Abstract
Solute carrier family 20 member 2 (SLC20A2) and xenotropic and polytropic retrovirus receptor 1 (XPR1) are transporters with phosphate uptake and efflux functions, respectively. Both are associated with primary familial brain calcification (PFBC), a genetic disease characterized by cerebral calcium-phosphate deposition and associated with neuropsychiatric symptoms. The association of the two transporters with the same disease suggests that they jointly regulate phosphate fluxes and cellular homeostasis, but direct evidence is missing. Here, we found that cross-talk between SLC20A2 and XPR1 regulates phosphate homeostasis, and we identified XPR1 as a key inositol polyphosphate (IP)-dependent regulator of this process. We found that overexpression of WT SLC20A2 increased phosphate uptake, as expected, but also unexpectedly increased phosphate efflux, whereas PFBC-associated SLC20A2 variants did not. Conversely, SLC20A2 depletion decreased phosphate uptake only slightly, most likely compensated for by the related SLC20A1 transporter, but strongly decreased XPR1-mediated phosphate efflux. The SLC20A2-XPR1 axis maintained constant intracellular phosphate and ATP levels, which both increased in XPR1 KO cells. Elevated ATP levels are a hallmark of altered inositol pyrophosphate (PP-IP) synthesis, and basal ATP levels were restored after phosphate efflux rescue with WT XPR1 but not with XPR1 harboring a mutated PP-IP-binding pocket. Accordingly, inositol hexakisphosphate kinase 1-2 (IP6K1-2) gene inactivation or IP6K inhibitor treatment abolished XPR1-mediated phosphate efflux regulation and homeostasis. Our findings unveil an SLC20A2-XPR1 interplay that depends on IPs such as PP-IPs and controls cellular phosphate homeostasis via the efflux route, and alteration of this interplay likely contributes to PFBC.
Highlights
The solute carrier (SLC) SLC20 and SLC53 family genes encode three cell surface multitransmembrane proteins known as PiT1/SLC20A1, PiT2/SLC20A2, and polytropic retrovirus receptor 1 (XPR1)/SLC53A1, which were shown to transport phosphate [1,2,3] and to serve as retroviral receptors for virus infection [4,5,6,7,8]
We found that SLC20A2 and xenotropic and polytropic retrovirus receptor 1 (XPR1) regulated the rates of phosphate fluxes in a concerted and PP-inositol polyphosphate (IP)-dependent manner and controlled intracellular phosphate and ATP levels, providing evidence for a specific SLC20A2, XPR1, and inositol pyrophosphate (PP-IP) interplay in phosphate homeostasis and metabolism, with new insights on the role of phosphate metabolic disorders in Primary familial brain calcification (PFBC)
With the exception of SLC20A2 p.DA145_V205, which most likely encoded a defective transporter, as it was undetectable by flow cytometry (Fig. 1A) or immunoblotting (Fig. 1B), all of the other SLC20A2 mutants were expressed at the expected size, were present at the plasma membrane as efficiently as WT SLC20A2, and did not affect cell surface expression of endogenous SLC20A1 and XPR1, as observed by flow cytometry using specific retroviral ligands derived from receptor-binding domains (RBDs) of gammaretrovirus envelope (Env) glycoproteins [1, 25, 40, 41]. 33P-radiolabeled phosphate flux assays showed that overexpression of human SLC20A2 led to a significant increase in phosphate uptake, as expected, compared with control parental cells (Fig. 1C)
Summary
The solute carrier (SLC) SLC20 and SLC53 family genes encode three cell surface multitransmembrane proteins known as PiT1/SLC20A1, PiT2/SLC20A2, and polytropic retrovirus receptor 1 (XPR1)/SLC53A1, which were shown to transport phosphate [1,2,3] and to serve as retroviral receptors for virus infection [4,5,6,7,8]. The intracellular level of phosphate is tightly regulated, given its central role in many key functions in cells. The deposition of calcium phosphate crystals in the brain and the association of two phosphate transporter genes with PFBC indicate a direct link between phosphate regulation dysfunction and the disease. Loss of function of either SLC20A2 or XPR1 results in the same PFBC disorder despite opposite phosphate transport directions. We found that SLC20A2 and XPR1 regulated the rates of phosphate fluxes in a concerted and PP-IP-dependent manner and controlled intracellular phosphate and ATP levels, providing evidence for a specific SLC20A2, XPR1, and PP-IP interplay in phosphate homeostasis and metabolism, with new insights on the role of phosphate metabolic disorders in PFBC
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