The mechanism by which a novel major histocompatibility complex class I protein, HFE, regulates iron uptake into the body is not known. HFE is the product of the gene that is mutated in >80% of hereditary hemochromatosis patients. Immunofluorescence experiments indicate that they also endocytose into transferrin-positive compartments. Combined, these results suggest a role for the transferrin receptor in HFE trafficking. Cells expressing HFE have modestly increased levels of transferrin receptor and drastically reduced levels of ferritin. These results implicate HFE further in the modulation of iron levels in the cell.

pp120/HA4 is a hepatocyte membrane glycoprotein phosphorylated by the insulin receptor tyrosine kinase. In this study, we have investigated the role of pp120/HA4 in insulin action. Transfection of antisense pp120/HA4 cDNA in H35 hepatoma cells resulted in inhibition of pp120/HA4 expression and was associated with a 2-3-fold decrease in the rate of insulin internalization. Furthermore, insulin internalization in NIH 3T3 fibroblasts co-transfected with insulin receptors and pp120/HA4 was increased 2-fold compared with cells expressing insulin receptors alone. In contrast, no effect on internalization was observed in cells overexpressing a naturally occurring splice variant of pp120/HA4 that lacks the phosphorylation sites in the intracellular domain. Insulin internalization was also unaffected in cells expressing three site-directed mutants of pp120/HA4 in which the sites of phosphorylation by the insulin receptor kinase had been removed (Y488F, Y488F/Y513F, and S503A). Our data suggest that pp120/HA4 is part of a complex of proteins required for receptor-mediated internalization of insulin. It is possible that this function is regulated by insulin-induced phosphorylation of the intracellular domain of pp120/HA4.Insulin binding to its receptor triggers the rapid endocytosis of the ligand-receptor complex (1, 2). Internalization of the insulin-insulin receptor complex constitutes the major mechanism of insulin degradation and down-regulation of cell surface receptors (3-5). The molecular events involved in the internalization process, however, are yet to be well defined. Specific sequences in the submembranous portion of the receptor are required for internalization to occur. Furthermore, activation of the receptor kinase is required for ligand-induced receptor internalization (6 -15). Following insulin-induced receptor autophosphorylation, several intracellular substrates are phosphorylated, the best characterized of which is insulin receptor substrate-1 (IRS-1) (16). However, the role of substrate phosphorylation in receptor internalization is not established.In the present study, we have investigated the role of pp120/ HA4, a substrate of the insulin receptor kinase that is predominantly expressed in liver, in the internalization process. pp120/HA4 is a transmembrane glycoprotein that is phosphorylated by the insulin receptor tyrosine kinase in intact cells (17)(18)(19)(20) and in cell-free systems (21). It is composed of a large extracellular domain containing 16 sites of potential N-linked glycosylation and a 71-amino acid cytoplasmic domain (22). Studies of site-directed mutants indicate that the intracellular domain contains a site of constitutive serine phosphorylation (Ser 503 ) as well as one tyrosine residue that is phosphorylated in response to insulin (Tyr 488 ) (20,23). Alternative splicing of pp120/HA4 mRNA generates two isoforms, one of which lacks 61 amino acids at the C terminus of the cytoplasmic domain, including all the potential phosphorylation sites (24). The function of pp120/HA4 is still un...

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Among patients with hepatic iron overload, the distinction between hereditary hemochromatosis (HH), a common yet treatable genetic disease, and other causes of siderosis remains problematic. The recent discovery of a specific homozygous mutation (C282Y) in a novel major histocompatibility complex class I-like gene (named HLA-H or HFE) in 80% to 100% of well-characterized cases of HH suggests that direct DNA-based mutation analysis may help resolve this dilemma. To assess the clinical utility of direct HLA-H mutation analysis in a typical diagnostic setting, we measured genotypic and phenotypic parameters of iron overload in 37 subjects with biopsy-proven hepatic siderosis (2+ or greater) and in 127 healthy control subjects. The prevalence of C282Y homozygotes was significantly greater in the hepatic siderosis group (32%) than in the control group (0%), confirming the association between this homozygous mutation and hepatic iron overload. In the hepatic siderosis group, C282Y homozygotes had significantly higher hepatic iron and ferritin levels, a significantly lower prevalence of hepatitis C virus or alcoholic liver disease, but no significant difference in the saturation of serum transferrin. Of the 20 subjects with a hepatic iron index (HII) in the previously defined "hemochromatosis range" (>1.9), 9 (45%) were C282Y homozygotes. Of the 11 nonhomozygous subjects with an HII greater than 1.9 (presumed false-positive HIIs), 10 (91%) had hepatic cirrhosis compared with 3 of 9 (33%) homozygotes with an HII greater than 1.9 who had cirrhosis (P<.02). The HII thus has poor diagnostic specificity for predicting genotypic HH in patients with cirrhosis. We conclude that direct determination of the HLA-H C282Y genotype may be the single best diagnostic test for HH, particularly in patients with cirrhosis, for whom the HII is quite nonspecific.