Abstract
The ATPase 6 accessory protein 2 (ATP6AP2)/(pro)renin receptor (PRR) is essential for the biogenesis of active vacuolar H+-ATPase (V-ATPase). Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD) and transmembrane domain (TM) of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M) and causes X-linked mental retardation Hedera type (MRXSH) and X-linked parkinsonism with spasticity (XPDS) in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.
Highlights
Binding of prorenin to therenin receptor (PRR) generates local angiotensin I due to the non-proteolytic activation of prorenin, and induces intracellular signaling [1]
We systematically constructed a series of retroviral vectors harboring full-length mouse ATPase 6 accessory protein 2 (ATP6AP2)/PRR or one of the following deletion mutants: carboxylterminal fragment (CTF), DCD, NTF, and R276A/KT/ R279A (AKTA, mutation within the putative furin recognition site), human full-length ATP6AP2/PRR, and human Dexon 4 mutant (D4M) (Figure 1A, B)
Expressed full-length ATP6AP2/PRR was detected in three forms, the full-length protein and the N- and C-terminal fragments produced by furin cleavage
Summary
Binding of prorenin to the (pro)renin receptor (PRR) generates local angiotensin I due to the non-proteolytic activation of prorenin, and induces intracellular signaling [1]. PRR is cleaved in the trans-Golgi network by the protease furin, generating an amino-terminal fragment (NTF) and a carboxylterminal fragment (CTF) [7]. Prior to the cloning of PRR, the encoding gene was named ATP6ap (ATPase 6 accessory protein 2) due to the demonstrated association of part of the CTF, termed M8.9, with the vacuolar H+-ATPase (V-ATPase) [9]. We and other groups recently demonstrated that ATP6AP2/PRR is essential for the biogenesis of active V-ATPase [10,11,12]. The tissue expression levels of ATP6AP2/PRR are closely correlated with other V-ATPase subunits in mice, implicating an essential function for ATP6AP2/ PRR as a mammalian V-ATPase-related protein [13]
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