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The Wnt receptor CRD domain is also found in MuSK and related orphan receptor tyrosine kinases
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The Wnt receptor CRD domain is also found in MuSK and related orphan receptor tyrosine kinases Piotr Masiakowski and George D. Yancopoulos The receptor tyrosine kinase (RTK) MuSK is part of the receptor complex that initiates neuromuscular junction formation in response to agrin [1–3]. MuSK, as well as the related orphan RTK-like proteins Ror1 and Ror2 [4], and their homologs in lower organisms, have an extracellular region composed of varying combinations of immunoglobulin and Kringle domains, but they all also have a domain defined by a pattern of 10 cysteine residues. We have noticed that the spacing of cysteines, and a number of other amino acids, is shared between this domain and a cysteine-rich domain (CRD) that serves as the ligandbinding portion of the Frizzled (Fz) family of seven-pass transmembrane receptors for the Wnt signaling molecules [5]. As seen in other receptor families, the Wnt-binding
domain can also be a part of secreted Frizzled-related proteins (sFRPs) [6]. The Fz CRD-like sequences have also been identified in Smoothened (Smo), as well as in the α1 chain of type XVIII collagen (Col18), and carboxypeptidase Z (CPZ). Smo is the signaling partner in the Sonic hedgehog (Shh) receptor system. Smo is inactivated by unoccupied Patched (Ptc) protein. Binding of Shh to Ptc relieves this inhibition. The similarity between Smo and Fz, including the CRD, prompted a suggestion [7] that the activity of Smo may in addition be modulated by a Wnt-related ligand, though no support for this possibility has been presented. Alternatively, the 10-cysteine domain found in these diverse proteins may be thought of as a general module for protein–protein interactions, evolving in different cases to bind very different counterparts (just like immunoglobulin domains). A computer alignment of the CRDs from representative proteins (Figure 1) shows that the Wntbinding Fz receptors and sFRPs form a distinct group, well separated from the Smo and MuSK/Ror receptors. Thus, determination of the binding specificity of CRDs in
R407
these receptors, as well as in Col18 and CPZ, seems likely to reveal new binding capabilities of the 10cysteine module. References 1.
2.
3.
4.
5.
6.
7. 8.
Valenzuela DM, Stitt TN, DiStefano PS, Rojas E, Mattsson K, Compton DL, et al.: Receptor tyrosine kinase specific for the skeletal muscle lineage: expression in embryonic muscle, at the neuromuscular junction, and after injury. Neuron 1995, 15:573-584. DeChiara TM, Bowen DC, Valenzuela DM, Simmons MV, Poueymirou WT, Thomas S, et al.: The receptor tyrosine kinase MuSK is required for neuromuscular junction formation in vivo. Cell 1996, 85:501-512. Glass DJ, Bowen DC, Stitt TN, Radziejewski C, Bruno J, Ryan TE, et al.: Agrin acts via a MuSK receptor complex. Cell 1996, 85:513-523. Masiakowski P, Carroll RD: A novel family of cell surface receptors with tyrosine kinase-like domain. J Biol Chem 1992, 267:26181-26190. Bhanot P, Brink M, Samos CH, Hsieh JC, Wang Y, Macke JP, et al.: A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature 1996, 382:225-230. Rattner A, Hsieh JC, Smallwood PM, Gilbert DJ, Copeland NG, Jenkins NA, Nathans J: A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors. Proc Natl Acad Sci USA 1997, 94:2859-2863. Nusse R: Patching up Hedgehog. Nature 1996, 384:119-120. Program Manual for the Wisconsin Package V.9. 1996, Genetics Computer Group. http://www.gcg.com.
Address: Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, New York 10591-6707, USA.
Figure 1
(a) hRor1 hRor2 rMuSK dSmo hSmo msFRP-1 msFRP-2 cFz1 mFz4 mFz3 mFz6 hFz5 mFz8 dFz2 mFz7 rFz2 rFz1 dFz1 msFRP-3 hCPZ mCol18 Consensus
(b) 1 CqPyrgia.. CqPyrgia.. Caqyrgev.. CyPtsnatnt CePlry..nv Cvdipvdlrl CkPipanlql Cqkvd..hem CdPir..iam CePit..lrm CePit..vpr Cqeit..vpm Cqeit..vpl Ceeit..ipm CqPis..ipl CqPis..ipl CqPis..ipl CePit..isi CePvr..ipl C..vdlqlrt ClPlpptltl
C.arfignrt C.arfignrt Cdavlvkdsl Cfgsklpye. Clgsvlpyga ChnvgYkkmv ChgieYqnmr CndlpYnlts CqnlgYnvtk CqdlpYnttf CmkmtYnmtf CrgigYnlth CkgigYnyty CrgigYnmts CtdiaYnqti CtdiaYnqti CtdiaYnqti CknipYnmti Ckslpwnmtk CsdaaYnhtt Csrlgighfw
vymeslHmqg iyvdslqmqg vffntsypdp lssldltdfh tstllagdsd lPNlleHetm lPNllgHetm fPNlvdeesw mPNlvgHelq mPNllnHydq fPNlmgHydq mPNqfnHdtq mPNqfnHdtq fPNemnHetq lPNllgHtnq mPNllgHtnq mPNllgHtnq mPNligHtkq mPNhlhHstq fPNllqHrsw lPNhlhHtds
eienqitaaf eienritaaf e.eaqellih tekelndkln sqeeahgklv aevk.....q kevl.....e kdas.....e tdae.....l qtaa.....l giaa.....v deag.....l deag.....l deag.....l edag.....l edag.....l edag.....l eeag.....l anai.....l evveasseyi vevea.....
tmigtsshls tmigtsthls tawnelkavs dyyalkhvpk lwsglrnapr qasswvpLln qagawipLvm siltykpLls qlttftpLiq amepfhpmvn emghflhLan evhqfwpLve evhqfwpLve evhqfwpLve evhqfypLvk evhqfypLvk evhqfypLvk evhqfapLvk ameqfegLlg llsvlhqLle tvqawgrflh
dkCSqfaips dqCSqfaips plCrpaaeal ..CwaaiqpF ..Cwaviqpl knChmgtqvF kqChpdtkkF vvCSeqlkfF ygCSsqlqfF ldCSrdfrpF leCSpniemF iqCSpdlrfF iqCSpdlkfF ikCSpdlkfF vqCSpelrfF vqCSpelrfF vqCSaelkfF igCSddlqlF thCSpdllfF gqCnpdlrll tnChpflawF
LChyafPyCd fChfvfPlCd LCnhlfqeC. LCavfkPkCe LCavymPkCe LCslfaPvC. LCslfaPvCl LCsvyfPmCn LCsvyvPmCt LCalyaPiCm LCqafiPtCt LCtmytPiCl LCsmytPiCl LCsmytPiCl LCsmyaPvC. LCsmyaPvC. LCsmyaPvC. LCslyvPvC. LCamyaPiCt gCavlaPrCe fClllaPsCg
etssvpkprd arsrapkpre spgvlptpmp kingedmvyl ....ndrvel ..l.drpi.y ddl.deti.q ekl.anpi.g eki.nipi.g e..ygrvt.l e..qihvv.l pdy.hkpl.p edy.kkpl.p edy.hkpl.p tvl.dqai.p tvl.eqai.p tvl.eqal.p til.erpi.p idfqhepi.k ggwvrr.... pg.ppppl.p
lCRdeCEile lCRdeCEvle iCReyClavk PsyemCritm PsRtlCqatr PCRwlCEavr PChslCvqvk PCRplClsvq PCggmClsvk PCRrlCqray PCRklCEkiv PCRsvCErak PCRsvCErak vCRsvCErar PCRslCErar PCRsiCErar PCRslCEra. PCRslCEsar PCksvCErar PCRhiCEglr PCRqfCEale
nvlCqteyif sdlCrqeyti elfCakewla ep.Cri.... gp.Cai.... ds.Cep.... dr.Cap.... ek.Clp.... rr.Cep.... se.Csk.... sd.Ckk.... ag.Csp.... ag.Cap.... sg.Cap.... qg.Cea.... qg.Cea.... qg.Cea.... .v.Cek.... qg.Cep.... ev.Cqp.... de.Cwn....
arsnpm.... arsnpl.... megkthrgly .......lyn .......ver .......vmq .......vms .......vle .......vlr .......lme .......lmd .......lmr .......lmr .......imq .......lmn .......lmn .......lmn .......lmk .......ili .......afd .......yla
..ilmrlklp ..ilmrlqlp rsgmhflpvp ttf..fPkfl erg..WPdfl ffgfyWPeml afgfpWPdml sfgfkWPdvi efgfaWPdtl mfgvpWPedm tfgirWPeel qygfaWPerm qygfaWPdrm qysfeWPerm kfgfqWPerl kfgfqWPerl kfgfqWPdtl tynfnWPenl kyrhsWPesl aidmaWPyfl gdrlp....v
nCedlPqpes kCealPmpes eCsklP.smh rCn.....et rCt.....pd kCdkfP.eg. eCdrfPqdn. rCdkfPlen. nCskfPpqn. eCsrfPd... eCnrlPh... sCdrlPvlgr rCdrlPeqg. aCehlPlhg. rCenfPvhg. rCehfPrhg. kCekfPvhg. eCskfPvhg. aCdelPvydr dChryftre. vCaslPsqe.
136 peaanC pdaanC qdptaC lfptkC rfpegC ...dvC ...dlC nrekmC dhnhmC .....C .....C daevlC npdtlC dpdnlC .ageiC .aeqiC .agelC .gedlC ...gvC ..degC ..dgyC
C-P------- C----Y---- -PN---H--- ---------- -------L-- --CS-----F LC----P-C- ---------- PCR--CE--- ---C------ ---------- -----WP--- -C---P---- -----C
hRor1 hRor2 rMuSK dSmo hSmo msFRP-1 msFRP-2 cFz1 mFz4 mFz3 mFz6 hFz5 mFz8 dFz2 mFz7 rFz2 rFz1 dFz1 msFRP-3 hCPZ mCol18
Current Biology
(a) Multiple sequence alignment of 10-cysteine domains and (b) a phylogenetic tree showing the clustering relationships used to create the alignment. The sequences were downloaded through NCBI Entrez and processed using the programs in the GCG Wisconsin Package [8].
The Wnt receptor CRD domain is also found in MuSK and related orphan receptor tyrosine kinases Piotr Masiakowski and George D. Yancopoulos The receptor tyrosine kinase (RTK) MuSK is part of the receptor complex that initiates neuromuscular junction formation in response to agrin [1–3]. MuSK, as well as the related orphan RTK-like proteins Ror1 and Ror2 [4], and their homologs in lower organisms, have an extracellular region composed of varying combinations of immunoglobulin and Kringle domains, but they all also have a domain defined by a pattern of 10 cysteine residues. We have noticed that the spacing of cysteines, and a number of other amino acids, is shared between this domain and a cysteine-rich domain (CRD) that serves as the ligandbinding portion of the Frizzled (Fz) family of seven-pass transmembrane receptors for the Wnt signaling molecules [5]. As seen in other receptor families, the Wnt-binding
domain can also be a part of secreted Frizzled-related proteins (sFRPs) [6]. The Fz CRD-like sequences have also been identified in Smoothened (Smo), as well as in the α1 chain of type XVIII collagen (Col18), and carboxypeptidase Z (CPZ). Smo is the signaling partner in the Sonic hedgehog (Shh) receptor system. Smo is inactivated by unoccupied Patched (Ptc) protein. Binding of Shh to Ptc relieves this inhibition. The similarity between Smo and Fz, including the CRD, prompted a suggestion [7] that the activity of Smo may in addition be modulated by a Wnt-related ligand, though no support for this possibility has been presented. Alternatively, the 10-cysteine domain found in these diverse proteins may be thought of as a general module for protein–protein interactions, evolving in different cases to bind very different counterparts (just like immunoglobulin domains). A computer alignment of the CRDs from representative proteins (Figure 1) shows that the Wntbinding Fz receptors and sFRPs form a distinct group, well separated from the Smo and MuSK/Ror receptors. Thus, determination of the binding specificity of CRDs in
R407
these receptors, as well as in Col18 and CPZ, seems likely to reveal new binding capabilities of the 10cysteine module. References 1.
2.
3.
4.
5.
6.
7. 8.
Valenzuela DM, Stitt TN, DiStefano PS, Rojas E, Mattsson K, Compton DL, et al.: Receptor tyrosine kinase specific for the skeletal muscle lineage: expression in embryonic muscle, at the neuromuscular junction, and after injury. Neuron 1995, 15:573-584. DeChiara TM, Bowen DC, Valenzuela DM, Simmons MV, Poueymirou WT, Thomas S, et al.: The receptor tyrosine kinase MuSK is required for neuromuscular junction formation in vivo. Cell 1996, 85:501-512. Glass DJ, Bowen DC, Stitt TN, Radziejewski C, Bruno J, Ryan TE, et al.: Agrin acts via a MuSK receptor complex. Cell 1996, 85:513-523. Masiakowski P, Carroll RD: A novel family of cell surface receptors with tyrosine kinase-like domain. J Biol Chem 1992, 267:26181-26190. Bhanot P, Brink M, Samos CH, Hsieh JC, Wang Y, Macke JP, et al.: A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature 1996, 382:225-230. Rattner A, Hsieh JC, Smallwood PM, Gilbert DJ, Copeland NG, Jenkins NA, Nathans J: A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors. Proc Natl Acad Sci USA 1997, 94:2859-2863. Nusse R: Patching up Hedgehog. Nature 1996, 384:119-120. Program Manual for the Wisconsin Package V.9. 1996, Genetics Computer Group. http://www.gcg.com.
Address: Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, New York 10591-6707, USA.
Figure 1
(a) hRor1 hRor2 rMuSK dSmo hSmo msFRP-1 msFRP-2 cFz1 mFz4 mFz3 mFz6 hFz5 mFz8 dFz2 mFz7 rFz2 rFz1 dFz1 msFRP-3 hCPZ mCol18 Consensus
(b) 1 CqPyrgia.. CqPyrgia.. Caqyrgev.. CyPtsnatnt CePlry..nv Cvdipvdlrl CkPipanlql Cqkvd..hem CdPir..iam CePit..lrm CePit..vpr Cqeit..vpm Cqeit..vpl Ceeit..ipm CqPis..ipl CqPis..ipl CqPis..ipl CePit..isi CePvr..ipl C..vdlqlrt ClPlpptltl
C.arfignrt C.arfignrt Cdavlvkdsl Cfgsklpye. Clgsvlpyga ChnvgYkkmv ChgieYqnmr CndlpYnlts CqnlgYnvtk CqdlpYnttf CmkmtYnmtf CrgigYnlth CkgigYnyty CrgigYnmts CtdiaYnqti CtdiaYnqti CtdiaYnqti CknipYnmti Ckslpwnmtk CsdaaYnhtt Csrlgighfw
vymeslHmqg iyvdslqmqg vffntsypdp lssldltdfh tstllagdsd lPNlleHetm lPNllgHetm fPNlvdeesw mPNlvgHelq mPNllnHydq fPNlmgHydq mPNqfnHdtq mPNqfnHdtq fPNemnHetq lPNllgHtnq mPNllgHtnq mPNllgHtnq mPNligHtkq mPNhlhHstq fPNllqHrsw lPNhlhHtds
eienqitaaf eienritaaf e.eaqellih tekelndkln sqeeahgklv aevk.....q kevl.....e kdas.....e tdae.....l qtaa.....l giaa.....v deag.....l deag.....l deag.....l edag.....l edag.....l edag.....l eeag.....l anai.....l evveasseyi vevea.....
tmigtsshls tmigtsthls tawnelkavs dyyalkhvpk lwsglrnapr qasswvpLln qagawipLvm siltykpLls qlttftpLiq amepfhpmvn emghflhLan evhqfwpLve evhqfwpLve evhqfwpLve evhqfypLvk evhqfypLvk evhqfypLvk evhqfapLvk ameqfegLlg llsvlhqLle tvqawgrflh
dkCSqfaips dqCSqfaips plCrpaaeal ..CwaaiqpF ..Cwaviqpl knChmgtqvF kqChpdtkkF vvCSeqlkfF ygCSsqlqfF ldCSrdfrpF leCSpniemF iqCSpdlrfF iqCSpdlkfF ikCSpdlkfF vqCSpelrfF vqCSpelrfF vqCSaelkfF igCSddlqlF thCSpdllfF gqCnpdlrll tnChpflawF
LChyafPyCd fChfvfPlCd LCnhlfqeC. LCavfkPkCe LCavymPkCe LCslfaPvC. LCslfaPvCl LCsvyfPmCn LCsvyvPmCt LCalyaPiCm LCqafiPtCt LCtmytPiCl LCsmytPiCl LCsmytPiCl LCsmyaPvC. LCsmyaPvC. LCsmyaPvC. LCslyvPvC. LCamyaPiCt gCavlaPrCe fClllaPsCg
etssvpkprd arsrapkpre spgvlptpmp kingedmvyl ....ndrvel ..l.drpi.y ddl.deti.q ekl.anpi.g eki.nipi.g e..ygrvt.l e..qihvv.l pdy.hkpl.p edy.kkpl.p edy.hkpl.p tvl.dqai.p tvl.eqai.p tvl.eqal.p til.erpi.p idfqhepi.k ggwvrr.... pg.ppppl.p
lCRdeCEile lCRdeCEvle iCReyClavk PsyemCritm PsRtlCqatr PCRwlCEavr PChslCvqvk PCRplClsvq PCggmClsvk PCRrlCqray PCRklCEkiv PCRsvCErak PCRsvCErak vCRsvCErar PCRslCErar PCRsiCErar PCRslCEra. PCRslCEsar PCksvCErar PCRhiCEglr PCRqfCEale
nvlCqteyif sdlCrqeyti elfCakewla ep.Cri.... gp.Cai.... ds.Cep.... dr.Cap.... ek.Clp.... rr.Cep.... se.Csk.... sd.Ckk.... ag.Csp.... ag.Cap.... sg.Cap.... qg.Cea.... qg.Cea.... qg.Cea.... .v.Cek.... qg.Cep.... ev.Cqp.... de.Cwn....
arsnpm.... arsnpl.... megkthrgly .......lyn .......ver .......vmq .......vms .......vle .......vlr .......lme .......lmd .......lmr .......lmr .......imq .......lmn .......lmn .......lmn .......lmk .......ili .......afd .......yla
..ilmrlklp ..ilmrlqlp rsgmhflpvp ttf..fPkfl erg..WPdfl ffgfyWPeml afgfpWPdml sfgfkWPdvi efgfaWPdtl mfgvpWPedm tfgirWPeel qygfaWPerm qygfaWPdrm qysfeWPerm kfgfqWPerl kfgfqWPerl kfgfqWPdtl tynfnWPenl kyrhsWPesl aidmaWPyfl gdrlp....v
nCedlPqpes kCealPmpes eCsklP.smh rCn.....et rCt.....pd kCdkfP.eg. eCdrfPqdn. rCdkfPlen. nCskfPpqn. eCsrfPd... eCnrlPh... sCdrlPvlgr rCdrlPeqg. aCehlPlhg. rCenfPvhg. rCehfPrhg. kCekfPvhg. eCskfPvhg. aCdelPvydr dChryftre. vCaslPsqe.
136 peaanC pdaanC qdptaC lfptkC rfpegC ...dvC ...dlC nrekmC dhnhmC .....C .....C daevlC npdtlC dpdnlC .ageiC .aeqiC .agelC .gedlC ...gvC ..degC ..dgyC
C-P------- C----Y---- -PN---H--- ---------- -------L-- --CS-----F LC----P-C- ---------- PCR--CE--- ---C------ ---------- -----WP--- -C---P---- -----C
hRor1 hRor2 rMuSK dSmo hSmo msFRP-1 msFRP-2 cFz1 mFz4 mFz3 mFz6 hFz5 mFz8 dFz2 mFz7 rFz2 rFz1 dFz1 msFRP-3 hCPZ mCol18
Current Biology
(a) Multiple sequence alignment of 10-cysteine domains and (b) a phylogenetic tree showing the clustering relationships used to create the alignment. The sequences were downloaded through NCBI Entrez and processed using the programs in the GCG Wisconsin Package [8].