Most cells in our body have an organelle called the primary cilium. The first cellular organelle to be described in biology (Wallingford and Mitchell, 2011), the primary cilium was long mistaken as a vestigial appendage.
The primary cilia are now considered as vital sensory organelles for detection and transmission of a broad range of chemical and mechanical signals. Signaling mediated by the primary cilia plays fundamental roles in cellular differentiation, polarity and cell cycle control. Not surprisingly, defects in cilia result in a heterogeneous group of newly described diseases known as “ciliopathies”, that frequently present with brain malformations, neural tube defects, retinopathy, renal and hepatic cysts, polydactyly, bone deformities, mental retardation, and obesity (Hildebrandt et al., 2011).
Why do the cells require a primary cilium for organizing signaling? The primary cilium is composed of a microtubule-based axoneme, extending from the basal body, and is ensheathed by membrane. The axoneme is assembled and maintained by a conserved process called intraflagellar transport (IFT) (Rosenbaum and Witman, 2002). The ciliary membrane acts as a privileged domain for concentration of signaling receptors fundamental for sensation.
This is apparent in the cases of the modified cilia; for example, olfactory neuronal cilia house olfactory G-protein coupled receptors (GPCRs) and respond to odorants, whereas the vertebrate photoreceptor outer segments mediate phototransduction by opsins. Similarly, primary cilia in neurons respond to a wide variety of neuroendocrine signals through ciliary GPCRs. The full complement of ciliary receptors and their downstream effectors are thus critical for determining the sensory modalities of cells in their respective milieu. However, our understanding of the signaling pathways, and the principles underlying the spatio-temporal organization of signaling in this organelle is in its infancy. The main focus on my research is to address the central question of why and how cells utilize these singular organelles as detection and signal-transducing machines.
Background of current research projects
A new function of a conserved IFT-A complex in ciliary GPCR trafficking.
The intraflagellar transport (IFT) machinery is fundamental for assembly and maintenance of primary cilia (Rosenbaum and Witman, 2002). The primary cilium is required for optimal Shh signaling in vertebrates, and mutants encoding components of the IFT-B complex (implicated in anterograde IFT), as well as the IFT motors exhibit decreased Shh signaling in the neural tube (Goetz and Anderson, 2010).
Paradoxically, mutations in the ciliary IFT-A complex, which is implicated in retrograde IFT, cause increased Shh signaling. Mutations in the tubby-like protein 3, Tulp3, also exhibit increased Shh signaling in the neural tube (Norman et al., 2009). While generating the tubby family interactome using tandem affinity purification (TAP) and mass spectrometry (MS)-based proteomics, we discovered that Tulp3 binds to the IFT-A complex.
Furthermore, systematic depletion of the IFT-A subunits suggested that three subunits of the IFT-A complex are organized as a core IFT-A subcomplex, and associates with Tulp3. This observation led us to discover that in addition to its known role in retrograde transport, the core IFT-A sub-complex has a preciliary role in recruiting Tulp3 to the cilia. Tulp3 in turn promotes trafficking of certain class A GPCRs to cilia, and both the IFT-A- and phosphoinositide-binding properties of Tulp3 are required for this function (Figure 1) (Mukhopadhyay et al., 2010).
Discovery of an IFT-A regulated ciliary GPCR in repression of Shh signaling.
The function of IFT-A/Tulp3 in mediating GPCR trafficking to the cilia could explain their paradoxical role as negative regulators of the Shh pathway. The criteria for such an unidentified GPCR mediating the IFT-A/Tulp3 regulation of Shh signaling would be that it is:
- Localized to cilia
- Expressed early during neural tube development
- Conserved among vertebrates
- Regulated by IFT-A/Tulp3
- A negative regulator of the Shh pathway
Using a candidate approach for ciliary GPCRs expressed early during development, we identified an “orphan” class A GPCR, Gpr161 that is ciliary (Figure 2a). Actually, this is the only GPCR known to date to be expressed endogenously in a wide variety of ciliated cells. Furthermore, depletion of Tulp3 and IFT-A prevents ciliary localization of Gpr161. Most importantly, a null knockout mice model for this receptor results in embryonic lethality by E10.5 (Figure 2b), and phenocopies the IFT-A/Tulp3 mutants by showing increased Shh signaling in the developing neural tube (Figure 2c, d), suggesting that Gpr161 functions as a negative regulator of the Shh pathway. The cAMP-activated kinase protein kinase A (PKA) is pivotal in the processing of Gli transcription factors into the GliRs (Goetz and Anderson, 2010); however, the cAMP regulating pathways that mediate this activation of PKA remain unknown.
The processing of Gli3 into Gli3R is blocked in the Gpr161 knockout (Figure 2d), and constitutive Gpr161 activity results in increased cAMP levels. This suggests that Gpr161 activity could regulate PKA-mediated processing of Gli3R via cAMP signaling (Figure 2a). Simultaneously, Shh signaling directs Gpr161 to be internalized from cilia, suggesting that a feedback loop regulates functioning of Gpr161 in the Shh pathway.
We have thus discovered a fundamentally new IFT-A/Tulp3-mediated trafficking and signaling pathway for a GPCR in the basal repression machinery of Shh signaling (Mukhopadhyay et al., 2013).
Mukhopadhyay S., Wen X., Ratti N., Loktev A., Rangell L., Scales S., Jackson P. (2013) The ciliary G-protein-coupled receptor Gpr161 negatively regulates the sonic hedgehog pathway via cAMP signaling. Cell.
Mukhopadhyay, S., Wen, X., Chih, B., Nelson, C.D., Lane, W.S., Scales, S.J., and Jackson, P.K. (2010). "TULP3 bridges the IFT-A complex and membrane phosphoinositides to promote trafficking of G protein-coupled receptors into primary cilia." Genes Dev 24, 2180-2193.
Norman, R.X., Ko, H.W., Huang, V., Eun, C.M., Abler, L.L., Zhang, Z., Sun, X., and Eggenschwiler, J.T. (2009). "Tubby-like protein 3 (TULP3) regulates patterning in the mouse embryo through inhibition of Hedgehog signaling." Hum Mol Genet 18, 1740-1754.