CA2306776A1

CA2306776A1 - Methods for modulating nerve cell function

Info

Publication number: CA2306776A1
Application number: CA002306776A
Authority: CA
Inventors: Corey Goodman; Thomas Kidd; Guy Tear; Claire Russell; Kevin J. Mitchell
Original assignee: Individual
Current assignee: University of California San Diego UCSD
Priority date: 1997-11-14
Filing date: 1998-11-13
Publication date: 1999-05-27
Also published as: AU1409499A; AU745762B2; EP1030920A1; JP2001523458A; WO1999025833A1

Abstract

The amount of active Robo expressed on a cell is modified by modulating the effective amount of a Comm polypeptide in contact with the cell, whereby the amount of expressed active Robo is modulated inversely with the modulation of the effective amount of the Comm polypeptide in contact with the cell. In a particular embodiment, the Comm polypeptide is provided to the cell exogenously in a pharmaceutically acceptable composition. In another aspect, the invention provides methods of screening for agents which modulate RoboComm interactions. These methods generally involve forming a mixture of a Roboexpressing cell, a Comm polypeptide and a candidate agent, and determining the effect of the agent on the amount of Robo expressed by the cell.

Description

Methods for Modulating Nerve Cell Function The research carried out in the subject application was supported in part by NIH grant NS 18366. The government may have rights in any patent issuing on this application.
INTRODUCTION
Field of the Invention The field of this invention is methods for modulating nerve cell function.
Back rg ound In the developing CNS, most growth cones confront the midline at one or multiple times during their journey and make the decision of whether to cross or not to cross. This decision is not a static one but rather changes according to the growth cone's history. For example, in the Drosophila ventral nerve cord, about 10% of the interneurons project their axons only on their own side, in some cases extending near the midline without crossing it.
The other 90% of the interneurons first project their axons across the midline and then turn to project longitudinally on the other side, often extending near the midline.
These growth cones, having crossed the midline once, never cross it again, in spite of their close proximity to the midline and the many commissural axons crossing it. This decision to cross or not to cross is not unique to Drosophila but is common to a variety of midline structures in all bilaterally symmetric nervous systems.
What midline signals and growth cone receptors control whether growth cones do or do not cross the midline? After crossing once, what mechanism prevents these growth cones from crossing again? A related issue concerns the nature of the midline as an intermediate target. If so many growth cones find the midline such an attractive structure, why do they cross over it rather than linger? Why do they leave the midline?
One approach to find the genes encoding the components of such a system is to screen for mutations in which either too many or too few axons cross the midline.
Such a large-scale mutant screen was previously conducted in Drosophila, and led to the identification of two key genes: commissureless (comm) and roundabout (robo) (Seeger et al., 1993;
reviewed by Tear et al., 1993). In comm mutant embryos, commissural growth cones initially orient toward the midline but then fail to cross it and instead recoil and extend on their own side. robo mutant embryos, on the other hand, display the opposite phenotype in that too many axons cross the midline; many growth cones that normally extend only on their own side instead now project across the midline and axons that normally cross the midline only once instead appear to cross and recross multiple times (Seeger et al, 1993; present disclosure). Double mutants of comm and robo display a robo-like phenotype.
How do comm and robo function to control midline crossing? Neither the initial paper on these genes (Seeger et al., 1993) nor the cloning of comm (Tear et al., 1996) resolved this question. comm encodes a novel surface protein expressed on midline cells. In fact, the comm paper (Tear et al., 1996) ended with the hope that future work would "... help shed some light on the enigmatic function of Comm."
A copending application {Robo, A Novel Family of Polypeptides and Nucleic Acids, by inventors: Corey S. Goodman, Thomas Kidd, Kevin J. Mitchell and Guy Tear, and filed herewith) discloses the cloning and characterization of robo in various species including Drosophila. robo encodes a new class of guidance receptor with 5 immunoglobulin (Ig) domains, 3 fibronectin type III domains, a transmembrane domain, and a long cytoplasmic domain. Robo defines a new subfamily of Ig superfamily proteins that is highly conserved from fruit flies to mammals. The Robo ectodomains, and in particular the first two Ig domains, are highly conserved from fruit fly to human, while the cytoplasmic domains are more divergent. Nevertheless, the cytoplasmic domains contain three highly conserved short proline-rich motifs which may represent binding sites for SH3 or other binding domains in linker or signaling molecules.
For those axons that never cross the midline, Robo is expressed on their growth cones from the outset; for the majority of axons that do cross the midline, Robo is expressed at high levels on their growth cones only after they cross the midline. Transgenic rescue experiments in Drosophila reveal that Robo can function in a cell autonomous fashion, consistent with it functioning as a receptor. Thus, in Drosophila, Robo appears to function as the gatekeeper controlling midline crossing; growth cones expressing high levels of Robo are prevented from crossing the midline. Robo proteins in mammals function in a similar manner in controlling axon guidance.

Here we disclose ectopic and overexpression studies revealing that Comm down-regulates Robo expression, demonstrating that Comm functions to suppress the Robo-mediated midline repulsion. These results show that the levels of Comm at the midline and Robo on growth cones are tightly intertwined and dynamically regulated to assure that only certain growth cones cross the midline, that those growth cones that cross do not linger at the midline, and that once they cross they never do so again.
SLIMMARY OF THE INVENTION
The invention provides methods and compositions for modulating the amount of active Robo expressed on a cell. The general method involves modulating the effective amount of a Comm polypeptide in contact with a cell expressing an amount of active Robo polypeptide, whereby the amount of expressed active Robo is modulated inversely with the modulation of the effective amount of the Comm polypeptide in contact with the cell. For example, where the effective amount of the Comm polypeptide is increased, the amount of expressed Robo is decreased. The Robo polypeptide is preferably a human, mouse, C. elegans or Drosophila Robo I or II sequence or a polypeptide domain thereof having a Robo-specific activity, and the Comm polypeptide specifically modulates Robo expression and (a) comprises SEQ
ID N0:14 or a deletion mutant thereof which specifically modulates Robo expression andlor (b) is encoded by a nucleic acid comprising SEQ ID N0:13 or a nucleic acid which hybridizes with SEQ ID N0:13, preferably under stringent conditions. In a particular embodiment, the Comm polypeptide is provided to the cell exogenously in a pharmaceutically acceptable composition.
In another aspect, the invention provides methods of screening for agents which modulate Robo-Comm interactions. These methods generally involve forming a mixture of a Robo-expressing cell, a Comm polypeptide and a candidate agent, and determining the effect of the agent on the amount of Robo expressed by the cell.
DETAILED DESCRIPTION OF THE INVENTION
The subject methods involve modulating the effective amount of a Comm polypeptide in contact with a cell expressing an amount of active Robo polypeptide, whereby the amount of expressed active Robo is modified inversely with the modulation of the effective amount of the Comm polypeptide in contact with the cell. Robo expression is found to regulate a wide WO 99/25833 PCT/US98/2432'I
variety of cell functions, including cell-cell interactions, cell mobility, morphology, etc.
Accordingly, the invention provides methods for modulating targeted cell function comprising the step of modulating Robo expression by contacting the cell with a Comm polypeptide.
The targeted Robo polypeptide is generally naturally expressed on the targeted cells.
The nucleotide sequences of exemplary natural cDNAs encoding drosophila 1, drosophila 2, C. elegans, human l, human 2 and mouse 1 Robo polypeptides are shown as SEQ ID
NOS:1, 3, S, 7, 9 and 11, respectively, and the full conceptual translates are shown as SEQ ID NOS:2, 4, 6, 8, 10 or 12. The targeted Robo polypeptides comprise at least a functional domain of SEQ ID NOS:2, 4, 6, 8, 10 and 12, which domain has Robo-specific amino acid sequence and binding specificity or function. Preferred Robo domains comprise at least 8, preferably at least 16, more preferably at least 32, most preferably at least 64 consecutive residues of one of these SEQ ID NOS. In a particular embodiment, the domains comprise one or more structural/functional Robo immunoglobulin, fibronectin or cytoplasmic motif domains described herein. The subject domains provide Robo-specific antigens and/or immunogens, especially when coupled to Garner proteins. For example, peptides corresponding to Robo-and human Robo-specific domains are covalently coupled to keyhole limpet antigen (KLH) and the conjugate is emulsified in Freunds complete adjuvant. Laboratory rabbits are immunized according to conventional protocol and bled. The presence of Robo-specific antibodies is assayed by solid phase immunosorbant assays using immobilized Robo polypeptides of SEQ ID N0:2, 4, 6, 8, 10 or 12. Generic Robo-specific peptides are readily apparent as conserved regions in the aligned Robo polypeptide sequences of Table 1.
Table 1. Sequence Alignment of Robo Family Members: The complete amino acid alignment of the predicted Robo proteins encoded by drosophila robo 1 (D1, SEQ ID N0:2) and Human robo 1 (H1, SEQ ID N0:8) are shown. The extracellular domain of C.elegans robo (CE, SEQ
ID N0:6; Sax-3; Zallen et al., 1997), the extracellular domain of Drosophila robo 2 (D2, SEQ
ID N0:4), and partial sequence of Human robo 2 (H2, SEQ ID NO:10) are also aligned. The D2 sequence was predicted by the gene-finder program Grail. The position of immunoglobulin domains (Ig), fibronectin domains (FN), the transmembrane domain (TM), and conserved cytoplasmic motifs are indicated. The extracelluiar domain of rat robo 1 is nearly identical to H 1.

mH.............PMHpENHAIaRSTSTTNNPSrsRSSRMWLIpAWLLLVLVASNGLP 47 D1 m.FNRKTLICTi.11V1QA..............vIrsFCEDASN1A........,..... 30 CE
mKWKHVPFIVMiS11S1S~~NHLFLaQLIPDPEDvErG.NDHGTPIpTSDNDDNSLGYTGS 59 H1 $
>IG #1 AVrGQYQSpriiehpTdlwKknepatlnckVegKpEptiewfkdgepvStn..EKKahr 105 D1 GENpriiehpMdTTvPknDpFtFncQaegNptptiQwfkdgRELKt...dTGshr D2 ........pViiehpldVwsRgSpatlncGaK.PStAKiTwykdgQpvItnkEQVNshr 81 CE
RLrQEDFPpriVehpSdlIvskgepatlnckaegRptptiewykGgeRvEtDkDdPRshr 119 H1 >IG #2 VQFKDgAlffYriMQgkkeQ..dGgEywcvaknRVgQavsrFiaslqIavlrddfrvepKd 163 D1 iMlpAgGlfflkvIhSrReS..dagTywcEakneFgVaRsrnaTlqvavlrdEfrLepAN D2 1$ iVlDTgslfLlkvNSgkNGKDSdagAyYcvaSneHgeVKsNEGsIKLaMlrEdfrvRpRT 141 CE
MLlpSgslfflriVhgrkSRP.dEgVyVcvaRnYLgeavsHnaslEvallrddfrQNpSd 178 H1 trvaKgeTallecgppKgIpeptLIwIkdgVplddLKAmSFGASSrVrivdggnlLiSNv 223 D1 trvaQgeValmecgAprg3pepQiswrkNgQTINL......VGNKririvdggnlAiQEA D2 vQALGgeMavlecSpprgFpepWswrkdDKEIRI.QDmP.....rYTLHSDgnIIiDPv 195 CE
vMvaVgePavmecQpprgHpeptiswKkdgSpldd.......KDEri.TIRggKIMiTYT 230 H1 >IG #3 EPIdEgNyKcIaQnLvgtresSYaKlIvQvkpYfMkepkdqVMLYgQTaTfHcSvggdpP 283 D1 2$ rQsdDgRyqcvVKnVvgtresATaFlKvHvrpFLIRGpQnqtAVvgSsvVfQcrIggdpL D2 DRsdSgTyqcvaNnmvgerVsNPaRlSvFekpKfEQepkdMtvDvgAAvLfDcrvTgdpQ 255 CE
rKsdAgKyVcvGTnmvgeresEVaElTvLerpSfVkRpSnLAvTvDDsaEfKcEARgdpV 290 H1 pKvlwkk..EEgnIpvsrA..........RiLHdEKslEiSNItpTdegTyvceaHnNvg 331 D1 pDvlwrrTASGgnmpLRKFSWLHSASGRVHVI.EdrslkLDDvtLEdmgeytceaDnAvg D2 pQITwkr..KNEPmpvTra..........YiAKdNrGIRiERvQpSdegeyvcYaRnPAg 303 CE
pTvRwrk..DDgELpKsrY..........Ei.RddHTlkiRKvtAGdmgSytcVaEnMvg 337 H1 >IG #'1 3$ QiSaRaSlIvhappNfTK:-pSnKKvGlNgVvQLPcMaSgnpPpSvfwTkegVSTlMfpn. 388 D1 GiTaTGIltvhappKfvIrpKnqLvEIgDEvLfecQaNgHpRpTLYwsVegNSSIILpGy D2 TLeasaHlRvqappSfQTkpAdqSvPAggtAtfecTLVgQpSpaYfwskegQqDllfpsy 363 CE

KAeasaTltvqEppHfvVkpRdqVvalgrtvtfQceaTgnpqpaIfwRRegsqnllf.sy 396 H1 qIvaQgrtvtfPceTKgnpqpavfwQkegsqnllfpn. H2 ...SsHGrQYvAADgtlQitDvrqedegyyv.cSaFSv~rDssTVrVFIQvSS..vD.... 440 Dl $ RDGRMEVTLTPEGRSVISiARFAredSgKVvTcNalnAvgsVSsrTWSvDt..QF.... D2 VSADGRTK..vsptgtltiEEvrqVdegAyv.cAGMnSagsslskaAlKvttKAvTGNTP 420 CE
qpPQsSsrFsvsQtgdltitnvqrsdVgyyi.cqTlnvagsiITkaYlevtd..vIA... 450 H1 qpQQPNsrCsvsptgdltitnIqrsdAgyyi.cqalTvagsilAkaQlevtd..vLT... H2 >IG #5 erpppiiQIgpAnqtlpKgsVaTlpcratgNpSpRiKwFHdgHAvQA.GNRYSi.iqG.. 496 D1 eLpppiieqgpvnqtlpvKsIVvlpcrTLgTpvpQVswYLdgIpidVqEHERrNLsDA.. D2 AKpppTieHgHQnqtlMvgsSaIlpcQaSgKpTpGiswlRdgLpidITd..sri.sqHST 477 CE
drpppViRqgpvnqtVavdgtFvlScVatgSpvpTiLwRkdgVLvSTqd..sriK.qLeN 507 H1 1$ drpppiiLqgpAnqtlavdgtaLcKcKatgDpLpViswlkEgFTFPGRd..PrATiq.eQ H2 >FN #1 SsIRVDdlq.lsdSgtytciasGeRgeTswAaTltveKpgs..TSLHraAdpstypAppg 553 D1 gAlTiSdlqrHEdEgLytcvasnRNgKsswsGylRLDTptNpNiKfFrapElstypgppg D2 gslHiAdl.kKPdtgVytciaKneDgestwsaSltveDHtsN.AqfVrMpdpsNFpsSpT 535 CE
gvlqiR.YAklGdtgRytciasTPsgeatwsayIEvQeFgVp.VqPPrPTdpNLIpsAps 565 H1 gTlqiKNl.rIsdtgtytcvaTSSsgeaswsaVlDvTeSgAT.i..SKNYdIsDLpgpps H2 TpKvLnvsrtsISlRwAKSqEKPGAVgpIi.gyTVeyfspdlQTgwIVAaHrvGDtQVti 612 D1 2$ kpqMvEKGEnsvtlsw...TRSNKVggSSLVgyVieMfGKNETDgwVAvGTrvQNttFtQ D2 QpIIvnvtDtEvElHw...NAPSTsgaGpitgyiiQyYspdlgQTwFNIPDYvAStEyRi 592 CE
kpEvtdvsrnTvtlsw...qpNLNsgaTp.tSyiieafsHASgSswqtvaENvktEtSAi 621 H1 kpqvtdvtKnsvtlsw...qpGTPGTLpA.SAyiieafsQSVSNswqtvaNHvkttLytV H2 >FN #2 SglTpgtsyVflvraenTQgisvpsGLsNViktIEA....DfDAASANdlsAarT.llTg 667 D1 TglLpgVNyFfliraenSHgLsLpsPMsEpitVGTR....YfNS..gLdlsEarASllsg D2 kglkpSHsyMfViraenEkgiGTpsVSsALvttSKPAAQVA1SDKNKMdMAIaEKRITsE 652 CE
kglkpnAiylflvraAnAYgisDpsqIsDpvktQDV.....1PTSQgVdHKQVQRE.1GN 675 H1 3$ RglRpntiylfMvraInPkV.svT.q H2 KSvelIDasAinAsavrlEwMLHvSADEkyvegLRiAyK..DaSVPSAQYHSITvMDAsa 725 D1 DvvelSnasvVDstsMKlTwQI...INGkyvegFyVYArQLpNPLNTKyRMLTILNGGGa D2 QLIKlEEVKTinstavrlFwKKR..KLEELiDgyyiKWrGPpRTNDNQyVN...vTSpsT 707 CE
AvLHIHnPTvLSsssIEVHwT..vDQQSQyiQgyKiLyrPSGaNHGESDWLVFEvRTpAK 733 H1 $ >FN #3 esFwGnlKkytKyeffLTpf...fETiegQpsnskTaltYedvpsappDNIQiGmYn.. 780 D1 SsCTiTGIVQytLyeffIVpf...YKsVegKpsnsRIaRtledvpsEApYgMEALLln.. D2 eNYwSnIMPFtnyeffVIpYHSGVHsiHgapsnsMDVltAeAPpsLppEDvRiRmlnL. 766 CE
NsVviPDIRkGVnyeIKARpf...fNEFQgaDsEIkFaKtleEApsappQgvTVSKNDGN 790 H1 QtaGWvRwTpppSQHHngNlYgykiEVSAgnTM.....KVIAnMtLnaTtTsvLlNnltt 835 Dl SSaVFLKwkapELKDRHgVILNyH.vivRgIDtAHNFSRIITnVtIdaASPTLvIAnItE D2 .tTLRIswkapKAdGIngIIKgFQiviv.gQAPNNNR.....nItTnERAAsvTIFHIVt 819 CE
GtaILvswQpppEdTQngMVQEykV.WCLgnEtR.....YHInKtVdGStFsvvIPFIVP 844 H1 1$
gAVysvrLNSFtKagDgpysKpISlFMdpTHHVHPpRAHPsGTHDGRHEGqDLTYHNNgN 895 D1 gVMyTvGvaaGNnagvgpyCVpATIRIdpITKRLDpFINQRDHVND.............. D2 gMTyKIrvAARSnGgvgv..........ShgTSEVIMNqDTIEKHL.AAQqENESFLYgL 868 CE
gIRysvEvaaStGagSgvKsEpQFIQldAhgNPVSpEDqVsIAQQI.............. 890 H1 > TM <
iPPGDINPTTHKKTTdYISGpwLMViVCiVILvlVisAAIsM.vyFkrkhQmTKEIGHLS 954 D1 ................vlTqpwFIiiLgAilavlMLs..fGAMvFVkrkhMm..MkQsAL D2 2$ iNK..............SHVpVIViVaILiIFvViiIAY.CYwRNS.rNSD...gkDRSF 909 CE
..............SdvVKqp..AFiagiGAaCWiiLMVfsIwLyRHrkKR..NgITsTY 932 H1 VVSDNEIT.......................AlniNSKESL.wIDHHRGwRTADTDKD.. 988 D1 AGIRKVPSFTFTPTVTYQRGGEAVSSGGRPGLIniSEPAAQPwLAD..TwPNTGNNHNDC 990 H1 ........SgLsEsKILSHVNSSQ..SnynnS..........DGGtDyAEvd....TRNL 1024 D1 SISCCTAGNgNsDsNITTYSRPADCIAnynnQLDNKQTNLMLPEStVyGDvdLSNKINEM 1050 H1 CYTOPLASMIC MOTIF #1 3$ TtfYNCR.......KSPDNptpyattMIiGTS........sSETCTkT.TSISADkDSGT 1068 D1 KtfNSPNLKDGRFVNPSGQptpyattQLiQSNLSNNMNNGsGDSGEkHWKPLGQQkQEVA 1110 H1 HSPyS........DAFAG(2VPAVpW..KSNyLqYPVEP......... " ,. " " " " 1097 D1 PVQyNIVEQNKLNKDYRANDTVPpTIPYNQSyDqNTGGSYNSSDRGSSTSGSQGHKKGAR 1170 H1 CYTOPLASMIC MOTIF #2 S .........InwSEFlppppEhppp...sSTy......GyAqGSp............ " . 1124 D1 TPKVPKQGGMnwADLlppppAhpppHSNsEEyNISVDESyDqEMpCPVPPARMYLQQDEL 1230 H1 ..eSSRKSSKSAGSgISTNQSILNAsIHsSSSGGFsAWGVSPQYAVAcp.......,... 1171 D1 EEeEDERGPTPPVRgAASSPAAVSYsHQsTATLTPsPQEELQPMLQDcpEETGHMQHQPD 1290 H1 ................pENVy...sNpl.....SAVAGGTQNRYQITPTNQHPPQ1.... 1203 D1 RRRQPVSPPPPPRPISpPHTyGYIsGpIVSDMDTDAPEEEEDEADMEVAKMQTRR1LLRG 1350 H1 ....paY..............,.FATTGPGGAVPPNHLP.............faTQRHaa 1230 D1 IS LEQTpaSSVGDLESSVTG~MINGWGSASEEDNISSGRSSVSSSDGSFFTDADfaQAVAaa 1410 H1 SeyQaglNAar................cAQSRACNsCdALATPSPmq............. 1261 D1 Aey.aglKVarRQMQDAAGRRHFHASQcPRPTSPVsTdSNMSAAVmqKTRPAKKLKHQPG 1469 H1 2O CYTOPLASMIC MOTIF #3 ...........ppppvpVpEGWYQPVHPNSH.PMHpTS.SNHQIYQCSSECsDHSRSsQS 1307 D1 HLRRETYTDDLppppvpPpAIKSPTAQSKTQLEVRpVWPKLPSMDARTDRsSDRKGsSY 1529 H1 HKrQL.................QLEeHGSSAkQrgGHHRRrA.pWQPCMESeN......ENM D1 2S KGrEVLDGRQVVDMRTNPGDPREAQeQQNDGkGrgNKAAKrDLpPAKTHLIQeDILPYCRPTF H1 LAEYEQrQYTsDCCNssrEGDTC..........SCSeGSCI..yAeAgePAPRQMTAKNT 1395 D1 PTSNNPrDPSsSSSMssrGSGSRQREQANVGRRNIAeMQVIGGy.eRgeDNNEELEETES 1651 H1 30 Exemplary such Robo specific immunogenic and/or antigenic peptides are shown in Table 2.
Table 2. Immunogenic Robo polypeptides eliciting Robo-specific rabbit polyclonal antibody:
Robo polypeptide-KLH conjugates immunized per protocol described below.
Robo Polypeptide. Sequence Immunogenicitv 3S SEQ ID N0:2, residues 68-77 +++
SEQ ID N0:2, residues 79-94 +++

SEQ ID N0:2, residues 95-103 +++

SEQ ID N0:2, residues 122-129 +++

SEQ ID N0:2, residues 165-176 +++

SEQ ID N0:2, residues 181-191 +++

SEQ ID N0:2, residues 193-204 +++

SEQ ID N0:2, residues 244-251 +++

SEQ ID N0:2, residues 274-290 +++

SEQ ID N0:2, residues 322-331 +++

SEQ ID N0:2, residues 339-347 +++

SEQ )D N0:2, residues 407-417 +++

SEQ ID N0:2, residues 44I-451 +++

SEQ ID N0:2, residues 453-474 +++

SEQ ID N0:2, residues 502-516 +++

SEQ ID N0:2, residues 541-553 +++

SEQ ID N0:2, residues 617-629 +++

In addition, species-specific antigenic and/or immunogenic peptides are readily apparent as diverged extracellular or cytosolic regions in Table 1. Human Robo-specific antibodies are characterized as uncross-reactive with non-human Robo polypeptides (SEQ )D
NOS:2, 4, 6 and 12). Exemplary such human specific peptides are shown in Table 3.
Table 3. Immunogenic Robo polypeptides eliciting human Robo-specific rabbit polyclonal antibody: Robo polypeptide-KLH conjugates immunized per protocol described below (some antibodies show cross-reactivity with corresponding mouse/rat Robo polypeptides).
Robo Polype~tide, Sequence Immuno enicitv SEQ ID N0:8, residues 1-12 +++
SEQ ID N0:8, residues 18-28 +++
SEQ )D N0:8, residues 31-40 +++
SEQ ID N0:8, residues 45-65 +++
SEQ ID N0:8, residues 106-116 +++
SEQ >D N0:8, residues 137-145 +++

SEQ ID N0:8, residues 174-184 +++

SEQ ID N0:8, residues 214-230 +++

SEQ ID N0:8, residues 274-286 +++

SEQ ID N0:8, residues 314-324 +++

SEQ ID N0:8, residues 399-412 +++

SEQ ID N0:8, residues 496-507 +++

SEQ ID N0:8, residues 548-565 +++

SEQ ID N0:8, residues 599-611 +++

SEQ ID N0:8, residues 660-671 +++

SEQ ID N0:8, residues 717-730 +++

SEQ ID N0:8, residues 780-791 +++

SEQ ID N0:8, residues 835-847 +++

SEQ ID N0:8, residues 877-891 +++

SEQ ID N0:8, residues 930-942 +++

SEQ ID N0:8, residues 981-998 +++

SEQ ID N0:8, residues 1040-1051+++

SEQ ID N0:8, residues 1080-1090+++

SEQ ID N0:8, residues 1154-1168+++

SEQ ID N0:8, residues 1215-1231+++

SEQ ID N0:8, residues 1278-1302+++

SEQ ID N0:8, residues 1378-1400++-+

SEQ ID N0:8, residues 1460-1469-i-f-+

SEQ ID N0:8, residues 1497-1519+++

SEQ ID N0:8, residues 1606-1626+++

SEQ ID N0:8, residues 1639-1651+++

SEQ ID NO:10, residues 5-16 +++

SEQ ID NO:10, residues 38-47 +++

SEQ ID NO:10, residues 83-94 +++

SEQ ID NO:10, residues 112-125 +++

SEQ ID NO:10, residues 168-180 +++

SEQ ID NO:10, residues 195-209 +++

SEQ ID NO:10, residues 222-235 +++
SEQ ID NO:10, residues 241-254 +++
The subject domains provide Robo domain specific activity or fimction, such as Robo-specific cell, especially neuron modulating or modulating inhibitory activity, Robo-ligand-binding or binding inhibitory activity. Robo-specific activity or fimction may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals (e.g. gene therapy, transgenics, etc.), etc. The binding target may be a natural intracellular binding target, a Robo regulating protein or other regulator that directly modulates Robo activity or its localization; or non-natural binding target such as a specific immune protein such as an antibody, or a Robo specific agent such as those identified in screening assays such as described below. Robo-binding specificity may be assayed by binding equilibrium constants (usually at least about 10' M-', preferably at least about 108 M-', more preferably at least about 109 M-'), by the ability of the subject polypeptide to function as 1 S negative mutants in Robo-expressing cells, to elicit Robo specific antibody in a heterologous host (e.g a rodent or rabbit), etc.
Similarly, the Comm polypeptide is conveniently selected from Comm polypeptides which specifically modulate Robo expression. Exemplary suitable Comm polypeptides (a) comprise SEQ ID N0:14 or a deletion mutant thereof which specifically modulates Comm expression and/or (b) are encoded by a nucleic acid comprising SEQ ID N0:13 or a nucleic acid which hybridizes with SEQ ID N0:13 under stringent conditions. Suitable deletion mutants are readily screened in Robo down-regulations assays as described below. Preferred Comm domains comprise at least 8, preferably at least 16, more preferably at least 32, most preferably at least 64 consecutive residues of SEQ ID N0:14 and provide a Comm specific activity, such as Comm-specific antigenicity and/or immunogenicity, especially when coupled to carrier proteins as described above for Robo. Exemplary such Conun specific immunogenic and/or antigenic peptides are shown in Table 4.
Table 4. Immunogenic Comm polypeptides eliciting Comm-specific rabbit polyclonal antibody: Comm polypeptide-KLH conjugates immunized per protocol described above.
Comm Polypeptide. Sequence Immuno enicitv SEQ ID N0:14, residues 1-11 +++

SEQ ID N0:14, residues 6-17 +++

SEQ ID N0:14, residucs 18-34 +++

SEQ ID N0:14, residues 35-44 +++

SEQ ID N0:14, residues 45-63 +++

SEQ ID N0:14, residues 64-73 +++

SEQ ID N0:14, residues 74-891 +++

SEQ ID N0:14, residues 92-109 +++

SEQ ID N0:14, residues 110-126 +++

SEQ ID N0:14, residues 127-136 +++

SEQ ID N0:14; residues 137-151 +++

SEQ ID N0:14, residues 152-171 +++

SEQ ID N0:14, residues 172-185 +++

SEQ ID N0:14, residues I86-199 +++

SEQ ID N0:14, residues 200-215 +++

SEQ ID N0:14, residues 216-235 ++~

SEQ ID N0:14, residues 236-250 +++

SEQ ID N0:14, residues 251-260 +++

SEQ ID N0:14, residues 261-275 +++

SEQ ID N0:14, residues 276-288 +++

SEQ ID N0:14, residues 289-307 +++

SEQ ID N0:14, residues 308-317 +++

SEQ ID N0:14, residues 318-331 +++

SEQ ID N0:14, residues 332-344 +++

SEQ ID N0:14, residues 345-356 +++

SEQ ID N0:14, residues 357-370 +++

SEQ ID N0:14, residues 41-153 +++

SEQ ID N0:14, residucs 117-329 +++

The subject domains provide Comm domain specific activity or fimction, such as Comm-specific cell, especially neuron modulating or modulating inhibitory activity, Comm-ligand-binding or binding inhibitory activity. Comm-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals (e.g. gene therapy, transgenics, etc.), etc.
The binding target rnay be a natural intracellular binding target, a Comm regulating protein or other regulator that directly modulates Comm activity or its localization; or non-natural binding target such as a specific immune protein such as an antibody, or a Comm specific agent such as those identified in screening assays such as described below. Comm-binding specificity may be assayed by binding equilibrium constants (usually at least about 10' M'', preferably at least about 10g M'', more preferably at least about 109 M''), by the ability of the subject polypeptide to function as negative mutants in Comm-expressing cells, to elicit Comm specific antibody in a heterologous host (e.g a rodent or rabbit), etc.
In one embodiment, the Comm polypeptides are encoded by a nucleic acid comprising SEQ ID N0:13 or a nucleic acid which hybridizes with a full-length strand of SEQ ID N0:13, preferably under stringent conditions. Such nucleic acids are at least 36, preferably at least 72, 1 S more preferably at least 144 and most preferably at least 288 bases in length. Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5 x SSPE (0.18 M NaCI, 0.01 M NaP04, pH7.7, 0.001 M EDTA) buffer at a temperature of 42°C and remaining bound when subject to washing at 42°C with 0.2 x SSPE Conditions I); preferably hybridizing in a buffer comprising 50%
formamide in 5 x SSPE buffer at a temperature of 42°C and remaining bound when subject to washing at 42°C with 0.2 x SSPE buffer at 42°C (Conditions II).
Exemplary nucleic acids which hybridize with a strand of SEQ ID N0:13 are shown in Table 5.
Table 5. Exemplary nucleic acids which hybridize with a strand of SEQ ID N0:13 under Conditions I andlor II.
Comm Nucleic Acids Hybridization SEQ ID N0:13, nucleotides 1-47 +
SEQ ID N0:13, nucleotides 58-99 +
SEQ ID N0:13, nucleotides 95-138 +
SEQ ID N0:13, nucleotides 181-220 +
SEQ )D N0:13, nucleotides 261-299 +

SEQ ID N0:13, nucleotides 274-315 +
SEQ ID N0:13, nucleotides 351-389 +
SEQ TD N0:13, nucleotides 450-593 +
SEQ ID N0:13, nucleotides 524-546 +
SEQ ID N0:13, nucleotides 561-608 +
SEQ ID N0:13, nucleotides 689-727 +
SEQ ID N0:13, nucleotides 708-737 +
SEQ ID N0:13, nucleotides 738-801 +
SEQ ID N0:13, nucleotides 805-854 +
SEQ ID N0:13, nucleotides 855-907 +
SEQ ID N0:13, nucleotides 910-953 +
SEQ ID N0:13, nucleotides 1007-1059 +
A wide variety of cell types express Robo polypeptides subject to regulation by the disclosed methods, including many neuronal cells, transformed cells, infected (e.g. virus) cells, etc. Ascertaining Robo expression is readily effected by antibody staining.
Accordingly, indications for the subject methods encompass a wide variety of cell types and function, including axon outgrowth, tumor cell invasion or migration, etc. The target cell may reside in culture or in situ, i.e. within the natural host. For in situ applications, the compositions are added to a retained physiological fluid such as blood or synovial fluid. For CNS
administration, a variety of techniques are available for promoting transfer of the therapeutic across the blood brain barrier including disruption by surgery or injection, drugs which transiently open adhesion contact between CNS vasculature endothelial cells, and compounds which facilitate translocation through such cells. Comm polypeptides may also be amenable to direct injection or infusion, topical, intratracheal/nasal administration e.g. through aerosol, intraocularly, or within/on implants e.g. fibers e.g. collagen, osmotic pumps, grafts comprising appropriately transformed cells, etc. A particular method of administration involves coating, embedding or derivatizing fibers, such as collagen fibers, protein polymers, etc. with therapeutic polypeptides. Other useful approaches are described in Otto et al.
(1989) J
Neuroscience Research 22, 83-91 and Otto and Unsicker (1990) J Neuroscience 10, 1912-1921. Generally, the amount administered will be empirically determined, typically in the range of about 10 to 1000 ~,g/kg of the recipient and the concentration will generally be in the range of about SO to S00 ~.g/ml in the dose administered. Other additives may be included, such as stabilizers, bactericides, etc. will be present in conventional amounts.
In one embodiment, the invention provides administering the subject Comm S polypeptides in combination with a pharmaceutically acceptable excipient such as sterile saline or other medium, gelatin, an oil, etc. to form pharmaceutically acceptable compositions.
The compositions and/or compounds may be administered alone or in combination with any convenient carrier, diluent, etc. and such administration may be provided in single or multiple dosages. Useful Garners include solid, semi-solid or liquid media including water and non-lU toxic organic solvents. In another embodiment, the invention provides the subject compounds in the foam of a pro-drug, which can be metabolically converted to the subject compound by the recipient host. A wide variety of pro-drug formulations for polypeptide-based therapeutics are known in the art. The compositions may be provided in any convenient form including tablets, capsules, troches, powders, sprays, creams, etc. As such the compositions, in 15 pharmaceutically acceptable dosage units or in bulk, may be incorporated into a wide variety of containers. For example, dosage units may be included in a variety of containers including capsules, pills, etc. The compositions may be advantageously combined and/or used in combination with other therapeutic or prophylactic agents, different from the subject compounds. In many instances, administration in conjunction with the subject compositions 20 enhances the efficacy of such agents, see e.g. Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9'" Ed., 1996, McGraw-Hill.
In another aspect, the invention provides methods of screening for agents which modulate Robo-Comm interactions. These methods generally involve forming a mixture of a Robo-expressing cell, a Comm polypeptide and a candidate agent, and determining the effect 25 of the agent on the amount of Robo expressed by the cell. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds.
Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development. Cell and animal 30 based neural guidance/repulsion assays are described in detail in the experimental section below.

The following experimental section and examples are offered by way of illustration and not by way of limitation.
EXPERIMENTAL
roundabout is Required to Prevent Ipsilateral Axons from Crossing the Midline.
Mutations in robo lead to an increase in the number of embryonic CNS axons in the commissures, coincident with a reduction of the number of axons in the longitudinal connectives as observed with MAb BP102. The two commissures are thicker than normal and partially fuse as they spill over into one another; the longitudinals are thinner and pulled closer together toward the midline. We analyzed the robo mutant phenotype in more detail using the 1 D4 MAb (anti-Fas II) which at stage 13 stains a subset of growth cones (including aCC, pCC, vMP2, MP1, dMP2) and from stages 14-17 stains three major longitudinal axon tracts, including (from medial to lateral) the pCC pathway (pioneered by the pCC
growth cone), the MP 1 pathway (pioneered by the MP 1 growth cone), and a 3rd lateral pathway (Lin et al., 1 S 1994; Hidalgo and Brand, 1997). Previous analysis (Seeger et al., 1993) with MAb 1 D4 showed that the pCC growth cone, which normally projects anteriorly on its own side near the midline to pioneer the pCC pathway, in robo mutant embryos projects across the midline, fasciculating with its contralateral homologue at the midline. The axon pathway it pioneers --the pCC pathway -- which normally projects longitudinally on its own side near the midline, in robo mutant embryos projects back and forth across the midline. The pCC
pathway takes on a circular pattern as it joins with the same pathway from the other side and whirls back and forth across the midline, thus defining the phenotype for which the gene was named.
The fuzzy commissure phenotype observed in robo mutant embryos does not appear to be due to changes in cell fates at the midline or elsewhere in the CNS. All of the midline cells are present, and their fates appear normal as assayed with a variety of different markers (Seeger et al., 1993). All of the commissural and longitudinal axon pathways begin in their normal location, but the longitudinal pathways are pulled closer at the midline as axon bundles circle around the midline, and the commissures become fused and fuzzy as too many axons cross the midline. In contrast, in mutants in which all or some of the midline cells die or fail to properly differentiate, the longitudinal pathways either collapse onto the midline or from the outset form closer together than normal (Klambt et al., 1991; Mayer and Niisslein-Volhard, 1988). Thus, the defects observed in robo mutant embryos are not due to changes in cell fates but rather result from defects in axon guidance.
We examined in greater detail the behavior of the pCC growth cone in robo mutant compared to wild type embryos. In wild type embryos, the vMP2 cell body lies embedded at the edge of the midline. The pCC growth cone extends anterior to a point just lateral to vMP2's cell body. The pCC growth cone is then met by the lateral extension of vMP2's growth cone, and as pCC extends anteriorly and a bit laterally, the vMP2 growth cone wraps around pCC's axons and extends right behind it (Lin et al., 1994). This tight association of vMP2 and pCC is mediated by Fasciclin II (Fas II), a homophilic cell adhesion molecule (CAM) (Grenningloh et al., 1990, 1991 ) that is expressed from the beginning of axon outgrowth on the cell bodies, axons, and growth cones of a subset of neurons, including pCC
and vMP2. In Fasll mutant embryos, vMP2 and pCC no longer tightly associate, and their axons fail to fasciculate (Lin et al., 1994).
If the pCC and vMP2 neurons express Fas II, and their growth cones and axons are so attracted to each other in a Fas II-mediated fashion, why does not pCC's growth cone initially extend more medially toward vMP2's cell body which is a short distance away?
The answer appears to be because vMP2's cell body is partly embedded in other midline cells, and thus vMP2's cell body is partly surrounded by the putative midline repellent. In robo mutant embryos, pCC's initial trajectory is directly toward vMP2's cell body, where it adheres to vMP2; pCC's growth cone then crosses the midline, fasciculating with its contralateral homologue at the midline.
roundabout is Required to Prevent Commissural Axons from Recrossing the Midline.
The circular pathway taken by the pCC pathway as it crosses back and forth across the midline (as visualized with the anti-Fas II MAb) led us to suggest that some axons were freely recrossing the midline. Although Fas II is expressed on a relatively small subset of axons in the early embryo, and thus we can use it to observe pCC's growth cone abnormal crossing of the midline in robo mutants, the resulting pattern of expression in older embryos becomes quite complicated and it is difficult to resolve precisely which axons are crossing the midline.
To confirm that axons cross and recross the midline freely in robo mutants, we examined the expression of Connectin (Nose et al., 1992), a CAM expressed on a more restricted subset of CNS axons than is Fas II. Connectin is also expressed on motor axons in the segmental nerve. We used the C 1.427 MAb to follow Connectin expression (Meadows et al., 1994). Connectin is expressed on the SP 1 neuron whose cell body lies near the midline just anterior to the anterior commissure, and just medial to the longitudinal tracts. SP I's growth cone normally projects across the midline, fasciculating with the axon of its contralateral homologue. The growth cone then appears to adhere to the cell body of its contralateral homologue, grows around that cell body, and turns to project anteriorly in a medial sub-fascicle of the pCC pathway.
In robo mutant embryos as in wild type embryos, SP I's growth cone extends across the midline, adheres to the axon and then cell body of its contralateral homologue, and turns to project anteriorly. However, as it extends anteriorly into the next segment, it typically moves toward the midline, apparently attracted towards and adhering to the axon of its contralateral homologue just on the other side of the midline. The two SP I axons typically join together around the posterior commissure of the next anterior segment. Sometimes they extend together on the left side of the midline and sometimes on the right side, freely crossing and recrossing the midline while fasciculating with the SP 1 axons originating from both sides of neighboring segments. These results show that in addition to preventing ipsilaterally projecting axons from crossing the midline, Robo also functions to prevent contralaterally projecting axons from recrossing the midline.
roundabout Controls Crossing of the midline in a Dosage Sensitive Manner.
Another axonal marker which labels a very small subset of axons is the Tau-~galactosidase reporter gene expressed under control of the apterous promoter (called apC; Lundgren et al., 1995). In wild type embryos, the apC-tau-lacZ transgene labels three interneurons per abdominal hemisegment, here called the Ap neurons. The Ap neurons have lateral cell bodies and their growth cones initially project towards the midline. Upon nearing the midline, these growth cones then turn to project anteriorly on their own side along the edge of the midline, fasciculating with each other and with their homologues from neighboring segments; in wild type embryos, they never cross the midline in abdominal segments.
In robo mutant embryos, the Ap axons cross the midline in every segment, join up with their contralateral homologues, and often project anteriorly in one discrete longitudinal fasicle.
The Ap fascicle displays two behaviors, usually crossing and recrossing the midline multiple times as a single bundle, or occasionally separating into different bundles of axons which project on one side or the other and independently cross the midline.
We observed a partially penetrant Ap axon phenotype in robo heterozygous embryos.
In wild type, none of the 6 Ap axons in each segment ever cross the midline;
in robo homozygous mutants, all 6 Ap axons cross the midline. In robo heterozygous mutant embryos, one of the Ap axons is observed crossing the midline in about 30% of segments, which accounts for a penetrance of about 5% of all Ap axons (Table 1 ). This partially penetrant crossing with 50% of robo indicates a dose requirement for the robo gene product in these axons. Moreover, since the Ap axons extend midway through axonogenesis, once many axon pathways have already been pioneered, these results indicate that robo is required throughout axonogenesis, not just to establish the initial projections of the pioneer axons.
Underexpression of Comm Leads to Increased Levels of Robo Protein. The commissureless (comm) mutant has a complementary phenotype to that of robo in that too few axons cross the midline (Seeger et al., 1993). When visualised with MAb BP102, the axon commissures are noticeably absent. In certain hypomorphic comm alleles (e.g., comma; Tear et al., 1996), the commissures are not completely absent, but rather partial and highly abnormal axon commissures do form in a few segments (particularly in the thorax). We examined the expression of Robo protein in these comm hypomorphic alleles using the 13C9 anti-Robo MAb (Kidd et al, 1997). Normally, Robo is expressed at very low levels on commissural axons and at high levels on longitudinal axons. In comm mutant embryos, Robo expression in the longitudinal tracts appears even higher than normal.
Interestingly, in comm hypomorphic alleles, the occasional thin commissures express Robo protein at levels that are higher than normally seen in the commissures and closer to what is typically seen in the longitudinal tracts. This result was our first hint that Comm protein might function by suppressing Robo expression on commissural axons. Previous studies had shown that comm encodes a novel transmembrane protein that is expressed by the midline glia and that is apparently transferred to commissural axons (Tear et al., 1996). Given these results, we wondered whether expression of comm in all neurons might reduce Robo levels and lead to a robo phenotype.
Overexpression of Comm generates a robo-Like Phenotype. To test the hypothesis that increased expression of comm might lead to a robo-like phenotype, we used the UAS-GAL4 system (Brand and Perrimon, 1993) to change the pattern of comm expression. We generated UAS comm transgenic lines and drove expression pan-neurally using the sca-GAL4 line. Since flies carrying a copy of both the driver and effector transgenes are viable, we used them as parents and examined their progeny. A continuous range of robo-like phenotypes was observed with MAbs BP102 and 1D4. The range of phenotypes reveals the comm gain-of function phenotype to be dosage sensitive, as the severity increased in embryos carrying two copies of both transgenes as compared to embryos carrying only one copy of each.
Superficially, the robo phenotype can be mimicked by mutants causing inappropriate migration or cell death of the midline glia, both of which result in fuzzy commissures (Kl~tnbt et al., 1991). However, such phenotypes are not visible until midway through axonogenesis, and are easily detected by examining early axon behavior. In addition, we stained the embryos with a MAb raised against 8H11, a protein expressed specifically by the midline glia, and confirmed that the midline glia are still present In the embryos ectopically expressing comm, Fas II positive axons, suich as pCC, were found to behave identical to how they behave in robo mutants. When Comm is overexpressed, the pCC growth cone extends towards the vMP2 cell body, and then across the midline, just as it does in a robo mutant. In the comm gain-of function, the pCC fascicle freely crosses and midline and forms the same circles or whirls as it does in the robo loss-of function.
Overexpression of Comm Leads to Reduced Levels of Robo Protein. Having established that the comm overexpression generates a bona fide robo-like axon guidance phenotype, we next examined Robo expression in these embryos using the anti-Robo MAb 13C9. The sca-GAL4 driver begins driving expression in the neuroepithelium before axon outgrowth (stage 9) has begun and switches off by stage 13; sca-GAL4 does not express in the epidermis. In wild type embryos, the pattern of Robo protein expression begins in the neuroepithelium, as well in some lateral epidermal stripes, but is conspicuously absent from the midline region. In comm gain-of function embryos, Robo expression in the neuroepithelium is greatly reduced or absent, while the epidermal expression outside the nervous system is maintained. This same pattern can be observed around the time when the first growth cones are extending. In wild type embryos during stages 12 and 13, no Robo is seen at the midline, but there is a high level of Robo expression on ipsilaterally projecting growth cones such as pCC and a significant level throughout the neuroepithelium. In contrast, in comm gain-of function embryos, the pCC growth cone lacks Robo protein and the neuroepithelium expresses greatly reduced levels of Robo.
The dramatic reduction in the levels of Robo were observed until about stage 14, coincident with the sca-GAL4 driver ceasing expression. In the sca-GAL4; UAS
robo embryos, Robo protein begins to accumulate throughout the CNS after stage 14, reaching significant levels (but still below wild type) by stage 16. Interestingly, in these transgenic embryos, although we observe some Robo-positive axons in the commissures at later stages, Robo expression remains higher in longitudinal tracts. We interpret the Robo-positive axons in the commissures as later axons following misguided pioneer axons;
fasciculation with the pioneers allows these Robo-positive axons to cross the midline in spite of modest levels of Robo.
The elav-GAL4 line also expresses pan-neurally but only in post-mitotic neurons; it begins driving expression of UAS transgenes during stage 12 and remains expressed throughout the rest of embryogenesis. Ectopic expression of comm by elav-GAL4 led to a less severe version of the robo phenotype. We interpret this weaker phenotype as being due to either a weaker overall level of Comm expression or because increased Comm initiates after the pioneers have already established the initial pathways. In addition, since sca-GAL4 drives expression in midline glia, the source of normal comm expression, while elav-GAL4 does not drive expression in the midline glia, the possibility exists that the less severe phenotype of the elav transgene is due to this lack of midline comm expression.
To address this issue, we attempted to increase the level of Comm specifically at the midline using multiple GAL4 lines, including sim-GAL4, slit-GAL4, F63-GAL4, and p52A-GAL4, all of which express at the midline during the period of commissure formation. When UAS comm was expressed by any of these four lines, only very weak BP102 phenotypes were observed, although because most of these inserts are homozygous lethal, we have not been able to easily increase the dosage with these lines to comparable levels as with the sca-GAL4 line. None of these gain-of function phenotypes was as strong as that observed with the sca-GAL4 line. We also cannot rule out that these differences in the strength of the gain-of function phenotypes using different GAL4 lines do not reflect differences in timing, levels of expression, or location of expression within the CNS.
We conclude that the normal function of comm is to down-regulate the low level of Robo expression present on commissural axons, thereby allowing them to cross the midline.
Increasing levels of Comm in the CNS lead to more severe robo-Iike phenotypes, indicating a dosage sensitivity. This sensitivity to dosage is also reflected in the behavior of Ap axons in robo heterozygotes, thus showing a parallel dosage sensitivity by either decreasing Robo or increasing Coxnm.
These results indicate that control of Robo expression is complex and highly regulated from transcription to translation to post-translational. We show that there is an inverse correlation between Comm expression and Robo expression. In wild type embryos, Comm is expressed at the midline, and Robo expression is very low on commissural axons crossing the midline. In comm hypomorphic mutant embryos, those few axons that do cross the midline now express higher levels of Robo protein. In comm gain-of function embryos (using transgenic constructs that drive over- and ectopic expression of comm), the overall levels of Robo are dramatically decreased wherever increased Comm expression coincides with Robo expression. Furthenmcre, using certain GAL4 lines that drive transient comm expression, we observe that once Comm disappears in older embryos, Robo protein expression begins to increase towards its normal levels. Thus, Comm down-regulates Robo expression in a very fight fashion.
Only a small amount of Comm is normally expressed at the midline. The midline also expresses high levels of a putative repellent that is the Iigand for the Robo receptor. Growth cones that express high levels of Robo, such as ipsilaterally projecting growth cones from the outset or commissural growth cones once they cross the midline, are relatively immune to significant down-regulation by the normally low levels of midline Comm and thus are prevented from crossing the midline. Only abnormally high levels of Comm (using transgenes that drive overexpression) are sufficient to down-regulate this Robo expression to a level that allows these growth cones to cross the midline. In contrast, growth cones that normally express lower levels of Robo (i.e., those commissural growth cones that cross the midline in the presence of Comm) are highly sensitive to Comm, in that the normal low levels of Comm can further reduce their levels of Robo and thus allow them to cross the midline. In the absence of Comm, these growth cones can not cross the midline, due to their Iow levels of Robo; in the robo; comm double mutant they all freely cross.
Genetic Stocks. All robo alleles were isolated on chromosomes deficient for Fasciclin III as described in Seeger et al., 1993. The robo phenotype is independent of the absence of Faslll.
Protein Immunocytochemistry. Immunocytochemistry was performed as described by Patel (1994). For anti-Robo staining, MAb 13C9 was diluted 1:10 in PBS with 0.1% Tween-20, and the embryos were fixed and cracked so as to minimize exposure to methanol. The presence of triton and storage of embryos in methanol were both found to destroy the activity of MAb 13C9. For anti-Connectin staining with MAb C1.427, the embryos were fixed in 3.7% formaldehyde/PEM buffer (100mM PIPES, 2mM EGTA, 1mM MgS04); C1.427 was diluted 1:10 in PBS with 0.1 % Triton. The apterous-tau-IacZ embryos were hand devitellinized and dissected on poly-lysine coated slides and subsequently fixed for 20 minutes with 3.7% formaldehyde; rabbit anti-~3-galactosidase (Cappell) was used at 1:10,000 and biotinylated anti-rabbit secondary was used at 1:1000 and enhanced with the Vectastain Elite ABC kit (Vector Laboratories).
Transformation of Drosophila, robo rescue and overexpression. The comm cDNA
was inserted as a 1.7kb XhoI-XbaI fragment into the XhoI and Xba sites of pUAST
(Brand and Perrimon, 1993). Transformant lines were generated and mapped by standard procedures.
References Brand, A. H. and Pernmon, N. (1993) Development 118, 401-415.
Hidalgo, A., and Brand, A.H. (1997) Development 124, 3253-3262.
Kidd, T., Brose, K., Mitchell, K., Fetter, R., Tessier-Lavigne, M., Goodman, C.S., and Tear, G. (1997). Roundabout controls axon crossing of the CNS midline and defines a new subfamily of evolutionarily conserved guidance receptors. Cell, in review.
Kl~mbt, C., Jacobs, J. R., and Goodman, C. S. (1991) Cell 64, 801-815.
Lundgren, S.E., et al. (1995) Development 121, 1769-1773.
Mayer, U. and Niisslein-Volhard, C. (1988) Genes Dev. 2, 1496 - 1511.
Meadows, L.A., et al. (1994) J. Cell Sci. 107, 321-328.
Nose, A., Mahajan, V. B., and Goodman, C. S. (1992) Cell 70, 553-567.
Patel, N. H. (1994) In "Methods in Cell Biology, Vol 44. Drosophila melanogaster: Practical Uses in Cell Biology" (L. S. B. Goldstein and E. Fyrberg, eds) Academic Press, New York.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A
Laboratory Manual (Cold Spring ITarbor, New York: Cold Spring Harbor Laboratory).

Seeger, M., Tear, G., Ferres-Marco, D. and Goodman C.S. (1993) Neuron 10, 409 -426.
Tear G., et al. (1996) Neuron 16, 501 - 514.
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

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3S (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4188 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear 4O (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

SUBSTITUTE SHEET (RULE 26) GATCCGCCGC CGAAAGTGTTGTGGAAAA.AGGAGGAGGGCAATATTCCGGTGTCCAGAGCG900 SUBSTITUTE SHEET (RULE 26) (2) INFORMATION SEQ ID
FOR N0:2:

(i) SEQUENCE TERISTICS:
CHARAC

(A) LENGTH : 95 acids 13 amino (B) TYPE: amino id ac (C) STRANDEDNE SS:single (D) TOPOLOGY: linear (ii) MOLECULE PE: peptide TY

IO (xi) SEQUENCE PTION: :2:
DESCRI SEQ
ID

Met His ProMetHis ProGluAsn HisAlaIle AlaArgSer ThrSer Thr Thr AsnAsnPro SerArgSer ArgSerSer ArgMetTrp LeuLeu IS Pro Ala TrpLeuLeu LeuValLeu ValAlaSer AsnGlyLeu ProAla Val Arg GlyGlnTyr GlnSerPro ArgIleIle GluHisPro ThrAsp Leu Val ValLysLys AsnGluPro AlaThrLeu AsnCysLys ValGlu Gly Lys ProGluPro ThrIleGlu TrpPheLys AspGlyGlu ProVal Ser Thr AsnGluLys LysSerHis ArgValGln PheLysAsp GlyAla 25 Leu Phe PheTyrArg ThrMetGln GlyLysLys GluGlnAsp GlyGly Glu Tyr TrpCysVal AlaLysAsn ArgValGly GlnAlaVal SerArg His Ala SerLeuGln IleAlaVal LeuArgAsp AspPheArg ValGlu Pro Lys AspThrArg ValAlaLys GlyGluThr AlaLeuLeu GluCys Gly Pro ProLysGly IleProGlu ProThrLeu IleTrpIle LysAsp 3S Gly Val ProLeuAsp AspLeuLys AlaMetSer PheGlyAla SerSer Arg Val ArgIleVal AspGlyGly AsnLeuLeu IleSerAsn ValGlu Pro Ile AspGluGly AsnTyrLys CysIleAla GlnAsnLeu ValGly Thr Arg GluSerSer TyrAlaLys LeuIleVal GlnValLys ProTyr Phe Met LysGluPro LysAspGln ValMetLeu TyrGlyGln ThrAla 45 Thr Phe HisCysSer ValGlyGly AspProPro ProLysVal LeuTrp Lys Lys GluGluGly AsnIlePro ValSerArg AlaArgIle LeuHis Asp Glu LysSerLeu GluIleSer AsnIleThr ProThrAsp GluGly Thr Tyr ValCysGlu AlaHisAsn AsnValGly GlnIleSer AlaArg Ala Ser LeuIleVal HisAlaPro ProAsnPhe ThrLysArg ProSer SS Asn Lys LysValGly LeuAsnGly ValValGln LeuProCys MetAla SUBSTITUTE SHEET (RULE 26) Ser GlyAsnPro ProProSer ValPheTrp ThrLysGluGly ValSer Thr LeuMetPhe ProAsnSer SerHisGly ArgGlnTyrVal AlaAla $ 385 390 395 400 Asp GlyThrLeu GlnIleThr AspValArg GlnGluAspGlu GlyTyr Tyr ValCysSer AlaPheSer ValValAsp SerSerThrVal ArgVal 1~ Phe LeuGlnVal SerSerVal AspGluArg ProProProIle IleGln Ile GlyProAla AsnGlnThr LeuProLys GlySerValAla ThrLeu Pro CysArgAla ThrGlyAsn ProSerPro ArgIleLysTrp PheHis Asp GlyHisAla ValGlnAla GlyAsnArg TyrSerIleIle GlnGly Ser SerLeuArg ValAspAsp LeuGlnLeu SerAspSerGly ThrTyr Thr CysThrAla SerGlyGlu ArgGlyGlu ThrSerTrpAla AlaThr Leu ThrValGlu LysProGly SerThrSer LeuHisArgAla AlaAsp Pro SerThrTyr ProAlaPro ProGlyThr ProLysValLeu AsnVal Ser ArgThrSer IleSerLeu ArgTrpAla LysSerGlnGlu LysPro Gly AlaValGly ProIleIle GlyTyrThr ValGluTyrPhe SerPro Asp LeuGlnThr GlyTrpIle ValAlaAla HisArgValGly AspThr Gln ValThrIle SerGlyLeu ThrProGly ThrSerTyrVal PheLeu Val ArgAlaGlu AsnThrGln GlyIleSer ValProSerGly LeuSer Asn ValIleLys ThrIleGlu AlaAspPhe AspAlaAlaSer AlaAsn Asp LeuSerAla AlaArgThr LeuLeuThr GlyLysSerVal GluLeu Ile AspAlaSer AlaIleAsn AlaSerAla ValArgLeuGlu TrpMet Leu HisValSer AlaAspGlu LysTyrVal GluGlyLeuArg IleHis Tyr LysAspAla SerValPro SerAlaGln TyrHisSerIle ThrVal 4$ 705 710 715 720 Met AspAlaSer AlaGluSer PheValVal GlyAsnLeuLys LysTyr Thr LysTyrGlu PhePheLeu ThrProPhe PheGluThrIle GluGly 50 Gln ProSerAsn SerLysThr AlaLeuThr TyrGluAspVal ProSer Ala ProProAsp AsnIleGln IleGlyMet TyrAsnGlnThr AlaGly Trp ValArgTrp ThrProPro ProSerGln HisHisAsnGly AsnLeu $5 785 790 795 800 SUBSTITUTE SHEET (RULE 26) Tyr Gly Tyr Lys Ile Glu Val Ser Ala Gly Asn Thr Met Lys Val Leu Ala AsnMet ThrLeu AsnAlaThrThr ThrSer ValLeuLeu AsnAsn Leu ThrThr GlyAla ValTyrSerVal ArgLeu AsnSerPhe ThrLys Ala GlyAsp GlyPro TyrSerLysPro IleSer LeuPheMet AspPro Thr HisHis ValHis ProProArgAla HisPro SerGlyThr HisAsp 1~ 865 870 875 8g0 Gly ArgHis GluGly GlnAspLeuThr TyrHis AsnAsnGly AsnIle Pro ProGly AspIle AsnProThrThr HisLys LysThrThr AspTyr Leu SerGly ProTrp LeuMetValLeu ValCys IleValLeu LeuVal Leu ValIle SerAla AlaIleSerMet ValTyr PheLysArg LysHis Gln MetThr LysGlu LeuGlyHisLeu SerVal ValSerAsp AsnGlu Ile ThrAla LeuAsn IleAsnSerLys GluSer LeuTrpIle AspHis His ArgGly TrpArg ThrAlaAspThr AspLys AspSerGly LeuSer 2$ Glu SerLys LeuLeu SerHisValAsn SerSer GlnSerAsn TyrAsn Asn SerAsp GlyGly ThrAspTyrAla GluVal AspThrArg AsnLeu Thr ThrPhe TyrAsn CysArgLysSer ProAsp AsnProThr ProTyr Ala ThrThr MetIle IleGlyThrSer SerSer GluThrCys ThrLys Thr ThrSer IleSer AlaAspLysAsp SerGly ThrHisSer ProTyr Ser AspAla PheAla GlyGlnValPro AlaVal ProValVal LysSer , Asn TyrLeu GlnTyr ProValGluPro IleAsn TrpSerGlu PheLeu Pro ProPro ProGlu HisProProPro SerSer ThrTyrGly TyrAla Gln GlySer ProGlu SerSerArgLys SerSer LysSerAla GlySer Gly IleSer ThrAsn GlnSerIleLeu AsnAla SerIleHis SerSer Ser SerGly GlyPhe SerAlaTrpGly ValSer ProGlnTyr AlaVal Ala CysPro ProGlu AsnValTyrSer AsnPro LeuSerAla ValAla Gly GlyThr GlnAsn ArgTyrGlnIle ThrPro ThrAsnGln HisPro Pro GlnLeu ProAla TyrPheAlaThr ThrGly ProGlyGly AlaVal Pro ProAsn HisLeu ProPheAlaThr GlnArg HisAlaAla SerGlu Tyr GlnAla GlyLeu AsnAlaAlaArg CysAla GlnSerArg AlaCys SUBSTITUTE SHEET (RULE 26) Asn Ser Cys Asp Ala Leu Thr Pro Ser Pro Met Gln Pro Ala Pro Pro Pro Val Pro Val Pro Glu Trp Tyr Gln Pro Val His Pro Gly Asn Ser His Pro Met His Pro Thr Ser Asn His Gln Ile Tyr Gln Ser Cys Ser Ser Glu Cys Ser Asp His Arg Ser Ser Gln Ser His Lys Ser Arg Gln lO Leu Gln Leu Glu Glu His Ser Ser Ala Lys Gln Arg Gly Gly Gly His His Arg Arg Arg Ala Pro Val Gln Pro Cys Met Glu Ser Val Glu Asn Glu Asn Met Leu Ala Glu Glu Gln Arg Gln Tyr Thr Ser Tyr Asp Cys Cys Asn Ser Ser Arg Glu Asp Thr Cys Ser Cys Ser Glu Gly Gly Ser Cys Leu Tyr Ala Glu Ala Glu Pro Ala Pro Arg Gln Met Gly Thr Ala Lys Asn Thr (2) INFORMATION FOR S EQ ID
N0:3:

(i) SEQUENCE CHARACTERISTICS:

2S (A) LENGTH: 4146 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYP E: cDNA

(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:3:

SUBSTITUTE SHEET (RULE 26) (2) INFORMATION
FOR SEQ
ID N0:4:

(i) SEQUENCE
CHARACTERISTICS:

(A) LENGTH:1381 aminoacids SO (B) TYPE:
amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY:
linear (ii) MOLECULE
TYPE: peptide (xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:4:

SS Gly Glu Glu His Asri Pro Pro Met Arg Ile Asp Thr Ile Thr Val Pro SUBSTITUTE SHEET (RULE 26~

Lys Asn AspProPhe ThrPheAsn CysGlnAla GluGlyAsn ProThr Pro Thr IleGlnTrp PheLysAsp GlyArgGlu LeuLysThr AspThr Gly Ser HisArgIle MetLeuPro AlaGlyGly LeuPhePhe LeuLys Val Ile HisSerArg ArgGluSer AspAlaGly ThrTyrTrp CysGlu 1~ Ala Lys AsnGluPhe GlyValAla ArgSerArg AsnAlaThr LeuGln Val Ala ValLeuArg AspGluPhe ArgLeuGlu ProAlaAsn ThrArg Val Ala GlnGlyGlu ValAlaLeu MetGluCys GlyAlaPro ArgGly Ser Pro GluProGln IleSerTrp ArgLysAsn GlyGlnThr LeuAsn Leu Val GlyAsnLys ArgIleArg IleValAsp GlyGlyAsn LeuAla Ile Gln GluAlaArg GlnSerAsp AspGlyArg TyrGlnCys ValVal Lys Asn ValValGly ThrArgGlu SerAlaThr AlaPheLeu LysVal His Val ArgProPhe LeuIleArg GlyProGln AsnGlnThr AlaVal Val Gly SerSerVal ValPheGln CysArgIle GlyGlyAsp ProLeu Pro Asp ValLeuTrp ArgArgThr AlaSerGly GlyAsnMet ProLeu 30 Arg Lys PheSerTrp LeuHisSer AlaSerGly ArgValHis ValLeu Glu Asp ArgSerLeu LysLeuAsp AspValThr LeuGluAsp MetGly Glu Tyr ThrCysGlu AlaAspAsn AlaValGly GlyIleThr AlaThr Gly Ile LeuThrVal HisAlaPro ProLysPhe ValIleArg ProLys Asn Gln LeuValGlu IleGlyAsp GluValLeu PheGluCys GlnAla 4U Asn Gly HisProArg ProThrLeu TyrTrpSer ValGluGly AsnSer Ser Leu LeuLeuPro GlyTyrArg AspGlyArg MetGluVal ThrLeu Thr Pro GluGlyArg SerValLeu SerIleAla ArgPheAla ArgGlu Asp Ser GlyLysVal ValThrCys AsnAlaLeu AsnAlaVal GlySer Val Ser SerArgThr ValValSer ValAspThr GlnPheGlu LeuPro Pro Pro IleIleGlu GlnGlyPro ValAsnGln ThrLeuPro ValLys Ser Ile ValValLeu ProCysArg ThrLeuGly ThrProVal ProGln Val Ser TrpTyrLeu AspGlyIle ProIleAsp ValGlnGlu HisGlu SUBSTITUTE SHEET (RULE 26) Arg ArgAsnLeu SerAspAla GlyAlaLeu ThrIleSer AspLeuGln Arg HisGluAsp GluGlyLeu TyrThrCys ValAlaSer AsnArgAsn $ Gly LysSerSer TrpSerGly TyrLeuArg LeuAspThr ProThrAsn Pro AsnIleLys PhePheArg AlaProGlu LeuSerThr TyrProGly Pro ProGlyLys ProGlnMet ValGluLys GlyGluAsn SerValThr Leu SerTrpThr ArgSerAsn LysValGly GlySerSer LeuValGly Tyr ValIleGlu MetPheGly LysAsnGlu ThrAspGly TrpValAla 1$ Val GlyThrArg ValG1nAsn ThrThrPhe ThrGlnThr GlyLeuLeu Pro GlyValAsn TyrPhePhe LeuIleArg AlaGluAsn SerHisGly Leu SerLeuPro SerProMet SerGluPro IleThrVal GlyThrArg Tyr PheAsnSex GlyLeuAsp LeuSerGlu AlaArgAla SerLeuLeu Ser GlyAspVal ValGluLeu SerAsnAla SerValVal AspSerThr 2$ Ser MetLysLeu ThrTrpGln IleIleAsn GlyLysTyr ValGluGly Phe TyrValTyr AlaArgGln LeuProAsn ProIleVal AsnAsnPro Ala ProValThr SerAsnThr AsnProLeu LeuGlySer ThrSerThr Ser AlaSerAla SerAlaSer AlaSerAla LeuIleSer ThrLysPro Asn IleAlaAla AlaGlyLys ArgAspGly GluThrAsn GlnSerGly 3$ Gly GlyAlaPro ThrProLeu AsnThrLys TyrArgMet LeuThrIle Leu AsnGlyGly GlyAlaSer SerCysThr IleThrGly LeuValGln Tyr ThrLeuTyr GluPhePhe IleValPro PheTyrLys SerValGlu Gly LysProSer AsnSerArg IleAlaArg ThrLeuGlu AspValPro Ser GluAlaPro TyrGlyMet GluAlaLeu LeuLeuAsn SerSerAla 4$ Val PheLeuLys TrpLysAla ProGluLeu LysAspArg HisGlyVal Leu LeuAsnTyr HisValIle ValArgGly IleAspThr AlaHisAsn Phe SerArgIle LeuThrAsn ValThrIle AspAlaAla SerProThr Leu ValLeuAla AsnLeuThr GluGlyVal MetTyrThr ValGlyVal Ala AlaGlyAsn AsnAlaGly ValGlyPro TyrCysVal ProAlaThr $$ Leu ArgLeuAsp ProIleThr LysArgLeu AspProPhe IleAsnGln SUBSTITUTE SHEET (RULE 26) Arg AspHis ValAsn AspValLeu ThrGlnProTrp PheIleIle Leu Leu GlyAla IleLeu AlaValLeu MetLeuSerPhe GlyAlaMet Val Phe ValLys ArgLys HisMetMet MetLysGlnSer AlaLeuAsn Thr Met ArgGly AsnHis ThrSerAsp ValLeuLysMet ProSerLeu Ser Ala ArgAsn GlyAsn GlyTyrTrp LeuAspSerSer ThrGlyGly Met Val TrpArg ProSer ProGlyGly AspSerLeuGlu MetGlnLys Asp His IleAla AspTyr AlaProVal CysGlyAlaPro GlySerPro Ala Gly GlyGly ThrSer SerGlyGly SerGlyGlyAla GlySerGly Ala Ser GlyGly AspAsp IleHisGly GlyHisGlySer GluArgAsn Gln Gln ArgTyr ValGly GluTyrSer AsnIleProThr AspTyrAla Glu Val SerSer PheGly LysAlaPro SerGluTyrGly ArgHisGly Asn Ala SerPro AlaPro TyrAlaThr SerSerIleLeu SerProHis Gln Gln GlnGln GlnGln GlnProArg TyrGlnGlnArg ProValPro Gly Tyr GlyLeu GlnArg ProMetHis ProHisTyrGln GlnGlnGln His Gln GlnGln GlnAla GlnGlnThr HisGlnGlnHis GlnAlaLeu Gln Gln HisGln GlnLeu ProProSer AsnIleTyrGln GlnMetSer Thr Thr SerGlu IleTyr ProThrAsn ThrGlyProSer ArgSerVal Tyr Ser GluGln TyrTyr TyrProLys AspLysGlnArg HisIleHis Ile Thr GluAsn LysLeu SerAsnCys HisThrTyrGlu AlaAlaPro Gly Ala LysGln SerSer ProIleSer SerGlnPheAla SerValArg Arg Gln GlnLeu ProPro AsnCysSer IleGlyArgGlu SerAlaArg Phe Lys ValLeu AsnThr AspGlnGly LysAsnGlnGln AsnLeuLeu Asp 1235 1240. 1245 Leu AspGly SerSer MetCysTyr AsnGlyLeuAla AspSerGly Cys Gly GlySer ProSer ProMetAla MetLeuMetSer HisGluAsp Glu His AlaLeu TyrHis ThrAlaAsp GlyAspLeuAsp AspMetGlu Arg Leu TyrVal LysVal AspGluGln GlnProProGln GlnGlnGln Gln Leu IlePro LeuVal ProGlnHis ProAlaGluGly HisLeuGln Ser SUBSTITUTE SHEET (RULE 26j WO 99/25833 PCT/US98l24327 Trp Arg Ser Ser Arg Lys Asn Gln Asn Gly Gln Glu Ser Thr Cys Arg Ile Lys Ile Tyr Ala Pro Glu Pro Gly Ser Val Ala Ser Glu Ser Leu S Glu Arg Ser Gly Ser Gly Ser Leu Thr Ser Ser Gln Leu Ser Pro Asn Ala G ly His Asn Val lO (2) INFORMATION
FOR SEQ
ID N0:5:

(i) S EQUENCE CHARACTERISTICS:

(A) LENGTH: 3894 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
double 1S (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: cDNA

(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:5:

GCTCCCTCCA CATCTGGCGC AGGACCAATCACTGGTTATA TCATTCAGTACTACAGTCCA 1$00 SUBSTITUTE SHEET (RULE 26j (2) INFORMATION
FOR
SEQ
ID N0:6:

3O (i) SEQUENCE
CHARACTERISTICS:

(Ay LENGTH: 1297 acids amino (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear 3$ (ii) MOLECULE
TYPE:
peptide (xi) SEQUENCE
DESCRIPTION:
SEQ
ID
N0:6:

Met Tyr Tyr Leu Gly Phe His ThrHisThr HisThrHis ThrTyr Tyr Ile Asn Phe Asp Lys Ile Asn AlaSerAsn LeuAlaPro ValIle Pro Ile Glu His Pro Ile Asp Val ValSerArg GlySerPro AlaThr Val Leu Asn Cys Gly Ala Lys Ser ThrAlaLys IleThrTrp TyrLys Pro 4$ Asp Gly Gln Pro Val Ile Asn LysGluGln ValAsnSer HisArg Thr Ile Val Leu Asp Thr Gly Leu PheLeuLeu LysValAsn SerGly Ser Lys Asn Gly Lys Asp Ser Ala GlyAlaTyr TyrCysVal AlaSer Asp $0 100 105 110 Asn Glu His Gly Glu Val Ser AsnGluGly SerLeuLys LeuAla Lys Met Leu Arg Glu Asp Phe Val ArgProArg ThrValGln AlaLeu Arg $$ Gly Gly Glu Met Ala Val Glu CysSerPro ProArgGly PhePro Leu SUBSTITUTE SHEET (RULE 26) Glu Pro Val Val Ser Trp Arg Lys Asp Asp Lys Glu Leu Arg Ile Gln Asp Met ProArg TyrThrLeuHis SerAspGly AsnLeuIle IleAsp Pro Val AspArg SerAspSerGly ThrTyrGln CysValAla AsnAsn Met Val GlyGlu ArgValSerAsn ProAlaArg LeuSerVal PheGlu Lys Pro LysPhe GluGlnGluPro LysAspMet ThrValAsp ValGly Ala Ala ValLeu PheAspCysArg ValThrGly AspProGln ProGln Ile Thr TrpLys ArgLysAsnGlu ProMetPro ValThrArg AlaTyr Ile Ala LysAsp AsnArgGlyLeu ArgIleGlu ArgValGln ProSer Asp Glu GlyGlu TyrValCysTyr AlaArgAsn ProAlaGly ThrLeu ZU Glu Ala SerAla HisLeuArgVal GlnAlaPro ProSerPhe GlnThr Lys Pro AlaAsp GlnSerValPro AlaGlyGly ThrAlaThr PheGlu Cys Thr LeuVal GlyGlnProSer ProAlaTyr PheTrpSer LysGlu Gly Gln GlnAsp LeuLeuPhePro SerTyrVal SerAlaAsp GlyArg Thr Lys ValSer ProThrGlyThr LeuThrIle GluGluVal ArgGln Val Asp GluGly AlaTyrValCys AlaGlyMet AsnSerAla GlySer Ser Leu SerLys AlaAlaLeuLys AlaThrPhe GluThrLys GlyArg Val Gln LysLys LysSerLysMet GlyLysGln LysGlnLys AsnVal Gln Ser IleIle LysTyrLeuIle SerAlaVal ThrGlyAsn ThrPro Ala Lys ProPro ProThrIleGlu HisGlyHis GlnAsnGln ThrLeu Met Val GlySer SerAlaIleLeu ProCysGln AlaSerGly LysPro Thr Pro GlyIle SerTrpLeuArg AspGlyLeu ProIleAsp IleThr Asp Ser ArgIle SerGlnHisSer ThrGlySer LeuHisIle AlaAsp Leu Lys LysPro AspThrGlyVal TyrThrCys IleAlaLys AsnGlu Asp Gly GluSer ThrTrpSerAla SerLeuThr ValGluAsp HisThr Ser Asn AlaGln PheValArgMet ProAspPro SerAsnPhe ProSer Ser Pro ThrGln ProIleIleVal AsnValThr AspThrGlu ValGlu Leu His TrpAsn AlaProSerThr SerGlyAla GlyProIle ThrGly SUBSTITUTE SHEET (RULE 26) Tyr Ile Ile Gln Tyr Tyr Ser Pro Asp Leu Gly Gln Thr Trp Phe Asn Ile ProAsp TyrValAla SerThrGlu TyrArgIle LysGlyLeu Lys $ Pro SerHis SerTyrMet PheValIle ArgAlaGlu AsnGluLys Gly Ile GlyThr ProSerVal SerSerAla LeuValThr ThrSerLys Pro Ala AlaGln ValAlaLeu SerAspLys AsnLysMet AspMetAla Ile Ala GluLys ArgLeuThr SerGluGln LeuIleLys LeuGluGlu Val Lys ThrIle AsnSerThr AlaValArg LeuPheTrp LysLysArg Lys 1$ Leu GluGlu LeuIleAsp GlyTyrTyr IleLysTrp ArgGlyPro Pro Arg ThrAsn AspAsnGln TyrValAsn ValThrSer ProSerThr Glu Asn TyrVal ValSerAsn LeuMetPro PheThrAsn TyrGluPhe Phe Val IlePro TyrHisSer GlyValHis SerIleHis GlyAlaPro Ser Asn SerMet AspValLeu ThrAlaGlu AlaProPro SerLeuPro Pro 2$ Glu AspVal ArgIleArg MetLeuAsn LeuThrThr LeuArgIle Ser Trp LysAla ProLysAla AspGlyIle AsnGlyIle LeuLysGly Phe Gln IleVal IleValGly GlnAlaPro AsnAsnAsn ArgAsnIle Thr 820 $25 830 Thr AsnGlu ArgAlaAla SerValThr LeuPheHis LeuValThr Gly Met ThrTyr LysIleArg ValAlaAla ArgSerAsn GlyGlyVal Gly 3$ Val SerHis GlyThrSer GluValIle MetAsnGln AspThrLeu Glu Lys HisLeu AlaAlaGln GlnGluAsn GluSerPhe LeuTyrGly Leu Ile AsnLys SerHisVal ProValIle ValIleVal AlaIleLeu Ile Ile PheVal ValIleIle IleAlaTyr CysTyrTrp ArgAsnSer Arg Asn SerAsp GlyLysAsp ArgSerPhe IleLysIle AsnAspGly Ser 4$ Val HisMet AlaSerAsn AsnLeuTrp AspValAla GlnAsnPro Asn Gln AsnPro MetTyrAsn ThrAlaGly ArgMetThr MetAsnAsn Arg Asn GlyGln AlaLeuTyr SerLeuThr ProAsnAla GlnAspPhe Phe $0 980 985 990 Asn AsnCys AspAspTyr SerGlyThr MetHisArg ProGlySer Glu His HisTyr HisTyrAla GlnLeuThr GlyGlyPro GlyAsnAla Met $$ Ser ThrPhe TyrGlyAsn GlnTyrHis AspAspPro SerProTyr Ala SUBSTITUTE SHEET (RULE 26) Thr ThrThrLeu ValLeuSer AsnGlnGln ProAlaTrp LeuAsnAsp Lys MetLeuArg AlaProAla MetProThr AsnProVal ProProGlu Pro ProAlaArg TyrAlaAsp HisThrAla GlyArgArg SerArgSer Ser ArgAlaSer AspGlyArg GlyThrLeu AsnGlyGly LeuHisHis Arg ThrSerGly SerGlnArg SerAspSer ProProHis ThrAspVal Ser TyrValGln LeuHisSer SerAspGly ThrGlySer SerLysGlu Arg ThrGlyGlu ArgArgThr ProProAsn LysThrLeu MetAspPhe Ile ProProPro ProSerAsn ProProPro ProGlyGly HisValTyr Asp ThrAlaThr ArgArgGln LeuAsnArg GlySerThr ProArgGlu Asp ThrTyrAsp SerValSer AspGlyAla PheAlaArg ValAspVal Asn AlaArgPro ThrSerArg AsnArgAsn LeuGlyGly ArgProLeu Lys GlyLysArg AspAspAsp SerGlnArg SerSerLeu MetMetAsp Asp AspGlyGly SerSerGlu AlaAspGly GluAsnSer GluGlyAsp Val ProArgGly GlyValArg LysAlaVal ProArgMet GlyIleSer Ala SerThrLeu AlaHisSer CysTyrGly ThrAsnGly ThrAlaGln Arg PheArgSer IleProArg AsnAsnGly IleValThr GlnGluGln Thr (2) INFORMATION FOR SEQ ID N0:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4956 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:

SUBSTITUTE SHEET (RULE 26) TGTCTTAGAG

CAGTGCAGAA

AGATGATGGA

TAGGAAGGTG

CAAAGCTGAA

CCGTGACCAG

TCCTCAACCA

ACCACCACAG

TGTCCAGCGA

lO TCTGATGTTG GTTATTACAT CTGCCAGACT TTAAATGTTG CTGGAAGCAT 1320 CATCACAAAG

TCGACAAGGT

GGCCACAGGC

CCAAGACTCT

GGGTGATACT

TGCTTACATT

AAATTTAATC

CACATTATCG

AGCCTTCAGC

AACATCTGCC

TAATGCATAT

CCTACCAACA

TCTGCACCTC

AGATCAACAG

CCACGGAGAA

AATCCCTGAT

ATTTCAAGGA

CCCACCCCAA

TTGGCAGCCA

TCTGGGCAAT

GGTCATTCCC

GGCTGGGTCT

TGTGTCACCT

GGCCTTCATA

GCTTTATCGA

AGTCCCGTCT

CAGTGGAGGG

AGACACGTGG

AGGCAATGGA

AAATTATAAC

TTATGGTGAT

TCTGAAGGAT

GCTCATCCAG

GCACTGGAAA

GGAGCAAAAC

ATACAACCAA

TAGTACATCT

GGGTGGCATG

CAATAGCGAA

CGTGCCACCA

AGGCCCCACT

TCAGTCCACT

TTGTCCAGAG

TCCTCCTCCA

CCTGGTCTCA

GGTAGCCAAG

SUBSTITUTE SHEET (RULE 26) (2) INFORMATION FORSEQID
N0:8:

(i) SEQUENCE TERISTICS:
CHARAC

ZO (A)LENGTH : 51 acids 16 amino (B)TY PE:amino acid (C)STRANDEDNE SS:
single (D)TO POLOGY: linear (ii) MOLECULE PE:peptide TY

ZS {xi} SEQUENCE SCRIPTION:
DE SEQ
ID
N0:8:

Met LysTrp LysHisVal ProPheLeu ValMetIle SerLeuLeu Ser Leu SerPro AsnHisLeu PheLeuAla GlnLeuIle ProAspPro Glu 30 Asp ValGlu ArgGlyAsn AspHisGly ThrProIle ProThrSer Asp Asn AspAsp AsnSerLeu GlyTyrThr GlySerArg LeuArgGln Glu Asp PhePro ProArgIle ValGluHis ProSerAsp LeuIleVal Ser Lys GlyGlu ProAlaThr LeuAsnCys LysAlaGlu GlyArgPro Thr Pro ThrIle GluTrpTyr LysGlyGly GluArgVal GluThrAsp Lys 4O Asp AspPro ArgSerHis ArgMetLeu LeuProSer GlySerLeu Phe Phe LeuArg IleValHis GlyArgLys SerArgPro AspGluGly Val Tyr ValCys ValAlaArg AsnTyrLeu GlyGluAla ValSerHis Asn Ala SerLeu GluValAla IleLeuArg AspAspPhe ArgGlnAsn Pro Ser AspVal MetValAla ValGlyGlu ProAlaVal MetGluCys Gln SO Pro ProArg GlyHisPro GluProThr IleSerTrp LysLysAsp Gly Ser ProLeu AspAspLys AspGluArg IleThrIle ArgGlyGly Lys Leu MetIle ThrTyrThr ArgLysSer AspAlaGly LysTyrVal Cys SUBSTITUTE SHEET (RULE 26j WO 99!25833 PCT/US98/24327 Val Gly Thr Asn Met Val Gly Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val LeuGluArg ProSerPhe ValLysArg ProSerAsn LeuAla $ Val Thr ValAspAsp SerAlaGlu PheLysCys GluAlaArg GlyAsp Pro Val ProThrVal ArgTrpArg LysAspAsp GlyGluLeu ProLys Ser Arg TyrGluIle ArgAspAsp HisThrLeu LysIleArg LysVal Thr Ala GlyAspMet GlySerTyr ThrCysVal AlaGluAsn MetVal Gly Lys AlaGluAla SerAlaThr LeuThrVal GlnGluPro ProHis 1$ Phe Val ValLysPro ArgAspGln ValValAla LeuGlyArg ThrVal Thr Phe GlnCysGlu AlaThrGly AsnProGln ProAlaIle PheTrp Arg Arg GluGlySer GlnAsnLeu LeuPheSer TyrGlnPro ProGln Ser Ser SerArgPhe SerValSer GlnThrGly AspLeuThr IleThr Asn Val GlnArgSer AspValGly TyrTyrIle CysGlnThr LeuAsn 2$ Val Ala GlySerIle IleThrLys AlaTyrLeu GluValThr AspVal Ile Ala AspArgPro ProProVal IleArgGln GlyProVal AsnGln Thr Val AlaValAsp GlyThrPhe ValLeuSer CysValAla ThrGly Ser Pro ValProThr IleLeuTrp ArgLysAsp GlyValLeu ValSer Thr Gln AspSerArg IleLysGln LeuGluAsn GlyValLeu GlnIle 3$ Arg Tyr AlaLysLeu GlyAspThr GlyArgTyr ThrCysIle AlaSer Thr Pro SerGlyGlu AlaThrTrp SerAlaTyr IleGluVal GlnGlu Phe Gly ValProVal GlnProPro ArgProThr AspProAsn LeuIle Pro Ser AlaProSer LysProGlu ValThrAsp ValSerArg AsnThr Val Thr LeuSerTrp GlnProAsn LeuAsnSer GlyAlaThr ProThr 4$ Ser Tyr IleIleGlu AlaPheSer HisAlaSer GlySerSer TrpGln Thr Val AlaGluAsn ValLysThr GluThrSer AlaIleLys GlyLeu Lys Pro AsnAlaIle TyrLeuPhe LeuValArg AlaAlaAsn AlaTyr $0 625 630 635 640 Gly Ile SerAspPro SerGlnIle SerAspPro ValLysThr GlnAsp Val Leu ProThrSer GlnGlyVal AspHisLys GlnVal-GlnArgGlu $$ Leu Gly AsnAlaVal LeuHisLeu HisAsnPro ThrValLeu SerSer SUBSTITUTE SHEET (RULE 26) Ser SerIleGluVal HisTrpThr ValAsp GlnGlnSer GlnTyrIle Gln GlyTyrLysIle LeuTyrArg ProSer GlyAlaAsn HisGlyGlu Ser AspTrpLeuVal PheGluVal ArgThr ProAlaLys AsnSerVal Val IleProAspLeu ArgLysGly ValAsn TyrGluIle LysAlaArg 1~ Pro PhePheAsnGlu PheGlnGly AlaAsp SerGluIle LysPheAla Lys ThrLeuGluGlu AlaProSer AlaPro ProGlnGly ValThrVal Ser LysAsnAspGly AsnGlyThr AlaIle LeuValSer TrpGlnPro Pro ProGluAspThr GlnAsnGly MetVal GlnGluTyr LysValTrp Cys LeuGlyAsnGlu ThrArgTyr HisIle AsnLysThr ValAspGly Ser ThrPheSerVal ValIlePro PheLeu ValProGly IleArgTyr Ser ValGluValAla AlaSerThr GlyAla GlySerGly ValLysSer Glu ProGlnPheIle GlnLeuAsp AlaHis GlyAsnPro ValSerPro Glu AspGlnValSer LeuAlaGln GlnIle SerAspVal ValLysGln Pro AlaPheIleAla GlyIleGly AlaAla CysTrpIle IleLeuMet 3~ Val PheSerIleTrp LeuTyrArg HisArg LysLysArg AsnGlyLeu Thr SerThrTyrAla GlyIleArg LysVal ProSerPhe ThrPheThr Pro ThrValThrTyr GlnArgGly GlyGlu AlaValSer SerGlyGly Arg ProGlyLeuLeu AsnIleSer GluPro AlaAlaGln ProTrpLeu Ala AspThrTrpPro AsnThrGly AsnAsn HisAsnAsp CysSerIle 4U Ser CysCysThrAla GlyAsnGly AsnSer AspSerAsn LeuThrThr Tyr SerArgProAla AspCysIle AlaAsn TyrAsnAsn GlnLeuAsp Asn LysGlnThrAsn LeuMetLeu ProGlu SerThrVal TyrGlyAsp Val AspLeuSerAsn LysIleAsn GluMet LysThrPhe AsnSerPro Asn LeuLysAspGly ArgPheVal AsnPro SerGlyGln ProThrPro Tyr AlaThrThrGln LeuIleGln SerAsn LeuSerAsn AsnMetAsn Asn GlySerGlyAsp SerGlyGlu LysHis TrpLysPro LeuGlyGln Gln LysGlnGluVal AlaProVal GlnTyr AsnIleVal GluGlnAsn SUBSTITUTE SHEET (RULE 26) Lys LeuAsnLys AspTyr ArgAlaAsn AspThrVal ProProThrIle Pro TyrAsnGln SerTyr AspGlnAsn ThrGlyGly SerTyrAsnSer S Ser AspArgGly SerSer ThrSerGly SerGlnGly HisLysLysGly Ala ArgThrPro LysVal ProLysGln GlyGlyMet AsnTrpAlaAsp Leu LeuProPro ProPro AlaHisPro ProProHis SerAsnSerGlu Glu TyrAsnIle SerVal AspGluSer TyrAspGln GluMetProCys Pro ValProPro AlaArg MetTyrLeu GlnGlnAsp GluLeuGluGlu Glu GluAspGlu ArgGly ProThrPro ProValArg GlyAlaAlaSer Ser ProAlaAla ValSer TyrSerHis GlnSerThr AlaThrLeuThr Pro SerProGln GluGlu LeuGlnPro MetLeuGln AspCysProGlu Glu ThrGlyHis MetGln HisGlnPro AspArgArg ArgGlnProVal Ser ProProPro ProPro ArgProIle SerProPro HisThrTyrGly Tyr IleSerGly ProLeu ValSerAsp MetAspThr AspAlaProGlu Glu GluGluAsp GluAla AspMetGlu ValAlaLys MetGlnThrArg Arg LeuLeuLeu ArgGly LeuGluGln ThrProAla SerSerValGly Asp LeuGluSer SerVal ThrGlySer MetIleAsn GlyTrpGlySer Ala SerGluGlu AspAsn IleSerSer GlyArgSer SerValSerSer Ser AspGlySer PhePhe ThrAspAla AspPheAla GlnAlaValAla Ala AlaAlaGlu TyrAla GlyLeuLys ValAlaArg ArgGlnMetGln Asp AlaAlaGly ArgArg HisPheHis AlaSerGln CysProArgPro Thr SerProVal SerThr AspSerAsn MetSerAla AlaValMetGln Lys ThrArgPro AlaLys LysLeuLys HisGlnPro GlyHisLeuArg Arg GluThrTyr ThrAsp AspLeuPro ProProPro ValProProPro Ala IleLysSer ProThr AlaGlnSer LysThrGln LeuGluValArg Pro ValValVal ProLys LeuProSer MetAspAla ArgThrAspArg Ser SerAspArg LysGly SerSerTyr LysGlyArg GluValLeuAsp Gly ArgGlnVal ValAsp MetArgThr AsnProGly AspProArgGlu SS Ala GlnGluGln GlnAsn AspGlyLys GlyArgGly AsnLysAlaAla SUBSTITUTE SHEET (RULE 26) Lys Arg Asp Leu Pro Pro Ala Lys Thr His Leu Ile Gln Glu Asp Ile Leu Pro Tyr Cys Arg Pro Thr Phe Pro Thr Ser Asn Asn Pro Arg Asp Pro Ser Ser Ser Ser Ser Met Ser Ser Arg Gly Ser Gly Ser Arg Gln Arg Glu Gln Ala Asn Val Gly Arg Arg Asn Ile Ala Glu Met Gln Val lO Leu Gly Gly Tyr Glu Arg Gly Glu Asp Asn Asn Glu Glu Leu Glu Glu Thr Glu Ser IS (2) INFORMATION FOR SEQ ID N0:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1300 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double 2O (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: misc_feature (B) LOCATION: 855..1187 2S (D) OTHER INFORMATION: /note= "N signifies gap in sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:

SO (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 434 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single SS (D) TOPOLOGY: linear SUBSTITUTE SHEET (RULE 26) (ii) MOLECULE TYPE:
peptide (ix) FEATURE:

(A)NAME/KEY:
Modified-site (B)LOCATION: 396 285..

$ (D)OTHER /note= signifies INFORMATION: "Xaa gap in sequence"

(xi) SEQUENCE
DESCRIPTION:
SEQ
ID
NO:10:

Gln IleVal AlaGlnGly ArgThrValThr PhePro CysGluThr Lys Gly AsnPro GlnProAla ValPheTrpGln LysGlu GlySerGln Asn Leu LeuPhe ProAsnGln ProGlnGlnPro AsnSer ArgCysSer Val Ser ProThr GlyAspLeu ThrIleThrAsn IleGln ArgSerAsp Ala 1$ Gly TyrTyr IleCysGln AlaLeuThrVal AlaGly SerIleLeu Ala Lys AlaGln LeuGluVal ThrAspValLeu ThrAsp ArgProPro Pro Ile IleLeu GlnGlyPro AlaAsnGlnThr LeuAla ValAspGly Thr loo l05 110 Ala LeuLeu LysCysLys AlaThrGlyAsp ProLeu ProValIle Ser Trp LeuLys GluGlyPhe ThrPheProGly ArgAsp ProArgAla Thr 2$ Ile GlnGlu GlnGlyThr LeuGlnIleLys AsnLeu ArgIleSer Asp Thr GlyThr TyrThrCys ValAlaThrSer SerSer GlyGluAla Ser Trp SerAla ValLeuAsp ValThrGluSer GlyAla ThrIleSer Lys Asn TyrAsp LeuSerAsp LeuProGlyPro ProSer LysProGln Val Thr AspVal ThrLysAsn SerValThrLeu SerTrp GlnProGly Thr 3$ Pro GlyThr LeuProAla SerAlaTyrIle IleGlu AlaPheSer Gln Ser ValSer AsnSerTrp GlnThrValAla AsnHis ValLysThr Thr Leu TyrThr ValArgGly LeuArgProAsn ThrIle TyrLeuPhe Met Val ArgAla IleAsnPro LysValSerVal ThrGln XaaLysPro Gln Lys AsnAsn GlySerThr TrpAlaAsnVal ProLeu ProProPro Pro 4$ Val GlnPro LeuProGly ThrGluLeuGlu HisTyr AlaValGlu Gln Gln GluAsn GlyTyrAsp SerAspSerTrp CysPro ProLeuPro Val Gln ThrTyr LeuHisGln GlyLeuGluAsp GluLeu GluGluAsp Asp $0 340 345 350 Asp ArgVal ProThrPro ProValArgGly ValAla SerSerPro Ala Ile SerPhe GlyGlnGln SerThrAlaThr LeuThr ProSerPro Arg $$ Glu GluMet GlnProMet LeuGlnAlaSer ProXaa PheThrSer Ser SUBSTITUTE SHEET (RULE 26) Gln Arg Pro Arg Pro Thr Ser Pro Phe Ser Thr Asp Ser Asn Thr Ser Ala Ala Leu Ser Gln Ser Gln Arg Pro Arg Pro Thr Lys Lys His Lys Gly Gly (2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 444 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

IS (xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:

2S (2) INFORMATION FOR SEQ ID N0:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 148 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single 3~ (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:

Ala Gln Ala Val Ala Ala Ala Ala Glu Tyr Ala Gly Leu Lys Val Ala 3S Arg Arg Gln Met Gln Asp Ala Ala Gly Arg Arg His Phe His Ala Ser Gln Cys Pro Arg Pro Thr Ser Pro Val Ser Thr Asp Ser Asn Met Ser Ala Val Val Ile Gln Lys Ala Arg Pro Ala Lys Lys Gln Lys His Gln Pro Gly His Leu Arg Arg Glu Ala Tyr Ala Asp Asp Leu Pro Pro Pro Pro Val Pro Pro Pro Ala Ile Lys Ser Pro Thr Val Gln Ser Lys Ala 4S Gln Leu Glu Val Arg Pro Val Met Val Pro Lys Leu Ala Ser Ile Glu Ala Arg Thr Asp Arg Ser Ser Asp Arg Lys Gly Gly Ser Tyr Lys Gly Arg Glu Ala Leu Asp Gly Arg Gln Val Thr Asp Leu Arg Thr Asn Pro S~ 130 135 140 Ser Asp Pro Arg (2) INFORMATION FOR SEQ ID N0:13:
SS (i) SEQUENCE CHARACTERISTICS:

SUBSTITUTE SHEET (RULE 26~

(A) LENGTH: 1781 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
double (D) TOPOLOGY: linear S (ii) MOLECULE
TYPE:
cDNA

(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:13:

lO ATAGAAACTCTACGGCAAAT AAAAAAACCCGAAATCGAACCCATGACTTATATATAGAGC 240 (2) INFORMATION
FOR SEQ
ID N0:14:

(i) SEQUENCE CHARACTERISTICS:

40 (A) LENGTH: 370 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 4S (xi) SEQUENCE DESCRIPTION:
SEQ ID N0:14:

Met Ile Ser Thr Thr Asp Thr ValGluThr ThrThrThr Ala Tyr Pro Glu Glu Leu Tyr Ala Glu Ser AlaProAla SerSerMet Ser Tyr Ile SO Pro Ala Ala Ile Ala Glu Gln GlnAsnGln IleThrPhe Glu His Leu Ile Pro Ser Ala His Asp His IleAspAla LeuAsnSer Phe Leu Arg Asn Ala Leu Leu Gln Arg Asn AlaAlaVal SerTyrAsp Pro Ile Gly SUBSTITUTE SHEET (RULE 26) Ala Pro Pro Ser Gly Trp Ser Pro Asp Gly Ser Ile Ser Thr Glu Gln Leu Ser LysSerVal ValLeuAsp LeuAlaAspLeu ArgAsp ArgSer Glu Glu SerGlyGlu SerSerTrp TrpSerGlnIle PheGly AspAla Asp Met HisValIle IleAsnTyr LeuTrpIleGly ValVal SerSer Leu Val IleLeuSer LeuValPhe IleLeuPheSer CysTyr PheTyr Arg Lys PheArgThr TrpLysLys CysAsnLysAsp IleArg AlaGln Ile His AlaAlaSer AspSerTyr SerSerHisLeu ValGly CysAsp Ala Ser ArgLeuLeu LeuHisGln GlnMetGlnHis ProHis HisArg Ser Ser GluAlaGly PheTyrGln IleGluSerPro ProCys TyrThr Ile Ala ThrGlyLeu ProSerTyr AspGluAlaLeu HisHis GlnPro Arg His PheAlaTyr GlyMetLys PheValTyrPro SerLeu AlaAla Val His HisHisHis HisCysIle SerAsnTrpGlu LysGln GluPro 2~ Leu Asn LysLeuGln LysCysLys LeuSerAlaAla AlaAla ValGlu Glu Asp LysAlaAsp SerSerSer SerThrSerAla SerAla SerPro Ser Ser SerGluSer SerAsnLeu AlaThrAlaThr ProAla IleCys Ile Asn MetProSer GlyArgGln AspGluGluVal AspAsn SerAsp Ser Asp SerAlaIle AlaValAla ValAlaValAla GlnSer LeuGln Pro Ala AlaProAla AspAspAsp CysAlaSerLeu ValVal ValVal Ala Ala SUBSTITUTE SHEET (RULE 26)

Claims

WHAT IS CLAIMED IS:

1. A method of modulating an amount of expressed active Robo on a cell, said method comprising the step of modulating the effective amount of a Comm polypeptide in contact with the cell, whereby the amount of expressed active Robo is specifically modulated inversely with the modulation of the effective amount of the Comm polypeptide in contact with the cell.

2. A method according to claim 1, wherein the effective amount of the Comm polypeptide is increased and the amount of expressed Robo is decreased.

3. The method of claim 1, in which the Comm polypeptide is provided to the cell exogenously in a pharmaceutically acceptable composition.

4. The method of claim 1, further comprising the steps of forming a mixture of the cell, the Comm polypeptide and a candidate agent, and determining the effect of the agent on the amount of expressed Robo.