Endothelins are potent vasoconstrictor peptides which mediate their activity through the endothelin receptor A (ETA) and B (ETB) belonging to the superfamily of G-protein-coupled receptors (GPCRs). In the myocardium endothelin-1 (ET1) has growth promoting and positive chronotropic as well as positive inotropic effects. Multiple phosphorylation sites have been reported for the ETA receptor. However, their functional roles for signaling and receptor desensitization are widely unknown.Methods
To analyze the impact of serine/threonine receptor phosphorylation on ET1-signaling we generated different phosphorylation-deficient alanine mutants of the ETA receptor: 1) ETA4PD with substitutions of the four distal C terminal amino acids T417, S420, S421 and S425 by alanine; 2) ETAPDZPD with substitutions of six amino acids belonging to the C-terminal PDZ-motif S391, S393, T396, S397, T403 and S404 and 3) ETA10PD with the combined substitutions of ETA4PD and ETAPDZPD. We analysed the ET1-induced inositol-trisphosphate (IP3) production in living cells transfected with ETA and mutant receptors by homogenous time resolved fluorescence (HTRF).Results
Stimulation of cells transfected either with ETA4PD and ETAPDZPD, showed the same second messenger production as the wild type form of ETA after 2 hours of ET1-treatment. By contrast, ETA10PD resulted in a significant (p < 0,001) increase of IP3 production, indicating that multiple phosphorylation of the C-terminal domain of the receptor modifies signal transduction of ETA.Conclusions
GRK phosphorylation of ETA receptor might play an important role for receptor desensitization after prolonged ET1 stimulus. We suggest that the desensitization process of the phosphorylation-deficient receptor mutant ETA10PD lacking 10 distal serine/threonine residues is significantly hampered, resulting in increased second messenger accumulation during permanent ET1 stimulation. Of note, the mutations in both receptors variants ETAPDZ-PD and ETA4PD each containing a sub-fraction of these 10 alanine substitutions cause no alteration in signaling. In summary, we suppose a cumulative phosphorylation effect at specific C-terminal sites for ETA-receptor desensitization.