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.

Mitochondria and the endoplasmic reticulum (ER) have an intimate functional relationship due to tethering proteins that bring their membranes in close (~30 nm) apposition. One function of this inter-organellar junction is to increase the efficiency of Ca2+ transfer into mitochondria, thus stimulating mitochondrial respiration. Here we show that the ER cation-permeant channel polycystin 2 (PC2) functions to reduce mitochondria-ER contacts. In cell culture models, PC2 knockdown led to a 50% increase in mitofusin-2 (MFN2) expression, an outer mitochondrial membrane GTPase. Live-cell super-resolution and electron microscopy analyses revealed enhanced MFN2-dependent tethering between the ER and mitochondria in PC2 knockdown cells. PC2 knockdown also led to increased ER-mediated mitochondrial Ca2+ signaling, bioenergetic activation, and mitochondrial density. Mutation or deletion of the gene encoding for PC2 results in autosomal dominant polycystic kidney disease (ADPKD), a condition characterized by numerous fluid-filled cysts. In cell culture models and mice with kidney-specific PC2 knockout, knockdown of MFN2 rescued defective mitochondrial Ca2+ transfer, and markedly diminished cell proliferation in kidney cysts. Consistent with these results, cyst-lining epithelial cells from human ADPKD kidneys had a 2-fold increase in mitochondria and MFN2 expression. Our data suggest that PC2 normally serves to limit key mitochondrial proteins at the ER-mitochondrial interface, and acts as a checkpoint for mitochondrial biogenesis and bioenergetics through a transcriptional mechanism. Loss of this regulation may contribute to the increased oxidative metabolism and aberrant cell proliferation typical of kidney cysts in ADPKD.