Pizzoni, A. | Search bioRxiv

bioRxiv2026

Rap1 Activates Endosomal AC9 to Drive the Second cAMP Wave

GsPCR signaling orchestrates cAMP production in three distinct spatial waves originating from the plasma membrane (PM), endosomes, and the nucleus. While the molecular drivers of the PM and nuclear waves are well defined, the regulation of the endosomal cAMP wave remains insufficiently understood. Here, we identify the small GTPase Rap1b as a direct activator of the endosomal adenylyl cyclase 9 (AC9), revealing a novel mechanism for intracellular cAMP synthesis. Like Gs, Rap1b-GTP interacts with the C2 domain of AC9, allosterically enhancing its catalytic activity both in vitro and in cells. Using AC9 mutations that selectively disrupt Rap1b versus Gs binding, we elucidate the role of the Rap1b-AC9 unit in mediating the endosomal cAMP wave, introducing a new layer of spatial regulation to cAMP signaling.

cell biology↗

bioRxiv2022

Soluble cyclase-mediated nuclear cAMP synthesis is sufficient for cell proliferation

cAMP is a key player in many physiological processes. Classically considered to originate solely from the plasma membrane, this view was recently challenged by observations showing that GPCRs can sustain cAMP signaling from intracellular compartments associated with nuclear PKA translocation and activation of transcriptional events. In this report we show that neither PKA translocation nor cAMP diffusion, but rather nuclear sAC activation represents the only source of nuclear cAMP accumulation, PKA activation, and CREB phosphorylation. Both pharmacological and genetic sAC inhibition, that did not affect the cytosolic cAMP levels, completed blunted nuclear cAMP accumulation, PKA activation and proliferation, while an increase in sAC nuclear expression significantly enhanced cell proliferation. Moreover, utilizing novel compartment-specific optogenetic actuators we showed that light-dependent nuclear cAMP synthesis can stimulate PKA, CREB and trigger cell proliferation. Thus, our results show that sAC-mediated nuclear accumulation is not only necessary but sufficient and rate-limiting for cAMP-dependent proliferation.

cell biology↗