Abstract
S-palmitoylation is a reversible posttranslational modification that plays an important role in regulating protein localization, trafficking, and stability. Recent studies have shown that some proteins undergo extremely rapid palmitoylation/depalmitoylation cycles after cellular stimulation supporting a direct signaling role for this posttranslational modification. Here we investigated whether β-adrenergic stimulation of cardiomyocytes led to stimulus-dependent palmitoylation of downstream signaling proteins. We found that β-adrenergic stimulation led to increased Gαs and Gαi palmitoylation. The kinetics of palmitoylation was temporally consistent with the downstream production of cAMP and positive inotropic responses. Additionally, we identified for the first time that G protein-coupled receptor kinase 2 (GRK2) is a palmitoylated protein in cardiomyocytes. The kinetics of GRK2 palmitoylation were distinct from those observed with Gαs and Gαi after β-adrenergic stimulation. Knockdown of the plasma membrane-localized palmitoyl acyltransferase DHHC5 revealed that this enzyme is necessary for palmitoylation of Gαs, Gαi, and GRK2 and functional responses downstream of β-adrenergic stimulation. Our results reveal that DHHC5 activity is required for signaling downstream of β-adrenergic receptors.
- Abbreviations
- β-AR
- Beta-adrenergic receptor
- GRK
- G protein-coupled receptor kinase
- PKA
- Protein Kinase A
- PAT
- palmitoyl acyltransferase
- NRVM
- neonatal rat ventricular myocytes
- ABE
- acyl-biotin exchange
- HA
- hydroxylamine
- ISO
- isoproterenol