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Large G protein ?-subunit XL?s limits clathrin-mediated endocytosis and regulates tissue iron levels in vivo.

ABSTRACT: Alterations in the activity/levels of the extralarge G protein ?-subunit (XL?s) are implicated in various human disorders, such as perinatal growth retardation. Encoded by GNAS, XL?s is partly identical to the ?-subunit of the stimulatory G protein (Gs?), but the cellular actions of XL?s 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 XL?s. Overexpression of XL?s 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 XL?s 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 XL?s 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, XL?s 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 XL?s mutant missing the G? portion, but not Gs? or an N-terminally truncated XL?s mutant unable to interact with SNX9 or dynamin. Thus, XL?s restricts clathrin-mediated endocytosis and plays a critical role in iron/transferrin uptake in vivo.

SUBMITTER: He Q

PROVIDER: S-EPMC5692581 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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

He Qing Q Bouley Richard R Liu Zun Z Wein Marc N MN Zhu Yan Y Spatz Jordan M JM Wang Chia-Yu CY Divieti Pajevic Paola P Plagge Antonius A Babitt Jodie L JL Bastepe Murat M

Proceedings of the National Academy of Sciences of the United States of America 20171023 45

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 <i>GNAS</i>, 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. Overexpressio ...[more]

PMID: 29078380

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Project description:BackgroundLymphotoxin β receptor (LTβR) is a member of tumor necrosis factor receptor (TNFR) superfamily which regulates the immune response. At the cellular level, upon ligand binding, the receptor activates the pro-inflammatory NF-κB and AP-1 pathways. Yet, the intracellular distribution of LTβR, the routes of its endocytosis and their connection to the signaling activation are not characterized. Here, we investigated the contribution of LTβR internalization to its signaling potential.MethodsIntracellular localization of LTβR in unstimulated and stimulated cells was analyzed by confocal microscopy. Endocytosis impairment was achieved through siRNA- or CRISPR/Cas9-mediated depletion, or chemical inhibition of proteins regulating endocytic routes. The activation of LTβR-induced signaling was examined. The levels of effector proteins of the canonical and non-canonical branches of the NF-κB pathway, and the phosphorylation of JNK, Akt, ERK1/2, STAT1 and STAT3 involved in diverse signaling cascades, were measured by Western blotting. A transcriptional response to LTβR stimulation was assessed by qRT-PCR analysis.ResultsWe demonstrated that LTβR was predominantly present on endocytic vesicles and the Golgi apparatus. The ligand-bound pool of the receptor localized to endosomes and was trafficked towards lysosomes for degradation. Depletion of regulators of different endocytic routes (clathrin-mediated, dynamin-dependent or clathrin-independent) resulted in the impairment of LTβR internalization, indicating that this receptor uses multiple entry pathways. Cells deprived of clathrin and dynamins exhibited enhanced activation of canonical NF-κB signaling represented by increased degradation of IκBα inhibitor and elevated expression of LTβR target genes. We also demonstrated that clathrin and dynamin deficiency reduced to some extent LTβR-triggered activation of the non-canonical branch of the NF-κB pathway.ConclusionsOur work shows that the impairment of clathrin- and dynamin-dependent internalization amplifies a cellular response to LTβR stimulation. We postulate that receptor internalization restricts responsiveness of the cell to subthreshold stimuli. Video Abstract.

Dataset Information

Large G protein ?-subunit XL?s limits clathrin-mediated endocytosis and regulates tissue iron levels in vivo.

Publications

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

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