Large G protein α-subunit XLαs limits clathrin-mediated endocytosis and regulates tissue iron levels in vivo

Abstract

Alterations in the activity/levels of the extralarge G protein α-subunit (XLαs) are implicated in various human disorders, such as perinatal growth retardation. Encoded by , XLαs is partly identical to the α-subunit of the stimulatory G protein (Gsα), but the cellular actions of XLαs remain poorly defined. Following an initial proteomic screen, we identified sorting nexin-9 (SNX9) and dynamins, key components of clathrin-mediated endocytosis, as binding partners of XLαs. Overexpression of XLαs in HEK293 cells inhibited internalization of transferrin, a process that depends on clathrin-mediated endocytosis, while its ablation by CRISPR/Cas9 in an osteocyte-like cell line (Ocy454) enhanced it. Similarly, primary cardiomyocytes derived from XLαs knockout (XLKO) pups showed enhanced transferrin internalization. Early postnatal XLKO mice showed a significantly higher degree of cardiac iron uptake than wild-type littermates following iron dextran injection. In XLKO neonates, iron and ferritin levels were elevated in heart and skeletal muscle, where XLαs is normally expressed abundantly. XLKO heart and skeletal muscle, as well as XLKO Ocy454 cells, showed elevated SNX9 protein levels, and siRNA-mediated knockdown of SNX9 in XLKO Ocy454 cells prevented enhanced transferrin internalization. In transfected cells, XLαs also inhibited internalization of the parathyroid hormone and type 2 vasopressin receptors. Internalization of transferrin and these G protein-coupled receptors was also inhibited in cells expressing an XLαs mutant missing the Gα portion, but not Gsα or an N-terminally truncated XLαs mutant unable to interact with SNX9 or dynamin. Thus, XLαs restricts clathrin-mediated endocytosis and plays a critical role in iron/transferrin uptake in vivo.

A G protein–coupled, IP3/protein kinase C pathway controlling the synthesis of phosphaturic hormone FGF23

“…Both the basal and PTH-induced levels of cAMP were higher in XLKO Ocy454 cells than in control cells (Supplemental Figure 2A). These enhanced cAMP levels in XLKO cells are consistent with our previous finding that Gsα protein level is moderately elevated in XLKO Ocy454 cells (38). We have previously demonstrated that ablation of XLαs represses IP3 generation and the total levels of several PKC isoforms, including PKCα and PKCδ, in renal proximal tubules (25).…”

“…XLαs, like Gαs, can stimulate generation of the second messenger cAMP. We thus examined the cAMP levels in Ocy454 cells, an osteocyte-like cell line (37), in which we have previously ablated XLαs by using CRISPR/Cas9 (38). Both the basal and PTH-induced levels of cAMP were higher in XLKO Ocy454 cells than in control cells (Supplemental Figure 2A).…”

“…In contrast, 1,25(OH) 2 D-and phosphate-induced FGF23 production may depend, at least partly, on the G protein/PKC pathway, given that the significantly elevated 1,25(OH) 2 D and phosphate levels in XLKO pups were insufficient to maintain the normal level of FGF23. Likewise, XLKO pups have been reported to have serum iron deficiency (38), and therefore, it appears that lack of XLαs also impedes the effect of iron deficiency on FGF23 production. Consistent with these conclusions, MAPK signaling has been implicated in FGF23 synthesis in response to both iron deficiency and phosphate (56)(57)(58).…”

“…The Ocy454 cells, which were previously described (37), were maintained in α-minimum essential medium (α-MEM, Gibco) with 10% FBS at 33°C and differentiated into osteocyte-like cells by growing at 37°C for 1 week (37,62). XLKO and control clonal Ocy454 cells were generated previously (38) and used here for protein and mRNA analyses. Saos-2 cells were obtained from ATCC (ATCC HTB-85) and cultured at 37°C in McCoy's 5a medium (Gibco) supplemented with 15% FBS.…”

Genome Editing Provides New Insights into Receptor-Controlled Signalling Pathways

“…The GNAS gene that encodes Gs can also generate a longer, N-terminally extended, variant described as XLαs. It was recently indicated that XLαs can interact with various intracellular sorting proteins that are key contributors to clathrin-mediated endocytosis, including nexin-9 and dynamins [30]. Elimination of XLαs from the osteocyte-like cell line Ocy454 using CRISPR/Cas9 increased internalisation of transferrin, whilst further studies indicated this to be a specific role for XLαs rather than for conventional Gs [30].…”