Differential Targeting of β-Adrenergic Receptor Subtypes and Adenylyl Cyclase to Cardiomyocyte Caveolae

TLDR

Spatial regulation of β1‑ and β2‑adrenergic receptor signaling in cardiomyocytes occurs in plasma membrane microdomains, with evidence of caveolin‑3 association with adenylyl cyclase V/VI. The study investigates whether caveolae serve as specialized subdomains that concentrate and organize β‑adrenergic signaling components in cardiomyocytes. Caveolae are highly enriched in β2‑adrenergic receptors, Gαi, PKA RIIα, caveolin‑3, and flotillins, while β1‑ARs, m2 receptors, Gαs, and adenylyl cyclase V/VI are more diffusely distributed; β2‑ARs remain caveolar across expression levels but are depleted upon agonist stimulation, and caveolae act as negative regulators of cAMP signaling, with caveolin dispensable for adenylyl cyclase targeting to low.

Abstract

Differential modes for beta(1)- and beta(2)-adrenergic receptor (AR) regulation of adenylyl cyclase in cardiomyocytes is most consistent with spatial regulation in microdomains of the plasma membrane. This study examines whether caveolae represent specialized subdomains that concentrate and organize these moieties in cardiomyocytes. Caveolae from quiescent rat ventricular cardiomyocytes are highly enriched in beta(2)-ARs, Galpha(i), protein kinase A RIIalpha subunits, caveolin-3, and flotillins (caveolin functional homologues); beta(1)-ARs, m(2)-muscarinic cholinergic receptors, Galpha(s), and cardiac types V/VI adenylyl cyclase distribute between caveolae and other cell fractions, whereas protein kinase A RIalpha subunits, G protein-coupled receptor kinase-2, and clathrin are largely excluded from caveolae. Cell surface beta(2)-ARs localize to caveolae in cardiomyocytes and cardiac fibroblasts (with markedly different beta(2)-AR expression levels), indicating that the fidelity of beta(2)-AR targeting to caveolae is maintained over a physiologic range of beta(2)-AR expression. In cardiomyocytes, agonist stimulation leads to a marked decline in the abundance of beta(2)-ARs (but not beta(1)-ARs) in caveolae. Other studies show co-immunoprecipitation of cardiomyocytes adenylyl cyclase V/VI and caveolin-3, suggesting their in vivo association. However, caveolin is not required for adenylyl cyclase targeting to low density membranes, since adenylyl cyclase targets to low buoyant density membrane fractions of HEK cells that lack prototypical caveolins. Nevertheless, cholesterol depletion with cyclodextrin augments agonist-stimulated cAMP accumulation, indicating that caveolae function as negative regulators of cAMP accumulation. The inhibitory interaction between caveolae and the cAMP signaling pathway as well as domain-specific differences in the stoichiometry of individual elements in the beta-AR signaling cascade represent important modifiers of cAMP-dependent signaling in the heart.