MAGED2 Is Required under Hypoxia for cAMP Signaling by Inhibiting MDM2-Dependent Endocytosis of G-Alpha-S

Mutations in MAGED2 cause transient Bartter syndrome characterized by severe renal salt wasting in fetuses and infants, which leads to massive polyhydramnios causing preterm labor, extreme prematurity and perinatal death. Notably, this condition resolves spontaneously in parallel with developmental increase in renal oxygenation. MAGED2 interacts with G-alpha-S (G alpha s). Given the role of G alpha s in activating adenylyl cyclase at the plasma membrane and consequently generating cAMP to promote renal salt reabsorption via protein kinase A (PKA), we hypothesized that MAGED2 is required for this signaling pathway under hypoxic conditions such as in fetuses. Consistent with that, under both physical and chemical hypoxia, knockdown of MAGED2 in renal (HEK293) and cancer (HeLa) cell culture models caused internalization of G alpha s, which was fully reversible upon reoxygenation. In contrast to G alpha s, cell surface expression of the beta 2-adrenergic receptor, which is coupled to G alpha s, was not affected by MAGED2 depletion, demonstrating specific regulation of G alpha s by MAGED2. Importantly, the internalization of G alpha s due to MAGED2 deficiency significantly reduced cAMP generation and PKA activity. Interestingly, the internalization of G alpha s was blocked by preventing its endocytosis with dynasore. Given the role of E3 ubiquitin ligases, which can be regulated by MAGE-proteins, in regulating endocytosis, we assessed the potential role of MDM2-dependent ubiquitination in MAGED2 deficiency-induced internalization of G alpha s under hypoxia. Remarkably, MDM2 depletion or its chemical inhibition fully abolished G alpha s-endocytosis following MAGED2 knockdown. Moreover, endocytosis of G alpha s was also blocked by mutation of ubiquitin acceptor sites in G alpha s. Thus, we reveal that MAGED2 is essential for the cAMP/PKA pathway under hypoxia to specifically regulate G alpha s endocytosis by blocking MDM2-dependent ubiquitination of G alpha s. This may explain, at least in part, the transient nature of Bartter syndrome caused by MAGED2 mutations and opens new avenues for therapy in these patients.