The primary cilium is a central signaling hub and provides a special environment that concentrates signaling proteins to generate adequate responses to external stimuli. Effective signaling of vertebrate primary cilia depends on the dynamic transport of central signaling components such as receptors and effectors into and out of primary cilia. Yet, apart from select factors, the extent of the proteomic remodeling of primary cilia during active signaling remains largely unknown.
We employ proximity labeling methods using cilia-localized ascorbate peroxidase (cilia-APEX) in combination with tandem-mass-tag-based quantitative mass spectrometry approaches to reveal the comprehensive proteomic alteration of primary cilia in response to signal in cells. By profiling the cilia proteome in a time-resolved manner after inducing the hallmark primary cilium signaling pathway by Sonic Hedgehog stimulation, we could reconcile previously described changes in the localization of known Hedgehog signaling components, including low abundant membrane receptors, such as GPCRs. Importantly, we revealed a rapid removal of the cAMP-dependent protein kinase (PKA) holoenzyme, including the orphan GPCR GPR161, which functions as the A-kinase anchoring protein (AKAP) in primary cilia. Hierarchical clustering identified the putative phosphatase PALD1 that accumulates in primary cilia in response to active Hedgehog signaling to dampen signaling in a cell type-specific manner. Surprisingly, we find PALD1 enriched in primary cilia also in response to other stimuli, suggesting a more general function in ciliary signal transduction.
Our unbiased analyses demonstrate that proximity labeling in combination with quantitative proteomics allows time-resolved proteomics of primary cilia and provide novel insights into how primary cilia orchestrate signaling processes.

Funding: Our research is funded by the German Research Foundation (DFG).