Mapping netrin receptor binding reveals domains of Unc5 regulating its tyrosine phosphorylation.
Netrin and its receptors Unc5 and deleted in colorectal carcinoma (DCC) regulate axon guidance and cell migration. We defined domains involved in the interactions between netrin-1, DCC, and Unc5c. We show that Unc5 requires both Ig domains to interact with netrin. DCC binds through the fourth fibronectin type III domain, whereas netrin binds through multiple domains to both receptors. We examined the functional consequences of removing the netrin binding and nonbinding domains from Unc5 in vitro and in vivo. In human embryonic kidney 293 cells, removal of the netrin binding second Ig domain causes an increase in basal tyrosine phosphorylation, whereas removal of the netrin nonbinding thrombospondin domains decreases tyrosine phosphorylation. Moreover, experiments in Caenorhabditis elegans indicate that both netrin binding and nonbinding domains are necessary for phenotypic rescue of an unc-5 loss of function mutation.
Kruger
Robert P
RP
Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, USA.
Lee
Jeeyong
J
Li
Weiquan
W
Guan
Kun-Liang
KL
D000818:Animals; D030801:Animals, Genetically Modified; D019556:COS Cells; D017173:Caenorhabditis elegans / Q000235:genetics; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D002645:Chickens; D002522:Chlorocebus aethiops; D000075389:DCC Receptor; D016163:Genes, DCC; D005816:Genetic Complementation Test; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000075386:Netrin Receptors; D000075388:Netrin-1; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D015815:Cell Adhesion Molecules; D000075389:DCC Receptor; C501909:DCC protein, human; C000617351:NTN1 protein, human; D009414:Nerve Growth Factors; D000075386:Netrin Receptors; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; C115727:UNC5C protein, human; C077588:UNC-5 protein, C elegans; D000075388:Netrin-1; D019000:Phosphotyrosine