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Proc Natl Acad Sci U S A. 2017 Nov 7;114(45):E9559-E9568. doi: 10.1073/pnas.1712670114. Epub 2017 Oct 23.

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

Author information

1
Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114.
2
Program in Membrane Biology, Department of Medicine and Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114.
3
Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, MA 02118.
4
Department of Cellular and Molecular Physiology, Institute of Translational Medicine University of Liverpool, Liverpool L69 3BX, United Kingdom.
5
Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; bastepe@helix.mgh.harvard.edu.

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 GNAS, 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.

KEYWORDS:

GNAS; endocytosis; heterotrimeric G proteins; stimulatory G protein; transferrin

PMID:
29078380
PMCID:
PMC5692581
DOI:
10.1073/pnas.1712670114
[Indexed for MEDLINE]
Free PMC Article

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