Faculty, Staff and Student Publications

Language

English

Publication Date

9-1-2025

Journal

Journal of Biological Chemistry

DOI

10.1016/j.jbc.2025.110539

PMID

40749829

PMCID

PMC12446771

PubMedCentral® Posted Date

7-31-2025

PubMedCentral® Full Text Version

Post-print

Abstract

cAMP influences multiple aspects of cardiac biology, including the regulation of contraction, relaxation, and overall stress responses. The functional outcomes of cAMP are driven by the spatial arrangement of enzymes that produce and degrade cAMP (adenylyl cyclase [AC] and phosphodiesterase, respectively), together with the downstream targets of cAMP. We performed proximity-dependent biotin identification (BioID) of near-neighbor interactions proteomics in cardiomyocytes to generate proximity maps for three cardiac AC isoforms. Processing and trafficking functions were the most common gene ontology terms for ACs, with AC5 and AC6 generating unique proximal protein sets compared to AC9. AC5/6 proximal proteins showed significant localization at the endo/sarcoplasmic reticulum with roles in calcium handling, whereas those near AC9 were more abundant at the plasma membrane. Upon treatment with the hypertrophic stimulus norepinephrine, only a few calcium handling proteins were differentially labeled for AC5/6, but not AC9. Endogenous AC activity copurified with vesicle transport, signaling, and muscle contraction proteins that were identified by BioID and/or pulldown of FLAG-tagged AC (FLAG-mass spectrometry) in cardiomyocytes, including ryanodine receptor 2, ATP2A2 (sarco/endoplasmic reticulum Ca2+-ATPase 2), epidermal growth factor receptor, syntrophin alpha 1, and vesicle-associated membrane protein 2. Finally, overlapping BioID and FLAG-mass spectrometry datasets suggested heterodimerization of AC5 and AC6, while super-resolution electron microscopy spatial mapping validated homodimerization and heterodimerization of these AC isoforms on the plasma membrane. Overall, our comprehensive network analysis has identified new binding partners and shed light on the spatial and functional significance of AC-containing macromolecular complexes in heart.

Keywords

Myocytes, Cardiac, Proteomics, Animals, Adenylyl Cyclases, Isoenzymes, Rats, adenylyl cyclase, cyclic AMP (cAMP), cardiomyocyte, intracellular trafficking, proximity proteomics, epidermal growth factor receptor (EGFR), ryanodine receptor (RYR2), sarcoplasmic reticulum (SR), Sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2), syntrophin alpha 1 (SNTA1), vesicle-associated membrane protein 2 (VAMP2)

Published Open-Access

yes

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