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

Abstract

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 (Gαs). Given the role of Gαs 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 Gαs, which was fully reversible upon reoxygenation. In contrast to Gαs, cell surface expression of the β2-adrenergic receptor, which is coupled to Gαs, was not affected by MAGED2 depletion, demonstrating specific regulation of Gαs by MAGED2. Importantly, the internalization of Gαs due to MAGED2 deficiency significantly reduced cAMP generation and PKA activity. Interestingly, the internalization of Gαs 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 Gαs under hypoxia. Remarkably, MDM2 depletion or its chemical inhibition fully abolished Gαs-endocytosis following MAGED2 knockdown. Moreover, endocytosis of Gαs was also blocked by mutation of ubiquitin acceptor sites in Gαs. Thus, we reveal that MAGED2 is essential for the cAMP/PKA pathway under hypoxia to specifically regulate Gαs endocytosis by blocking MDM2-dependent ubiquitination of Gαs. 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.

Reciprocal Regulation of MAGED2 and HIF-1α Augments Their Expression under Hypoxia: Role of cAMP and PKA Type II

“…To further support this notion, we also showed that forskolin increases the abundance of MAGED2 transcript under hypoxic conditions, which is in agreement with the presence of four CRE sites in the 2000 bp of the MAGED2 promoter. The lack of effect of isoproterenol in MAGED2-depleted cells on the hypoxic induction of HIF-1α concurs with our previous study showing that MAGED2 is essential for Gαs dependent activation of membrane-bound adenylate cyclase [ 9 ] thereby explaining that agents acting upstream of Gαs such isoproterenol are ineffective in MAGED2-depleted cells.…”

“…We have shown previously that MAGED2 depletion inhibits the functioning of Gαs by causing its MDM2 and ubiquitin-dependent internalization, which precludes activation of membrane-bound adenylate cyclase and hence cAMP generation [ 9 ]. To investigate if the effects of the MAGED2 knockdown on HIF-1α expression are indeed caused by inhibiting Gαs, we analyzed also the effect of GNAS knockdown on hypoxic HIF-1α induction by exposing cells to physical hypoxia ( Figure 3 a,b) or to the hypoxia mimetic CoCl 2 ( Figure 3 c,d).…”

“…Given that transient overexpression induces ER stress [ 40 ], especially when overexpressing transmembrane proteins [ 41 ], we speculate that this stress rendered the maturation of NKCC2 and NCC MAGED2 sensitive. Along these lines, it is conceivable that the effects of MAGED2 depletion on HIF-1 α induction and G α s localization seen with cobalt chloride results from redox stress [ 9 ]. Taken together, we speculate that MAGED2 protects against various forms of stress, namely hypoxic stress, ER stress, and redox stress.…”

“…We and others have recently shown, that truncating mutations in MAGED2 , which promotes expression and activity of salt-transporter NKCC2, cause a similar antenatal phenotype during gestation as those in the gene encoding NKCC2 [ 6 , 7 , 8 ]. More recently, we demonstrated that MAGED2 is required under hypoxia to allow for the generation of cAMP, which is essential for the cell surface expression and function of NKCC2, thus explaining, at least in part, the salt loss in patients with MAGED2 mutations [ 9 ]. The idea that MAGED2 shields renal salt transport from hypoxic stress by allowing cAMP generation needed for NKKC2 expression and function concurs with recent studies demonstrating that many members of the large family of MAGE proteins protect against various forms of stress [ 10 ] by modulating activity and substrate specificity of ubiquitin E3 ligases [ 11 ].…”

Case Report: Transient antenatal bartter syndrome in an extremely preterm infant with a novel MAGED2 variant

“…Bartter syndrome is a group of inherited renal tubular diseases characterised by secondary aldosteronism, hypokalaemia and hypochloraemic metabolic alkalosis; blood pressure is not affected ( 4 ). The MAGED2 encodes melanoma-associated antigen D2 (MAGE-D2), required for activation of the cyclic adenosine monophosphate/protein kinase A pathway under hypoxic conditions ( 5 ). MAGE-D2 participates in the biofunction of Na + -K + -2Cl− cotransporters (NKCC2) and Na + -2Cl− cotransporters (NCC), possibly by cooperating with Hsp40 and Gs α proteins, respectively.…”