Novel mechanisms in the control of cAMP dynamics

GPCRs are the largest family of mammalian membrane receptors with an established relevance in therapy,
representing a third of the currently FDA-approved drugs. Particularly GsPCRs via cAMP are involved in many
physiological and pathophysiological conditions. Classically considered to originate solely from the plasma
membrane, this view was recently challenged by observations showing that GsPCRs, upon internalization, can
sustain cAMP signaling from intracellular compartments. Most importantly, this second endocytic cAMP phase
was strictly associated with nuclear transcriptional events responsible for the generation of specific biological
responses, like chronic inflammation, chronic pain, and migraine, conditions that affect millions worldwide.
However, the molecular mechanisms involved in sustained signaling are still unknown. Their characterization
should deepen our understanding of the GPCRs’ spatiotemporal organization and function and may provide
novel avenues for therapeutic intervention. We have identified a novel GαS-independent CAP1-Rap1-AC9
regulatory unit and its potential involvement in sustained signaling will be addressed in this proposal.
The scientific premise for this proposal is based on the identification of a novel GαS-independent CAP1-Rap1
complex directly regulating AC9. Utilizing novel optogenetic actuators and targeting strategies, we will test the
hypothesis that upon the first plasma membrane-generated GαS-dependent cAMP wave, this new intracellular
GαS-independent CAP1-Rap1-AC9 regulatory unit is responsible for the sustained cAMP phase.
Four integrated specific aims are proposed to experimentally test this hypothesis. In SA #1 we will characterize
AC9 as the CAP1-Rap1-sensitive tmAC isoform involved in the potentiation of cAMP dynamics in cells. In SA #2
we will identify and characterize key residues in AC9 that discriminate GαS and Rap1 binding. In SA #3, we will
use the ALFA-tag targeting strategy to bring sensors (AC9-ALFA/Nb-ALFA-H188) close to AC9 to test the
hypothesis that the AC9-CAP1-Rap1 ternary complex establishes a compartmentalized positive feedback loop
responsible for the GPCR sustained cAMP wave. Finally, in SA #4, we will use GαS- and Rap1-targeted
disruptors to uncouple functional GαS- and Rap1-dependent cyclase activation events in the plasma membrane
and intracellular AC9 compartments.
Our laboratory’s long-standing interest is to understand the spatiotemporal regulation of cAMP-dependent
signaling events. A multidisciplinary approach combining biochemistry, optogenetics, and nanobody-targeted
strategies will be exploited to test the role of AC9-CAP1-Rap1 in compartmentalized cAMP signaling.
Successful completion of the proposed studies should provide new insights into the mechanism/s cells utilize
to compute (code/decode) the relay of the cAMP signal (i.e., fidelity, specificity, efficiency) and provide us
opportunities in the identification of new subcellular localized targets for pharmacological intervention of this
critical second messenger pathway.

Grant Number: 5R01GM148449-03
NIH Institute/Center: NIH
Principal Investigator: DANIEL ALTSCHULER