Cholesterol Derivatives Regulate Adenylyl Cyclase 7 Activity by Binding CARC and CRAC Motifs in the Cytosolic Subunits

Transmembrane adenylyl cyclases (tmACs; ACs) are enzymes that synthesize cyclic AMP (cAMP), which is a key molecule in cellular signaling. Disruptions in AC activity can lead to long-term shifts in cAMP levels associated with various pathologies. In our study, we analyzed AC primary sequences and identified cholesterol-binding CARC and CRAC motifs located in conserved cytosolic regions, a surprising finding for motifs that are typically membrane-associated. Focusing on AC7, we mapped these motifs within its predicted structure and performed docking studies with cholesterol derivatives (hydrocortisone, dexamethasone, and 25-hydroxycholesterol). Our results showed that these molecules predominantly bind to the forskolin (FSK) binding site, which contains two CARC motifs. Using membranes overexpressing AC7, we observed that all three derivatives significantly decreased FSK-mediated AC7 activity by up to 55%. This suggests that cholesterol derivatives might interact with CARC and CRAC motifs to regulate AC7 function and underscore the potential of cholesterol derivatives as natural modulators as well as provide a compelling basis for future exploration of cholesterol derivatives as possible therapeutic regulators of AC7.