Abstract 2283: Role of Posttranslational Arginylation in Cardiovascular Development and G-Protein Signaling
The conjugation of arginine (Arg) from Arg-tRNAArg to N-terminal Asp, Glu, or Cys is a eukaryotic protein modification that can lead to ubiquitylation and proteasomal degradation of the resulting Arg-conjugated proteins through the N-end rule pathway. We have previously identified ATE1 gene encoding R-transferases responsible for all known N-terminal arginylation activities and have shown that ATE1−/− embryos die owing to various cardiovascular defects including ventricular hypoplasia and septal defect and late angiogenesis. Through genomewide functional proteomic screening with 18,000 cDNAs, we have identified RGS4, RGS5, and RGS16 as ATE1 substrates. These structurally related RGS proteins act as regulators of Gq-activated cardiovascular signaling and have been implicated in myocardial growth and hypertrophy. Degradation of these substrates initiates from the cleavage of N-terminal Met, exposing Cys-2 at the N-terminus, a degron that undergoes oxidation in the presence of oxygen and subsequent arginylation by ATE1 to create an N-degron for ubiquitylation by the UBR E3 family. Here, we show that the proliferation of myocardium, in particular cardiomyocytes, is impaired in ATE1−/− embryos as early as E11.5, which precedes developmental defects observed in E12.5 hearts. ATE1 is prominently expressed in cardiomyocytes, compared with non-cardiomyocytes, where it is required for angiotensin II-induced proliferation. RGS4 is accumulated throughout the entire body of normally developing ATE1−/−, and its expression pattern in ATE1−/− embryos correlates to that of ATE1 in various embryonic tissues. ATE1−/− hearts are impaired in Gαq-activated signaling as determined by activities of protein kinase C and its downstream component, MEK1. Finally, we used transgenic mouse model to address the functional link between ATE1 and Gαq signaling. We demonstrate that cardiac defects, including thin myocardium and ventricular septal defect, but not vascular defects, are significantly rescued in mutants lacking ATE1 but overexpressing Gαq in the heart. These results together suggest that ATE1 controls cardiac development and signaling in part through regulated proteolysis of multiple cardiovascular regulators of Gαq-activated signaling pathways.