar, AMPA receptor in and Rev. Neurosci. PubMed Scopus Google Scholar). proteins in endocytosis at the phosphorylation and at the This of in the of the protein and important in neuronal The in and J. J. P.J. of I at 2012; PubMed Scopus Google Scholar). this specific was to in the of it was that with via and that this important for endocytosis in J. J. P.J. with to AMPA receptor internalization and long-term PubMed Scopus Google Scholar). This a for this in the and DHPG-induced group I mGluR activation, a and the kinase The of and has a in function J. J.T. of phosphorylation in and PubMed Scopus Google Scholar). the phosphorylation of to the phosphorylation of to level. Furthermore, and of a protein linked to receptor were significantly neurons, was to in J. G. of synaptic by and the 2010; PubMed Scopus Google Scholar, J. for the PubMed Scopus Google was to a postsynaptic function in internalization in glutamate receptor in a endocytosis PubMed Scopus Google Scholar). The of these in which for further kinases in the phosphorylation DHPG-induced group I mGluR we a phosphorylation of the This us to phosphorylation in kinase linked to proteins in The and the at the which a of the kinases of for 2010; 50: PubMed Scopus Google Scholar). we identified the which a of The the which of the for the protein kinases a Rev. PubMed Scopus Google Scholar, The and of protein protein J. PubMed Scopus Google Scholar). and a for these kinases in the This as a for as the which as the the activation of these as as kinases to activation and we quantitative of activation of several of these kinases of activation PubMed Scopus Google Scholar). this we were to the activation of phosphorylation of the activation of and and and we to the of with in hippocampal the we that with the DHPG-induced in the with mGluR-induced confirmed that the of and the DHPG-induced of the and kinase such as the for and of significantly Here, we signaling such as the kinase a specific of the protein and the protein The group and as as proteins of the phosphorylation from of which to by in and both by phosphorylation in on downstream to in of to in F. of in and and Scopus Google Scholar, for novel in by phosphoproteomics of and PubMed Scopus Google Scholar). proteins were to both newly synthesized and by phosphorylation proteins several proteins in and protein synthesis and as as protein neuronal protein and a that for in depression in the with a in and postsynaptic PubMed Scopus Google Scholar). that it has to by to The novel in AMPA receptor endocytosis and long-term PubMed Scopus Google Scholar). of and a of amino acids in this in a it to We validated changes in phosphorylation of of these and significantly We confirmed of of using which the and of this protein The for and Collingridge G.L. of in the synaptic activation of glutamate metabotropic PubMed Scopus Google a protein in and proteins in and which synthesized in the of protein and the of with a phosphorylation at by of the proteins of the phosphoproteomics and protein synthesis in with the from the kinase in a of molecular by group I mGluR activation in neurons the phosphorylation we identified that a for the protein via and PubMed Scopus Google was at and has a in we neurons with a both the and of development and endocytosis synaptic in hippocampal PubMed Scopus Google Scholar). of in neurons a as development and endocytosis synaptic in hippocampal PubMed Scopus Google and confirmed of in neurons was significantly in neurons that in the of Furthermore, we that the in in to was in neurons further that to stimulated The of in internalization the of this quantitative and we a of pulsed and labeling to study protein synthesis in to group I mGluR This for and of and newly synthesized This has been to to of cultured neurons J. of newly synthesized proteins for quantitative 2012; PubMed Scopus Google Scholar, J. of newly synthesized proteins in using amino PubMed Scopus Google Scholar, kinase the synthesis of proteins in PubMed Scopus Google Scholar, at hippocampal PubMed Scopus Google Scholar). of the of these pulsed labeling in with the to and in of proteins the of a of This to the of newly synthesized proteins mGluR activation, both and novel proteins linked to Here, we identified several proteins to in such as and synaptic Rev. Neurosci. PubMed Scopus Google and and proteins implicated in endocytosis such as G. J. G. Dysregulation of in the of by and synaptic and cognitive 2012; PubMed Scopus Google and we Group mGluR-dependent synaptic long-term and for and 2010; PubMed Scopus Google Scholar, in synaptic Fragile PubMed Scopus Google which by the of the of the This by was identified as a newly synthesized protein in the was for further to quantitative we of several proteins that in protein such as and and proteins in and in to the of the and of proteins in the of and of PubMed Scopus Google Scholar). of proteins to the of and several proteins with roles in these of proteins to the these on proteins with functions the in with and This and and in We the protein on the phosphorylation that the signaling pathways downstream of group I to the to to and protein This phosphorylation the in the of mGluR stimulated signaling pathways, and it therefore on the molecular receptor Here, we confirmed the of in mGluR-induced internalization in both and kinase on the of kinase and in the and of in Bear M.F. protein kinase activation for metabotropic glutamate long-term depression in hippocampal Neurosci. PubMed Scopus Google Scholar, Activation of the of signaling pathway for metabotropic glutamate long-term Neurosci. PubMed Scopus Google Scholar, C.M. Metabotropic glutamate long-term molecular Rev. PubMed Scopus Google the of phosphorylation in this of kinase has been and been to in the activation of protein via the and and pathways, and kinases to for for and G. with PubMed Scopus Google Scholar, of a on postsynaptic proteins mGluRs with and J. Neurosci. PubMed Scopus Google Scholar). to the signaling pathways that generally protein has been to signaling pathways Collingridge G.L. of long-term depression by metabotropic glutamate receptor activation in the in Scopus Google Scholar, Collingridge G.L. into in DHPG-induced in the of the PubMed Scopus Google Scholar). with these we of proteins of the and pathways to Furthermore, the kinase confirmed that of DHPG-induced This in with which that on Collingridge G.L. into in DHPG-induced in the of the PubMed Scopus Google Scholar). we of a of activation, as it phosphorylation of the of the protein to the of group I mGluRs. this activation with activation P.J. J. group I metabotropic glutamate Neurosci. 2012; PubMed Scopus Google Scholar). hippocampal with a DHPG-induced J. The roles of in and the development in PubMed Scopus Google that at of the functions in synaptic processes in hippocampal confirmed a for kinases in as further using the and specific which internalization the roles of and This for which to signaling of by phosphorylation was that group I mGluR activation in phosphorylation of were of which were J. metabotropic glutamate long-term depression and Neurosci. 2011; PubMed Scopus Google Scholar, of the and synaptic J. 2011; PubMed Scopus Google of both the and mGluR-induced been to in J. metabotropic glutamate long-term depression and Neurosci. 2011; PubMed Scopus Google Scholar). The of mGluR activation on proteins that mGluR activation on the in the this of of the protein synthesis and to on molecular functions of the identified proteins of of small of the were to newly synthesized group I mGluR activation, of the and and several of been on small in to several of synaptic on the of these of in as these and for the of small in and further Bear M.F. of hippocampal pathophysiology in and Neurosci. 35: PubMed Scopus Google Scholar, AMPA receptor Scopus Google Scholar). has been to in the of synaptic it was to of the in glutamate receptor in a endocytosis PubMed Scopus Google Scholar). Here, a for in at in as of the of in in the of the of internalization was in the neurons, a of in and The by which receptor to via such as the identified phosphorylation via of with that was for activation, and for protein via and PubMed Scopus Google Scholar). of identified phosphorylation of these in the of the the the we were to a of signaling and group I mGluR activation by changes in of protein phosphorylation and we activation of several signaling pathways, as as of protein signaling in This revealed several novel in of which we the of We that quantitative on protein phosphorylation and in to group I mGluR activation as a rich resource for further analyses. The been to the via the with the The been to the via the with the with metabotropic glutamate receptors receptors

Topics & Concepts