Almost every quiescent vertebrate cell has an organelle called the primary cilium. Cilia and flagella are among the earliest described subcellular structures; however, the primary cilium was long mistaken as vestigial. The primary cilia are now considered to function as cellular antennae for sensing a wide variety of signals, and defects in these organelles are increasingly being implicated in diverse developmental and degenerative human diseases called ciliopathies. Although, mechanisms driving ciliary assembly have been studied, the principles underlying ciliary signaling are largely unknown. The main focus of this proposal is to investigate the spatio-temporal organization of signaling in the primary cilium, and the unique features that have resulted in its widespread use as a signaling compartment. The primary cilium is indispensable for sonic hedgehog (Shh) signaling during neural tube development by regulating the downstream bifunctional Gli transcription factors. The cAMP activated protein kinase A (PKA) triggers proteolytic processing that generates Gli repressors in a cilia-dependent manner; however, pathways that promote PKA activation, and the exact function of cilia is unclear. We recently identified an orphan ciliary G-protein-coupled receptor (GPCR), Gpr161 that acts as a negative regulator of Shh signals and Gli processing via cAMP signaling. Active Shh signaling also results in removal of this receptor from the primary cilia. Thus, Gpr161 is likely to activate PKA by increasing ciliary cAMP levels, while also being regulated by Shh signaling in a positive feedback circuit. Our discovery of Gpr161 allows us to address the following outstanding questions in ciliary signaling. First, how are signaling molecules dynamically compartmentalized in the primary cilia? Second, what are the phenotypic consequences of disrupting signaling in the primary cilia? Our ability to uncouple distinct aspects of Gpr161's function, such as signaling, ciliary localization and removal from cilia, and study endogenous pools in cilia help us mechanistically dissect signaling in the context of intact cilia. Here, we propose to identify regulatory mechanisms underlying Gpr161-mediated ciliary signaling using integrative approaches. First, we will identify factors determining specificity of ciliary localization of GPCRs utilizing proteomic approaches with membrane-targeted ciliary localization motifs. We will test the role of these potential candidates in ciliar trafficking of endogenous Gpr161 using knockdown assays. Second, we will define mechanisms involved in Shh-dependent removal of Gpr161 from the cilia by identifying regions of the receptor that are necessary for this process, and study the cross-talk between Shh signaling, Gpr161 activity, desensitization, and endocytosis pathways. Third, we will study the developmental consequences of disrupting dynamic regulation of Gpr161 in the cilia, and test the role of Gpr161-mediated ciliary cAMP signaling during compartmentalized PKA activation in the Shh pathway. These experiments will establish a solid foundation for studying ciliary regulation and compartmentalization of GPCRs in developmental pathways.

Public Health Relevance

Defects in the primary cilium result in a heterogeneous group of newly described human diseases known as 'ciliopathies', and studying the principles underlying ciliary signaling is central to the understanding of the pathogenesis of these diseases. This project addresses several key issues in ciliary signaling, including the dynamic trafficking, compartmentalization, and signaling of receptors in the cilium, and will use orthologous mouse models to evaluate the role of cilia-generated signaling during neural tube development. Studying ciliary signaling also provides a more general paradigm for studying cellular sensory networks in regulating developmental pathways, and in understanding disease pathologies in humans.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM113023-01
Application #
8800856
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Dunsmore, Sarah
Project Start
2015-01-01
Project End
2019-12-31
Budget Start
2015-01-01
Budget End
2015-12-31
Support Year
1
Fiscal Year
2015
Total Cost
$276,165
Indirect Cost
$105,165
Name
University of Texas Sw Medical Center Dallas
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Badgandi, Hemant B; Hwang, Sun-Hee; Shimada, Issei S et al. (2017) Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins. J Cell Biol 216:743-760
Mukhopadhyay, Saikat; Badgandi, Hemant B; Hwang, Sun-Hee et al. (2017) Trafficking to the primary cilium membrane. Mol Biol Cell 28:233-239
Shimada, Issei S; Mukhopadhyay, Saikat (2017) G-protein-coupled receptor signaling and neural tube closure defects. Birth Defects Res 109:129-139
Shimada, Issei S; Badgandi, Hemant; Somatilaka, Bandarigoda N et al. (2017) Using Primary Neurosphere Cultures to Study Primary Cilia. J Vis Exp :
Pal, Kasturi; Hwang, Sun-Hee; Somatilaka, Bandarigoda et al. (2016) Smoothened determines ?-arrestin-mediated removal of the G protein-coupled receptor Gpr161 from the primary cilium. J Cell Biol 212:861-75
Mukhopadhyay, Saikat (2015) TCTEX1D2, a potential link to skeletal ciliopathies. Cell Cycle 14:293-4