Time-resolved proteomics profiling of the ciliary Hedgehog response

Abstract

The primary cilium is a signaling compartment that interprets Hedgehog signals through changes of its protein, lipid, and second messenger compositions. Here, we combine proximity labeling of cilia with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary proteome in response to Hedgehog. This approach correctly identifies the three factors known to undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together with the G protein–coupled receptor GPR161 in response to Hedgehog, and we propose that the GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, we identify the phosphatase Paladin as a cell type–specific regulator of Hedgehog signaling that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling malfunctions.

Ciliary and extraciliary Gpr161 pools repress hedgehog signaling in a tissue-specific manner

“…Production of cAMP by Gpr161 in the endomembrane compartment, as reported for other GPCRs (Calebiro et al, 2009;Crilly & Puthenveedu, 2020;Irannejad et al, 2013;Kotowski et al, 2011;Vilardaga et al, 2014), could be relevant in cumulatively contributing to Gli3R processing. Direct Gpr161 C-tail binding to type I PKA regulatory subunits (Bachmann et al, 2016) is likely to facilitate PKA activation in proximity to Gpr161-mediated cAMP production in cilia for GliR processing (May et al, 2021;Mick et al, 2015), but if such coupling happens in the endomembrane compartment is not known. Gpr161 mut1/mut1 limb buds showed decreased Gli3R processing compared to whole embryo lysates, suggesting limited compensation by extracellular Gpr161 pools in the limb buds and tissue specificity in contribution of extracellular Gpr161 pools to Gli3R processing.…”

Phosphorylation and Ubiquitylation Regulate Protein Trafficking, Signaling, and the Biogenesis of Primary Cilia

“…Apart from the IFT complexes, cilia require a multitude of additional factors to convey ciliary signals, which involves not only common protein trafficking components, such as β-arrestins, but also cilia-specific machinery, including the BBSome or the Tubby family of proteins (Mukhopadhyay and Jackson, 2011). While the inventory of primary cilia continues to expand (Mick et al, 2015;Kohli et al, 2017;May et al, 2021), the number of enzymes, which catalyze PTMs and have been unambiguously shown to localize to primary cilia, is limited. Nonetheless, we are beginning to unravel how ciliary signaling dynamics can be established as we identify more and more targets of PTMs in cilia.…”

“…GPR161's C-terminal tail not only contains the AKAP binding domain for PKA but also several protein kinase consensus sites, including one for PKA (Bachmann et al, 2016). Upon Hedgehog pathway activation, the C-terminal tail of GPR161 is phosphorylated by GRK2 and presumably PKA (Bachmann et al, 2016;Pal et al, 2016;May et al, 2021). GRK-mediated phosphorylation recruits the molecular sensor of activated GPCRs, specifically β-arrestin2, which is required for the removal of activated GPCRs from cilia (Figure 1B; Pal et al, 2016).…”

“…GRK-mediated phosphorylation recruits the molecular sensor of activated GPCRs, specifically β-arrestin2, which is required for the removal of activated GPCRs from cilia (Figure 1B; Pal et al, 2016). Consequently, GPR161 exits cilia together with its binding partner PKA (May et al, 2021). Thereby, PKA activity in cilia is inhibited by two mechanisms: (i) reducing cAMP levels and (ii) removing PKA itself.…”

Ciliary and extraciliary Gpr161 pools repress hedgehog signaling in a tissue-specific manner

“…Production of cAMP by Gpr161 in the endomembrane compartment, as reported for other GPCRs (Calebiro et al, 2009;Crilly & Puthenveedu, 2020;Irannejad et al, 2013;Kotowski et al, 2011;Vilardaga et al, 2014), could be relevant in cumulatively contributing to Gli3R processing. Direct Gpr161 C-tail binding to type I PKA regulatory subunits (Bachmann et al, 2016) is likely to facilitates PKA activation in proximity to Gpr161-mediated cAMP production in cilia for GliR processing (May et al, 2021;Mick et al, 2015), but if such coupling happens in the endomembrane compartment is not known.…”