Direct visualization of cAMP signaling in primary cilia reveals up-regulation of ciliary GPCR activity following Hedgehog activation

Abstract

The primary cilium permits compartmentalization of specific signaling pathways, including elements of the Hedgehog (Hh) pathway. Hh transcriptional activity is thought to be negatively regulated by constitutively high ciliary cAMP maintained by the Gα(s)-coupled GPCR, GPR161. However, cilia also sequester many other Gα(s)-coupled GPCRs with unknown potential to regulate Hh. Here we used biosensors optimized for ciliary cAMP and strategies to isolate signals in the cilium from the cell body and neighboring cells. We found that ciliary cAMP was not elevated relative to cellular cAMP, inconsistent with constitutive cAMP production. Gα(s)-coupled GPCRs (e.g., the 5-HT6 serotonin and D1R dopamine receptor) had reduced ability to generate cAMP upon trafficking to the ciliary membrane. However, activation of the Hh pathway restored or amplified GPCR function to permit cAMP elevation selectively in the cilium. Hh therefore enables its own local GPCR-dependent cAMP regulatory circuit. Considering that GPCRs comprise much of the druggable genome, these data suggest alternative strategies to modify Hh signaling.

Primary cilium-dependent cAMP/PKA signaling at the centrosome regulates neuronal migration

“…S1), similar to what was described recently (5). The PC is a cAMP-rich region (10)(11)(12)(13), raising the possibility that the cAMP hotspot at the centrosome could be PC-dependent. To test this hypothesis, we used two mouse lines in which ablation of the PC can be genetically-induced by Cre recombination (Kif3a lox/lox and Rpgrip1l lox/lox mice).…”

“…As the PC is a cAMP-rich source (10)(11)(12)(13), we asked whether it could regulate neuronal migration through cAMP dynamics, in the context of migration along the postnatal Rostral Migratory Stream (RMS) from the ventricular/subventricular zone to olfactory bulb (OB) ( Fig. 1A).…”

Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

“…However, this approach does not work for primary cilia. Genetically-encoded biosensors for cAMP and Ca 2+ have already been targeted to primary cilia (Delling, DeCaen et al, 2013, Delling, Indzhykulian et al, 2016, Jiang, Falcone et al, 2019, Moore et al, 2016, Mukherjee et al, 2016. These sensors have been fused to the C terminus of full-length GPCRs, e.g.…”

Hedgehog and Gpr161: Regulating cAMP Signaling in the Primary Cilium

“…The direct visualization of cAMP dynamics in the cilium has proved to be technically challenging, and measurements of resting cAMP levels in primary cilia vary [90][91][92][93][94]. Indirect evidence for activation of AC and elevation of cAMP levels in the primary cilium in the absence of Hh ligands is provided by studies on the involvement of GPCRs in the regulation of Hh signaling.…”

“…Cilia host a collection of GPCRs that are coupled to Gαs or Gαi. The following reviews summarize the identity of ciliary localized GPCRs [3,6,7] and one recent paper indicates the coupling of a selection of ciliary GPCRs [91]. Besides ligand availability, constitutive receptor activities, receptor abundance, and receptor desensitization, it is the import/export of the inventory of the ciliary cAMP platforms that affects Hh antagonizing effects of cAMP.…”