Heme-binding Protein HRG-1 Is Induced by Insulin-like Growth Factor I and Associates with the Vacuolar H+-ATPase to Control Endosomal pH and Receptor Trafficking

Katie M O'Callaghan,Veronica Ayllon,Jean O'Keeffe,Yanru Wang,Orla T Cox,Gary Loughran,Michael Forgac,Rosemary O'Connor

doi:10.1074/jbc.m109.063248

Katie M O'Callaghan, Veronica Ayllon + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m109.063248

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Abstract

Endocytosis and trafficking of receptors and nutrient transporters are dependent on an acidic intra-endosomal pH that is maintained by the vacuolar H(+)-ATPase (V-ATPase) proton pump. V-ATPase activity has also been associated with cancer invasiveness. Here, we report on a new V-ATPase-associated protein, which we identified in insulin-like growth factor I (IGF-I) receptor-transformed cells, and which was separately identified in Caenorhabditis elegans as HRG-1, a member of a family of heme-regulated genes. We found that HRG-1 is present in endosomes but not in lysosomes, and it is trafficked to the plasma membrane upon nutrient withdrawal in mammalian cells. Suppression of HRG-1 with small interfering RNA causes impaired endocytosis of transferrin receptor, decreased cell motility, and decreased viability of HeLa cells. HRG-1 interacts with the c subunit of the V-ATPase and enhances V-ATPase activity in isolated yeast vacuoles. Endosomal acidity and V-ATPase assembly are decreased in cells with suppressed HRG-1, whereas transferrin receptor endocytosis is enhanced in cells that overexpress HRG-1. Cellular uptake of a fluorescent heme analogue is enhanced by HRG-1 in a V-ATPase-dependent manner. Our findings indicate that HRG-1 regulates V-ATPase activity, which is essential for endosomal acidification, heme binding, and receptor trafficking in mammalian cells. Thus, HRG-1 may facilitate tumor growth and cancer progression.

Highlights

An increasingly acidic lumenal pH gradient from early to late endosomes is required for receptor trafficking and is maintained by the V-ATPase proton pump acting in concert with other pH regulatory channels [9, 10]
HRG-1 mRNA and Protein Are Induced by insulin-like growth factor I (IGF-I)—The mouse slc48a1/hrg-1 gene (NCBI accession number NM_ 026353) is expressed as two transcripts of 2.5 and 1.6 kb that are more abundant in Rϩ cells than in RϪ cells (Fig. 1A)
HRG-1 Is Required for Receptor Endocytosis, Cell Migration, and Survival—As HRG-1 is an endosomal protein, we investigated whether HRG-1 expression is important for endocytosis, and cell survival or migration, by suppressing its expression with Small Interfering RNA (siRNA) in HeLa cells

Summary

Introduction

An increasingly acidic lumenal pH gradient from early to late endosomes is required for receptor trafficking and is maintained by the V-ATPase proton pump acting in concert with other pH regulatory channels [9, 10]. In an effort to identify IGF-I-regulated proteins that are important for cancer progression, we isolated a series of genes that are differentially expressed in nontransformed cells (RϪ, derived from the IGF-IR knock-out mouse) and highly transformed cells (Rϩ and RϪ cells that overexpress the IGF-IR) [17,18,19]. Among these was a gene encoding an endosomal protein that we found to associate with the endosomal V-ATPase and to regulate V-ATPase activity in endosomes. Our data indicate that HRG-1 is the first known heme-binding protein that regulates function of the V-ATPase in endosome acidification and trafficking of receptors essential for cell metabolism

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