The deubiquitinase ubiquitin-specific protease 20 is a positive modulator of myocardial β-adrenergic receptor expression and signaling.

Reversible ubiquitination of G protein-coupled receptors regulates their trafficking and signaling; whether deubiquitinases regulate myocardial β-adrenergic receptors (βARs) is unknown. We report that ubiquitin-specific protease 20 (USP20) deubiquitinates and attenuates lysosomal trafficking of the βAR. βAR-induced phosphorylation of USP20 Ser-333 by protein kinase A-α (PKAα) was required for optimal USP20-mediated regulation of βAR lysosomal trafficking. Both phosphomimetic (S333D) and phosphorylation-impaired (S333A) USP20 possess intrinsic deubiquitinase activity equivalent to WT activity. However, unlike USP20 WT and S333D, the S333A mutant associated poorly with the βAR and failed to deubiquitinate the βAR. USP20-KO mice showed normal baseline systolic function but impaired βAR-induced contractility and relaxation. Dobutamine stimulation did not increase cAMP in USP20-KO left ventricles (LVs), whereas NKH477-induced adenylyl cyclase activity was equivalent to WT. The USP20 homolog USP33, which shares redundant roles with USP20, had no effect on βAR ubiquitination, but USP33 was up-regulated in USP20-KO hearts suggesting compensatory regulation. Myocardial βAR expression in USP20-KO was drastically reduced, whereas βAR expression was maintained as determined by radioligand binding in LV sarcolemmal membranes. Phospho-USP20 was significantly increased in LVs of wildtype (WT) mice after a 1-week catecholamine infusion and a 2-week chronic pressure overload induced by transverse aortic constriction (TAC). Phospho-USP20 was undetectable in βAR KO mice subjected to TAC, suggesting a role for USP20 phosphorylation in cardiac response to pressure overload. We conclude that USP20 regulates βAR signaling and Additionally, βAR-induced USP20 phosphorylation may serve as a feed-forward mechanism to stabilize βAR expression and signaling during pathological insults to the myocardium.