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

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