One signal, two pathways: Analysis of how a single Wnt ligand can initiate discrete signaling pathways through the activation of two distinct receptors

Cell to cell communication is vital throughout the development of multicellular organisms and during adult homeostasis. One way in which cell communication occurs is through the secretion of signaling molecules that are received by neighboring responding cells. Wnt ligands comprise a large family of secreted hydrophobic glycoproteins that control a variety of developmental and adult processes in all metazoan organisms. As inappropriate Wnt signaling contributes to cancer and other degenerative disorders, much effort has been made to understand the ways in which Wnt signaling is activated and regulated. In the past, controlled in vitro studies of Wnt signal activation were hampered by a lack of active, soluble protein. However, purified Wnt proteins can now be generated and questions regarding how various Wnt proteins exert their pleiotropic effects can finally be studied.;In this thesis I will address the fundamental question of whether all Wnt family members signal in the same manner or whether different Wnt family members initiate discrete signaling pathways by engaging different receptors. In the most well understood "canonical" Wnt signaling pathway, Wnt binding to Frizzled receptors induces beta-Catenin protein stabilization and entry into the nucleus, where it complexes with TCF/LEF transcription factors to affect the transcription of target genes. Through the use of purified Wnt proteins, I will show that Wnt5a protein can both activate and inhibit canonical Wnt signaling in the presence of the appropriate receptor. Wnt5a-mediated inhibition of canonical Wnt signaling requires the receptor tyrosine kinase Ror2 and occurs at the level of gene transcription. Through mutational analysis, I will also show which regions of the mRor2 receptor are necessary for mediating the Wnt5a inhibitory signal. Lastly, to gain insight into the role Ror2 plays in inhibiting canonical Wnt signaling in vivo, I will explore the expression pattern of the Ror2 receptor using mono- and polyclonal antibodies that I generated for that purpose.