mapping-cellular-distribution-an-optogenetic-protein-using-light

Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid

Our goal was to accurately track the cellular distribution of an optogenetic protein and evaluate its functionality within a specific cytoplasmic ...

University of Pittsburgh School of Medicine

CAP1 (Cyclase-Associated Protein 1) is highly conserved in evolution. Originally identified in yeast as a bifunctional protein involved in Ras-adenylyl cyclase and F-actin dynamics regulation, the adenylyl cyclase component seems to be lost in mammalian cells. Prompted by our recent identification of the Ras-like small GTPase Rap1 as a GTP-independent but geranylgeranyl-specific partner for CAP1, we hypothesized that CAP1-Rap1, similar to CAP-Ras-cyclase in yeast, might play a critical role in cAMP dynamics in mammalian cells. In this study, we report that CAP1 binds and activates mammalian adenylyl cyclase in vitro, modulates cAMP in live cells in a Rap1-dependent manner, and affects cAMP-dependent proliferation. Utilizing deletion and mutagenesis approaches, we mapped the interaction of CAP1-cyclase with CAP's N-terminal domain involving critical leucine residues in the conserved RLE motifs and adenylyl cyclase's conserved catalytic loops (e.g., C1a and/or C2a). When combined with a FRET-based cAMP sensor, CAP1 overexpression-knockdown strategies, and the use of constitutively active and negative regulators of Rap1, our studies highlight a critical role for CAP1-Rap1 in adenylyl cyclase regulation in live cells. Similarly, we show that CAP1 modulation significantly affected cAMP-mediated proliferation in an RLE motif-dependent manner. The combined study indicates that CAP1-cyclase-Rap1 represents a regulatory unit in cAMP dynamics and biology. Since Rap1 is an established downstream effector of cAMP, we advance the hypothesis that CAP1-cyclase-Rap1 represents a positive feedback loop that might be involved in cAMP microdomain establishment and localized signaling.

CAP1 binds and activates adenylyl cyclase in mammalian cells.

cAMP, a key player in many physiological processes, was classically considered to originate solely from the plasma membrane (PM). This view was recently challenged by observations showing that upon internalization GsPCRs can sustain signaling from endosomes and/or the trans-Golgi network (TGN). In this new view, after the first PM-generated cAMP wave, the internalization of GsPCRs and ACs generates a second wave that was strictly associated with nuclear transcriptional events responsible for triggering specific biological responses. Here, we report that the endogenously expressed TSHR, a canonical GsPCR, triggers an internalization-dependent, calcium-mediated nuclear sAC activation that drives PKA activation and CREB phosphorylation. Both pharmacological and genetic sAC inhibition, which did not affect the cytosolic cAMP levels, blunted nuclear cAMP accumulation, PKA activation, and cell proliferation, while an increase in nuclear sAC expression significantly enhanced cell proliferation. Furthermore, using novel nuclear-targeted optogenetic actuators, we show that light-stimulated nuclear cAMP synthesis can mimic the proliferative action of TSH by activating PKA and CREB. Therefore, based on our results, we propose a novel three-wave model in which the "third" wave of cAMP is generated by nuclear sAC. Despite being downstream of events occurring at the PM (first wave) and endosomes/TGN (second wave), the nuclear sAC-generated cAMP (third wave) is sufficient and rate-limiting for thyroid cell proliferation.

Article	Year
CAP1 binds and activates adenylyl cyclase in mammalian cells. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 34099549	2021
Soluble cyclase-mediated nuclear cAMP synthesis is sufficient for cell proliferation. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 36656863	2023

Alejandro Pizzoni

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