neurotrophins & nts receptors loredana lombardi ariadna laguna molecular mechanisms of...
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Neurotrophins & NTs Receptors
Loredana LombardiAriadna Laguna
Molecular Mechanisms of Development (EMBO),
Barcelona 2006
Neurotrophins: Ligands, Receptors and their pleiotropic role in Development
Cell Death Cell Survival, Growth and Differentiation
AimAnalyse the sequences of neurotrophins and their receptors using bioinformatic tools to better understand their biological properties.
Procedure1) Retrieve protein sequences from databases (mouse)
2) Align protein sequences
3) Search for known protein domains
4) Search for post-translational modifications in NTs
5) Search for phosphorylation sites and possible binding partners in Trks
1) Retrieve protein sequences from databases (mouse)
Name NCBI SwissProt Length pI/MW
Nerve Growth Factor (NGF) AAA37686.1 P01139 307 aa 9.56 / 27 kDa
Brain Derived Neurotrophic Factor
(BDNF)NP_031566 P21237 249 aa 8.91 / 28 kDa
Neurotrophin 3 (NT3) NP_032768 P20181 258 aa 9.24 / 29 kDa
Neurotrophin 4/5 (NT4/5) NP_937833 209 aa 9.17 / 22 kDa
Name NCBI SwissProt Length pI/MW
Tyrosine Receptor Kinase A (TrkA)
XP_283871 799 aa 5.87 / 88 kDa
Tyrosine Receptor Kinase B (TrkB)
NP_001020245 P15209 821 aa 6.08 / 92 kDa
Tyrosine Receptor Kinase C (TrkC)
NP_032772 825 aa 6.20 / 93 kDa
p75 NP_150086 427 aa 4.60 / 45 kDa
mouse_BDNF 1 ------MTILFLTMVISYFGCMKAAPMKEVNV-HGQGNLAYPGVRTHGTLESVNGPRAGS mouse_NT4/5 1 ------MLPRHSCSLLLFLFLLPSVPMEP-----------HP---PSSTLPPFLAP---- mouse_NT3 1 ------MSILFYVIFLAYLRGIQGNSMDQRSLPEDSLNSLIIKLIQADILKNKLSKQMVD mouse_NGF 1 SEVHSVMSMLFYTLITAFLIGVQAEPYTDSNVPEGDSVPEAHWTKLQHSLDTALRR---- mouse_BDNF 54 RGLTTTSLADTFEHVIEELLDEDQKVRPNEENHKDADLY-------TSRVMLSSQVPLEP mouse_NT4/5 37 ----------------------------------EWDLL-------SPRVALSRGAPAGP mouse_NT3 55 VKENYQSTLPKAEAPREPEQGEATRSEFQPMIATDTELLRQQRRYNSPRVLLSDSTPLEP mouse_NGF 57 ------ARSAPTAPIAARVTGQTRNITVDPRLFKKRRLH-------SPRVLFSTQPPPTS mouse_BDNF 107 PLLFLLEE--YKNYLDA-ANMSMR-VRRHSDPARRGELSVCDSISEWVTAADKKTAVDMS mouse_NT4/5 56 PLLFLLEAGAYGEPAGAPANRSRRGVSETAPASRRGELAVCDAVSGWVT--DRRTAVDLR mouse_NT3 115 PPLYLMED--YVGNPVVANRTSPRRKRYAEHKSHRGEYSVCDSESLWVT--DKSSAIDIR mouse_NGF 104 SDTLDLDFQAHGTIPFNRTHRSKR--SSTHPVFHMGEFSVCDSVSVWVG--DKTTATDIK mouse_BDNF 163 GGTVTVLEKVPVSKGQ-LKQYFYETKCNPMGYTKEG-------CRGIDKRHWNSQCRTTQ mouse_NT4/5 114 GREVEVLGEVPAAGGSPLRQYFFETRCKAESAGEGGPGVGGGGCRGVDRRHWLSECKAKQ mouse_NT3 171 GHQVTVLGEIKTGNSP-VKQYFYETRCKEARPVKNG-------CRGIDDKHWNSQCKTSQ mouse_NGF 160 GKEVTVLAEVNINNSV-FRQYFFETKCRASNPVESG-------CRGIDSKHWNSYCTTTH mouse_BDNF 215 SYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR-- mouse_NT4/5 174 SYVRALTADSQGRVGWRWIRIDTACVCTLLSRTGRA- mouse_NT3 223 TYVRALTSENNKLVGWRWIRIDTSCVCALSRKIGRT- mouse_NGF 212 TFVKALTTD-EKQAAWRFIRIDTACVCVLSRKATRRG
2-3) Align protein sequences and search for known protein domains
Clustalw, Multialign, Boxshade Prosite, Pfam, Motifscan,SMART
Signal Peptide
Propeptide
NGF domain
Glycosilation (Asparagine)
Disulfid Bonds
2-3) Analyse p75 protein sequence and search for known protein domains
Prosite, Pfam, Motifscan,SMART
MRRAGAACSAMDRLRLLLLLLLLLGVSFGGAKETCSTGMYTHSGECCKAC NLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECLGLQSMSA PCVEADDAVCRCSYGYYQDEETGRCEACSVCGVGSGLVFSCQDKQNTVCE ECPEGTYSDEANHVDPCLPCTVCEDTERQLRECTPWADAECEEIPGRWIT RSTPPEGSDVTTPSTQEPEAPPERDLIASTVADTVTTVMGSSQPVVTRGT ADNLIPVYCSILAAVVVGLVAYIAFKRWNSCKQNKQGANSRPVNQTPPPE GEKLHSDSGISVDSQSLHDQQTHTQTASGQALKGDGNLYSSLPLTKREEV EKLLNGDTWRHLAGELGYQPEHIDSFTHEACPVRALLASWGAQDSATLDA LLAALRRIQRADIVESLCSESTATSPV
Signal Peptide
Trans Membrane Domain
DEATH Domain
4 TNFR Domains
TMPRED
PSIPRED
2-3) Align protein sequences and search for known protein domains
Clustalw, Multialign, Boxshade Prosite, Pfam, Motifscan,SMART
Signal Peptide
IgG like domain
Leucine Rich
TrkB_isoformA 1 -----MSPWLKWHGPAMARLWGLCLLVLGFWRASLACPTSCKCSSARIWCTEPSPGIVAF TrkC_isoform1 1 -----MDVSLCPAKCSFWRIFLLGSVWLDYVGSVLACPANCVCSKTEINCRRPDDGN-LF TrkA_isoform1 1 MLRGQRLGQLGWHRPAAGLGSLMTSLMLACASAASCREVCCPVGPSGLRCTR-AGSLDTL TrkB_isoformA 56 PRLEPNSVD-------------PENITEILIANQKRLEIINEDDVEAYVGLRNLTIVDSG TrkC_isoform1 55 PLLEGQDSGNSNGNASINITDISRNITSIHIENWRGLHTLNAVDMELYTGLQKLTIKNSG TrkA_isoform1 60 RGLRG-----------------AGNLTELYVENQQHLQRLEFEDLQGLGELRSLTIVKSG TrkB_isoformA 103 LKFVAYKAFLKNSNLRHINFTRNKLTSLSRRHFRHLDLSDLILTGNPFTCSCDIMWLKTL TrkC_isoform1 115 LRNIQPRAFAKNPHLRYINLSSNRLTTLSWQLFQTLSLRELRLEQNFFNCSCDIRWMQLW TrkA_isoform1 103 LRFVAPDAFRFTPRLSHLNLSSNALESLSWKTVQGLSLQDLTLSGNPLHCSCALFWLQRW TrkB_isoformA 163 QET-KSSPDTQDLYCLNESSKNMPLANLQIPNCGLPSARLAAPNLTVEEGKSVTLSCSVG TrkC_isoform1 175 QEQGEARLDSQSLYCISADGSQLPLFRMNISQCDLPEISVSHVNLTVREGDNAVITCNGS TrkA_isoform1 163 EQEGLCGVHTQTLHDSGPGDQFLPLG--HNTSCGVPTVKIQMPNDSVEVGDDVFLQCQVE TrkB_isoformA 222 GDPLPTLYWDVGNLVSKHMNETSHTQG-----SLRITNISSDDSGKQISCVAENLVGEDQ TrkC_isoform1 235 GSPLPDVDWIVTGLQSINTHQTNLNWTNVHAINLTLVNVTSEDNGFTLTCIAENVVGMSN TrkA_isoform1 221 GLALQQADWILTELEGAATVKKFGDLP---SLGLILVNVTSDLNKKNVTCWAENDVGRAE TrkB_isoformA 277 DSVNLTVHFAPTITFLESPTSDHHWCIPFTVRGNPKPALQWFYNGAILNESKYICTKIHV TrkC_isoform1 295 ASVALTVYYPPRVVSLVEPEVRLEHCIEFVVRGNPTPTLHWLYNGQPLRESKIIHMDYYQ TrkA_isoform1 278 VSVQVSVSFP-ASVHLGLAVEQHHWCIPFSVDGQPAPSLRWLFNGSVLNETSFIFTQFLE TrkB_isoformA 337 ---TNHTEYHGCLQLDNPTHMNNGDYTLMAKNEYGKDERQISAHFMGRPGVDYETNPNYP TrkC_isoform1 355 ---EGEVS-EGCLLFNKPTHYNNGNYTLIAKNALGTANQTINGHFLKEP----------- TrkA_isoform1 337 SALTNETMRHGCLRLNQPTHVNNGNYTLLAANPYGQAAASVMAAFMDNP-----------
TrkB_isoformA 394 EVLYEDWTTPTDIGDTTNKSNEIPSTDVADQSNREHLSVYAVVVIASVVGFCLLVMLLLL TrkC_isoform1 400 ------FPESTDFFDFESDASPTPPITVTHKPEEDTFGVSIAVGLAAFACVLLVVLFIMI TrkA_isoform1 386 ---FEFNPEDPIPVSFSPVDGNSTSRDPVEKKDETPFGVSVAVGLAVSAALFLSALLLVL TrkB_isoformA 454 -KLARHSKFGMKGPASVISNDDDSASPLHHISNGSNTPSSSEGGPDAVIIGMTKIPVIEN TrkC_isoform1 454 NKYGRRSKFGMKGPVAVISGEEDSASPLHHINHGITTPSSLDAGPDTVVIGMTRIPVIEN TrkA_isoform1 443 NKCGQRSKFGINRP-AVLAPEDGLAMSLHFMTLGGSSLSPTEGKGSGLQG-----HIMEN TrkB_isoformA 513 PQYFGITNSQLKPDTFVQHIKRHNIVLKRELGEGAFGKVFLAECYNLCPEQDKILVAVKT TrkC_isoform1 514 PQYFRQGHNCHKPDTYVQHIKRRDIVLKRELGEGAFGKVFLAECYNLSPTKDKMLVAVKA TrkA_isoform1 497 PQYF--------SDTCVHHIKRQDIILKWELGEGAFGKVFLAECYNLLNDQDKMLVAVKA TrkB_isoformA 573 LKDASDNARKDFHREAELLTNLQHEHIVKFYGVCVEGDPLIMVFEYMKHGDLNKFLRAHG TrkC_isoform1 574 LKDPTLAARKDFQREAELLTNLQHEHIVKFYGVCGDGDPLIMVFEYMKHGDLNKFLRAHG TrkA_isoform1 549 LKEASENARQDFQREAELLTMLQHQHIVRFFGVCTEGGPLLMVFEYMRHGDLNRFLRSHG TrkB_isoformA 633 PDAVLMAEGNP---PTELTQSQMLHIAQQIAAGMVYLASQHFVHRDLATRNCLVGENLLV TrkC_isoform1 634 PDAMILVDGQPRQAKGELGLSQMLHIASQIASGMVYLASQHFVHRDLATRNCLVGANLLV TrkA_isoform1 609 PDAKLLAGGED-VAPGPLGLGQLLAVASQVAAGMVYLASLHFVHRDLATRNCLVGQGLVV TrkB_isoformA 690 KIGDFGMSRDVYSTDYYRVGGHTMLPIRWMPPESIMYRKFTTESDVWSLGVVLWEIFTYG TrkC_isoform1 694 KIGDFGMSRDVYSTDYYRVGGHTMLPIRWMPPESIMYRKFTTESDVWSFGVILWEIFTYG TrkA_isoform1 668 KIGDFGMSRDIYSTDYYRVGGRTMLPIRWMPPESILYRKFSTESDVWSFGVVLWEIFTYG TrkB_isoformA 750 KQPWYQLSNNEVIECITQGRVLQRPRTCPQEVYELMLGCWQREPHTRKNIKSIHTLLQNL TrkC_isoform1 754 KQPWFQLSNTEVIECITQGRVLERPRVCPKEVYDVMLGCWQREPQQRLNIKEIYKILHAL TrkA_isoform1 728 KQPWYQLSNTEAIECITQGRELERPRACPPDVYAIMRGCWQREPQQRLSMKDVHARLQAL TrkB_isoformA 810 AKASPVYLDILG TrkC_isoform1 814 GKATPIYLDILG TrkA_isoform1 788 AQAPPSYLDVLG
2-3) Align protein sequences and search for known protein domains
Clustalw, Multialign, Boxshade Prosite, Pfam, Motifscan,SMART
Predicted P-Tyr
TransMembrane Domain
Tyrosine Kinase Domain
TMPRED
PSIPRED
NetPhos 2.0 Server
ATP binding site
Activation Loop
5) Search for phosphorylation sites and possible binding partners in Trks
Src homology 2 group (SH2)
p85 SH2 Gene Card PIK3R1
Site Score Percentile Sequence SA
Y760 0.3680 0.156 % RACPPDVYAIMRGCW 0.312
Basophilic serine/threonine kinase group (Baso_ST_kin)
PKC alpha/beta/gamma Gene Card PRKCA
Site Score Percentile Sequence SA
T239 0.3881 0.143 % TELEGAATVKKFGDL 1.002
Kinase binding site group (Kin_bind)
PDK1 Binding Gene Card PDPK1
Site Score Percentile Sequence SA
T725 0.3973 0.068 % VVLWEIFTYGKQPWY 0.623
Phosphotyrosine binding group (PTB)
Shc PTB Gene Card SHC1
Site Score Percentile Sequence SA
Y499 0.4248 0.026 % HIMENPQYFSDTCVH 1.863
SCANSITE
TrkA
Conclusions
We found some discrepancies between different servers in the predicted protein domains essential to compare different predictions !!!
We found a good correspondance between different servers in the most conserved domains NGF domain in NT and PTyrK domain in Trks
It is useful to predict post-translational modifications and phosphorylation sites with bioinformatic tools to later manipulate the protein in the lab.