A-Kinase-Anchoring Protein Subtypes Differentially Regulate GPCR Signaling and Function in Human Airway Smooth Muscle

AKAPs (A-kinase-anchoring proteins) act as scaffold proteins that anchor the regulatory subunits of the cAMP-dependent PKA (protein kinase A) to coordinate and compartmentalize signaling elements and signals downstream of Gs-coupled GPCRs (G protein-coupled receptors). The β₂AR (β-2-adrenoceptor), as well as the Gs-coupled EP2 and EP4 (E-prostanoid) receptor subtypes of the EP receptor subfamily, are effective regulators of multiple airway smooth muscle (ASM) cell functions whose dysregulation contributes to asthma pathobiology. Here, we identify specific roles of the AKAPs Ezrin and Gravin in differentially regulating PKA substrates downstream of the β₂AR, EP2R (EP2 receptor) and EP4R. Knockdown of Ezrin, Gravin, or both in primary human ASM cells caused differential phosphorylation of the PKA substrates VASP (vasodilator-stimulated phosphoprotein) and HSP20 (heat shock protein 20). Ezrin knockdown, as well as combined Ezrin and Gravin knockdown, significantly reduced the induction of phospho-VASP and phospho-HSP20 by β₂AR, EP2R, and EP4R agonists. Gravin knockdown inhibited the induction of phospho-HSP20 by β₂AR, EP2R, and EP4R agonists. Knockdown of Ezrin, Gravin, or both also attenuated histamine-induced phosphorylation of MLC20. Moreover, knockdown of Ezrin, Gravin, or both suppressed the inhibitory effects of Gs-coupled receptor agonists on cell migration in ASM cells. These findings demonstrate the role of AKAPs in regulating Gs-coupled GPCR signaling and function in ASM and suggest the therapeutic utility of targeting specific AKAP family members in the management of asthma.