Uncovering the Landscape of Noncanonical Hedgehog Signaling
Hedgehog (HH) signaling is a highly regulated network that dictates animal development and homeostasis. A linear model of HH signaling that serves as the central dogma describes an inhibitory receptor, Patched1 (PTCH1), in an active state in the absence of the endogenous ligand HH. In its active state, PTCH1 inhibits the activity of the central transducer of the pathway, Smoothened (SMO). PTCH1 binds to HH, leading to the de-repression of SMO and the transmission of signals that ultimately lead to the activation of the glioma-associated oncogene transcription factors (GLI). GLI transcriptional activity is a canonical readout of HH signaling. The HH signaling network eminates from these prototypical proteins, resulting in diversity of signaling that depends on spatial and temporal regulation and is cell type specific. The transcriptional activity of GLI is intimately related to ciliogenesis. PTCH1 inhibits SMO activation by controlling its ciliary localization and, seemingly, through modulation of cholesterol levels. Furthermore, SMO must activate GLI at primary cilia for high-level signaling output. GLI transcriptional activity is the quintessential endpoint of ciliary HH signaling, but the primary cilium is not an absolute requirement for oncogenic/disease-driven HH signaling in which aberrant activation of upstream or downstream components override the requirement of primary cilia. Moreover, PTCH1 and SMO can initiate non-canonical HH signaling in which GLI transcriptional activity is not the endpoint. Unlike canonical HH signaling in which GLI is the main downstream effector, known downstream effectors of non-canonical HH signaling are diverse and can be activated within minutes of receptor activation. Growing evidence suggest that there must be a balance of canonical HH signaling and non-canonical HH signaling for normal animal development and adult cellular homeostasis. My thesis project uncovers novel mechanistic insight into the relationship between these two forms of HH signaling. Surprisingly, SMO has signaling competency in the presence and in the absence of primary cilia. We exploited the G protein-coupled receptor (GPCR) characteristics of SMO to show that SMO can activate inhibitory G proteins (Gi) and RHOA in a cilium-independent manner while failing completely to activate GLI in the absence of primary cilia. In the process of making parallels between SMO and a well-known family of Gi-coupled receptors, we uncovered the ability of SHH to induce the rapid phosphorylation of extracellular signal-regulated kinase (ERK); however, this phenomenon appears to be independent of SMO, G i, and primary cilia. While SMO was not involved, we found that depletion of β-arrestin 2 (βARR2) enhanced ERK phosphorylation. Further investigation into the mechanism revealed a novel interaction between PTCH1 and ?-arrestins 1 and 2 (βARR1/2). The classical targets of βARR1/2 are GPCRs, however classes of membrane receptors have been reported to interact to βARRs. Remarkably, we found that depletion of either or both βARR1/2 enhanced GLI1 induction and GLI-luciferase activity by SHH, opposite to some early published data. We reasoned that the correlation between the SHH-mediated phosphorylation of ERK, the ability of PTCH1 to interact with βARRs, and the enhanced ERK phosphorylation upon depletion of βARRs is in line with the tumor suppressor characteristics of PTCH1 that are under the extensive control of E3 ubiquitin ligases. In this line, we found two endogenous HECT-domain containing E3 ligases (ITCH and WWP2) co-immunoprecipitating with PTCH1, which we found control PTCH1 internalization and degradation in resting conditions in a cilia-independent manner. ^ Altogether, our research contributes greatly to a better understanding of the relationship between the membrane proteins of the HH pathway and primary cilia by focusing on their non-canonical signaling capabilities. We believe that our research will increase the understanding of how the balance of HH signaling is essential for life and is disrupted in cancer and other human diseases.^
Ho Wei, Lan, "Uncovering the Landscape of Noncanonical Hedgehog Signaling" (2018). ETD Collection for Thomas Jefferson University. AAI10974397.