Anja Mehlum

Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes.

Hrs and the endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are involved in the endosomal sorting of membrane proteins into multivesicular bodies and lysosomes or vacuoles. The ESCRT complexes are also required for formation of intraluminal endosomal vesicles and for budding of certain enveloped RNA viruses such as HIV. Here, we show that Hrs binds to the ESCRT-I subunit Tsg101 via a PSAP motif that is conserved in Tsg101-binding viral proteins. Depletion of Hrs causes a reduction in membrane-associated ESCRT-I subunits, a decreased number of multivesicular bodies and an increased size of late endosomes. Even though Hrs mainly localizes to early endosomes and Tsg101 to late endosomes, the two proteins colocalize on a subpopulation of endosomes that contain lyso-bisphosphatidic acid. Overexpression of Hrs causes accumulation of Tsg101 on early endosomes and prevents its localization to late endosomes. We conclude that Hrs mediates the initial recruitment of ESCRT-I to endosomes and, thereby, indirectly regulates multivesicular body formation.

Hrs recruits clathrin to early endosomes

The hepatocyte growth factor‐regulated tyrosine kinase substrate, Hrs, has been implicated in intracellular trafficking and signal transduction. Hrs contains a phosphatidylinositol 3‐phosphate‐binding FYVE domain that contributes to its endosomal targeting. Here we show that Hrs and EEA1, a FYVE domain protein involved in endocytic membrane fusion, are localized to different regions of early endosomes. We demonstrate that Hrs co‐localizes with clathrin, and that the C‐terminus of Hrs contains a functional clathrin box motif that interacts directly with the terminal β‐propeller domain of clathrin heavy chain. A massive recruitment of clathrin to early endosomes was observed in cells transfected with Hrs, but not with Hrs lacking the C‐terminus. Furthermore, the phosphatidylinositol 3‐kinase inhibitor wortmannin caused the dissociation of both Hrs and clathrin from endosomes. While overexpression of Hrs did not affect endocytosis and recycling of transferrin, endocytosed epidermal growth factor and dextran were retained in early endosomes. These results provide a molecular mechanism for the recruitment of clathrin onto early endosomes and suggest a function for Hrs in trafficking from early to late endosomes.

STAM and Hrs are subunits of a multivalent ubiquitin-binding complex on early endosomes

STAM1 and STAM2, which have been identified as regulators of receptor signaling and trafficking, interact directly with Hrs, which mediates the endocytic sorting of ubiquitinated membrane proteins. The STAM proteins interact with the same coiled-coil domain that is involved in the targeting of Hrs to endosomes. In this work, we show that STAM1 and STAM2, as well as an endocytic regulator protein, Eps15, can be co-immunoprecipitated with Hrs both from membrane and cytosolic fractions and that recombinant Hrs, STAM1/STAM2, and Eps15 form a ternary complex. We find that overexpression of Hrs causes a strong recruitment of STAM2 to endosome membranes. Moreover, STAM2, like Hrs and Eps15, binds ubiquitin, and Hrs, STAM2, and Eps15 colocalize with ubiquitinated proteins in clathrin-containing endosomal microdomains. The localization of Hrs, STAM2, Eps15, and clathrin to endosome membranes is controlled by the AAA ATPase mVps4, which has been implicated in multivesicular body formation. Depletion of cellular Hrs by small interfering RNA results in a strongly reduced recruitment of STAM2 to endosome membranes and an impaired degradation of endocytosed epidermal growth factor receptors. We propose that Hrs, Eps15, and STAM proteins function in a multivalent complex that sorts ubiquitinated proteins into the multivesicular body pathway.

Regulation and intracellular trafficking pathways of the endothelin receptors.

The effects of endothelin (ET) are mediated via the G protein-coupled receptors ETA and ETB. However, the mechanisms of ET receptor desensitization, internalization, and intracellular trafficking are poorly understood. The aim of the present study was to investigate the molecular mechanisms of ET receptor regulation and to characterize the intracellular pathways of ET-stimulated ETA and ETB receptors. By analysis of ETA and ETB receptor internalization in transfected Chinese hamster ovary cells in the presence of overexpressed βARK, β-arrestin-1, β-arrestin-2, or dynamin as well as dominant negative mutants of these regulators, we have demonstrated that both ET receptor subtypes follow an arrestin- and dynamin/clathrin-dependent mechanism of internalization. Fluorescence microscopy of Chinese hamster ovary and COS cells expressing green fluorescent protein (GFP)-tagged ET receptors revealed that the ETA and ETB subtypes were targeted to different intracellular routes after ET stimulation. While ETA-GFP followed a recycling pathway and colocalized with transferrin in the pericentriolar recycling compartment, ETB-GFP was targeted to lysosomes after ET-induced internalization. Both receptor subtypes colocalized with Rab5 in classical early endosomes, indicating that this compartment is a common early intermediate for the two ET receptors during intracellular transport. The distinct intracellular routes of ET-stimulated ETA and ETB receptors may explain the persistent signal response through the ETAreceptor and the transient response through the ETBreceptor. Furthermore, lysosomal targeting of the ETBreceptor could serve as a biochemical mechanism for clearance of plasma endothelin via this subtype.

1 kb of the lactase-phlorizin hydrolase promoter directs post-weaning decline and small intestinal-specific expression in transgenic mice

Adult‐type hypolactasia is a genetic condition making approximately one half of the human population intolerant to milk because of abdominal symptoms. The cause is a post‐weaning down‐regulation of the intestinal‐specific enzyme lactase‐phlorizin hydrolase (LPH) reducing the intestinal capacity to hydrolyze lactose. We here demonstrate that the stretch −17 to −994 in the pig LPH‐promoter carries cis‐elements which direct a small intestinal‐specific expression and a post‐weaning decline of a linked rabbit β‐globin gene. These data demonstrate that the post‐weaning decline of LPH is mainly due to a transcriptional down‐regulation.

Mice overexpressing human lecithin: cholesterol acvltransferase are not protected against diet-induced atherosclerosis

Lecithin: cholesterol acyltransferase (LCAT)(EC 2.3.1.43) is generally assumed to participate in reverse cholesterol transport, i.e., cholesterol transport from peripheral tissues to the liver. LCAT is secreted by the liver and transported in plasma mostly associated with high density lipoprotein. It catalyzes the esterification of cholesterol, mainly high density lipoprotein cholesterol, and produces cholesteryl ester and lysolecithin. Transgenic mice overexpressing human LCAT on a C57BL/6 background have elevated high density lipoprotein cholesterol and markedly reduced low and very low density lipoprotein cholesterol and triglyceride levels in plasma, suggesting that such mice may be less susceptible to diet‐induced atherosclerosis than isogenic nontransgenic controls. To determine if the apparent anti‐atherogenic lipoprotein profile of the LCAT transgenics reduced their susceptibility to atherogenesis, the atherosclerotic lesions developing in transgenic LCAT mice and controls when fed an atherogenic diet were compared by histology and morphometry. Histological examination of the aortas from mice fed a high fat diet for 12, 17 and 22 weeks revealed that the aortic lesions were no smaller or less developed in the transgenic LCAT mice than in the C57BL/6 controls. After 17 weeks there were significantly more “fatty streaks” in the transgenic mice than in the controls. Thus, overexpression of human LCAT in transgenic mice, in spite of their very favourable blood lipoprotein and lipid profile, does not protect against development of atherosclerosis.

Function of hrs in endocytic trafficking and signalling

The hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, becomes tyrosine-phosphorylated upon the binding of various growth factors and cytokines to their receptors. This protein is essential for ventral folding morphogenesis, and it shares structural similarity with Vps27p, which is involved in vacuolar protein sorting in yeast. Since Hrs is localized to endosomes and has been implicated in the regulation of signal transduction as well as membrane trafficking, it has been regarded as a potential co-ordinator of endosomal receptor sorting and signalling. Here we discuss the possible functions of Hrs in light of its interactions with phosphatidylinositol 3-phosphate and multiple proteins.

Overexpression of human lecithin:cholesterol acyltransferase in mice offers no protection against diet-induced atherosclerosis.

Human lecithin:cholesterol acyltransferase (LCAT) is a key enzyme in the metabolism of cholesterol. We have used homozygous transgenic mice overexpressing the human LCAT transgene to study the effect of a “Western‐type” atherogenic diet (30% fat, 5% cholesterol and 2% cholic acid) on their LCAT expression, activity, lipoprotein profile and tendency to develop atherosclerosis. The LCAT activity was 35‐fold higher in serum of the homozygous transgenic mice than in murine control serum, and decreased 11–20% in the transgenic mice when fed the atherogenic diet. The total cholesterol and high‐density lipoprotein cholesterol (HDL‐C) concentrations were approximately doubled in the transgenic mice compared with the controls when both groups were fed a regular chow diet. In mice on the atherogenic diet, the triglyceride concentration decreased about 50% to the same level in transgenic and control mice. Total cholesterol and HDL‐C concentrations increased and were 60–80% higher in the transgenic mice. The expression of LCAT mRNA in the liver was decreased by 49–60% in the transgenic mice when fed the atherogenic diet. The development of atherosclerosis was similar in transgenic and control mice. Thus, the 14‐ to 27‐fold higher LCAT activity and the higher HDL‐C concentrations in the homozygous LCAT transgenic mice had no significant protective influence on the development of diet‐induced atherosclerosis.

Isolation of serum amyloid A protein by small-scale hydrophobic interaction chromatography and two-dimensional electrophoresis

A recently introduced technique to isolate serum amyloid A protein is hydrophobic interaction chromatography combined with two-dimensional electrophoresis with immobilized pH gradients. A modification of the original version of this technique is presented. Mouse serum was subjected to hydrophobic interaction chromatography on a small scale, and the eluate was applied directly to two-dimensional electrophoresis. Simple electropherogramss with optimal resolution of serum amyloid A protein were obtained. The presented technique facilitates isolation of serum amyloid A protein from small blood volumes, and might also be adapted to alternative applications.