PI3P signaling regulates receptor sorting but not transport in the endosomal pathway

While evidence is accumulating that phosphoinositide signaling plays a crucial role in growth factor and hormone receptor down-regulation, this signaling pathway has also been proposed to regulate endosomal membrane transport and multivesicular endosome biogenesis. Here, we have followed the fate of the down-regulated EGF receptor (EGFR) and bulk transport (fluid phase) markers in the endosomal pathway in vivo and in vitro. We find that bulk transport from early to late endosomes is not affected after inhibition of the phosphatidylinositol-3-phosphate (PI3P) signaling pathway, but that the EGFR then remains trapped in early endosomes. Similarly, we find that hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is not directly involved in bulk solute transport, but is required for EGFR sorting. These observations thus show that transport and sorting can be uncoupled in the endosomal pathway. They also show that PI3P signaling does not regulate the core machinery of endosome biogenesis and transport, but controls the sorting of down-regulated receptor molecules in early endosomes via Hrs.     

Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes

After endocytosis, some membrane proteins recycle from early endosomes to the plasma membrane whereas others are transported to late endosomes and lysosomes for degradation. Conjugation with the small polypeptide ubiquitin is a signal for lysosomal sorting. Here we show that the hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, is involved in the endosomal sorting of ubiquitinated membrane proteins. Hrs contains a clathrin-binding domain, and by electron microscopy we show that Hrs localizes to flat clathrin lattices on early endosomes. We demonstrate that Hrs binds directly to ubiquitin by way of a ubiquitin-interacting motif (UIM), and that ubiquitinated proteins localize specifically to Hrs- and clathrin-containing microdomains. Whereas endocytosed transferrin receptors fail to colocalize with Hrs and rapidly recycle to the cell surface, transferrin receptors that are fused to ubiquitin interact with Hrs, localize to Hrs- and clathrin-containing microdomains and are sorted to the degradative pathway. Overexpression of Hrs strongly and specifically inhibits recycling of ubiquitinated transferrin receptors by a mechanism that requires a functional UIM. We conclude that Hrs sorts ubiquitinated membrane proteins into clathrin-coated microdomains of early endosomes, thereby preventing their recycling to the cell surface.     

EPR studies of the mitochondrial alternative oxidase. Evidence for a diiron carboxylate center

The alternative oxidase (AOX) is a ubiquinol oxidase found in the mitochondrial respiratory chain of plants as well as some fungi and protists. It has been predicted to contain a coupled diiron center on the basis of a conserved sequence motif consisting of the proposed iron ligands, four glutamate and two histidine residues. However, this prediction has not been experimentally verified. Here we report the high level expression of the Arabidopsis thaliana alternative oxidase AOX1a as a maltose-binding protein fusion in Escherichia coli. Reduction and reoxidation of a sample of isolated E. coli membranes containing the alternative oxidase generated an EPR signal characteristic of a mixed-valent Fe(II)/Fe(III) binuclear iron center. The high anisotropy of the signal, the low value of the g-average tensor, and a small exchange coupling (-J) suggest that the iron center is hydroxo-bridged. A reduced membrane preparation yielded a parallel mode EPR signal with a g-value of about 15. In AOX containing a mutation of a putative glutamate ligand of the diiron center (E222A or E273A) the EPR signals are absent. These data provide evidence for an antiferromagnetic-coupled binuclear iron center, and together with the conserved sequence motif, identify the alternative oxidase as belonging to the growing family of diiron carboxylate proteins. The alternative oxidase is the first integral membrane protein in this family, and adds a new catalytic activity (ubiquinol oxidation) to this group of enzymatically diverse proteins.     

Hypoxia-induced pulmonary artery adventitial remodeling and neovascularization: contribution of progenitor cells

Information is rapidly emerging regarding the important role of the arterial vasa vasorum in a variety of systemic vascular diseases. In addition, increasing evidence suggests that progenitor cells of bone marrow (BM) origin may contribute to postnatal neovascularization and/or vascular wall thickening that is characteristic in some forms of systemic vascular disease. Little is known regarding postnatal vasa formation and the role of BM-derived progenitor cells in the setting of pulmonary hypertension (PH). We sought to determine the effects of chronic hypoxia on the density of vasa vasorum in the pulmonary artery and to evaluate if BM-derived progenitor cells contribute to the increased vessel wall mass in a bovine model of hypoxia-induced PH. Quantitative morphometric analyses of lung tissue from normoxic and hypoxic calves revealed that hypoxia results in a dramatic expansion of the pulmonary artery adventitial vasa vasorum. Flow cytometric analysis demonstrated that cells expressing the transmembrane tyrosine kinase receptor for stem cell factor, c-kit, are mobilized from the BM in the circulation in response to hypoxia. Immunohistochemistry revealed an increase in the expression of c-kit+ cells together with vascular endothelial growth factor, fibronectin, and thrombin in the hypoxia-induced remodeled pulmonary artery vessel wall. Circulating mononuclear cells isolated from neonatal calves exposed to hypoxia were found to differentiate into endothelial and smooth muscle cell phenotypes depending on culture conditions. From these observations, we suggest that the vasa vasorum and circulating progenitor cells could be involved in vessel wall thickening in the setting of hypoxia-induced PH.     

Cbl-dependent ubiquitination is required for progression of EGF receptors into clathrin-coated pits

Ligand binding causes the EGF receptor (EGFR) to become ubiquitinated by Cbl upon association with the adaptor protein Grb2. We have investigated the role of ubiquitin and Grb2 in ligand-induced endocytosis of the EGFR. Incubation of cells with EGF on ice caused translocation of Grb2 and Cbl from the cytosol to the rim of coated pits. Grb2 with point mutations in both SH3 domains inhibited recruitment of the EGFR to clathrin-coated pits, in a Ras-independent manner. On overexpression of the Cbl-binding protein Sprouty, ubiquitination of the EGFR was inhibited, the EGFR was recruited only to the rim of coated pits, and endocytosis of the EGFR was inhibited. Conjugation-defective ubiquitin similarly inhibited recruitment of EGF-EGFR to clathrin-coated pits. Even though this does not prove that cargo must be ubiquitinated, this indicates the importance of interaction of ubiquitinated protein(s) with proteins harboring ubiquitin-interacting domains. We propose that Grb2 mediates transient anchoring of the EGFR to an Eps15-containing molecular complex at the rim of coated pits and that Cbl-induced ubiquitination of the EGFR allows relocation of EGFR from the rim to the center of clathrin-coated pits.     

Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells

Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane trafficking. In order to study the intracellular localization of the PI3K product, phosphatidylinositol 3-phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind specifically, and with high affinity, to PI(3)P both in vitro and in vivo. When expressed in fibroblasts, a tagged probe localized to endosomes, as detected by fluorescence microscopy. Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of multivesicular endosomes. While yeast cells deficient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of endosomes and vacuoles of wild-type yeast. vps27Delta yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endosome and the subsequent steps leading to multivesicular endosome formation.     

Endothelin receptor blockade decreases lung water in young rats exposed to viral infection and hypoxia

Viral respiratory infections may increase the susceptibility of young animals to hypoxia-induced pulmonary edema. Because hypoxia stimulates endothelin production, we hypothesized that an increase in lung endothelin contributes to these alterations in lung water. Weanling rats were infected with Sendai virus, causing a mild respiratory infection. At day 7 after infection, animals were exposed to hypoxia (inspired O(2) fraction = 0.1) for 24 h. Exposure to virus plus hypoxia led to increases in lung water compared with control groups (P < 0.001). Lung endothelin levels were significantly higher in the virus plus hypoxia group than in control groups (P < 0.001). A second group of infected animals received bosentan, a nonselective endothelin receptor antagonist, during exposure to hypoxia. Bosentan-treated animals showed less lung water accumulation, less lung lavage fluid protein, and less perivascular fluid cuffing than untreated animals (P < 0.01). We conclude that the combination of a recent viral respiratory infection and exposure to moderate hypoxia led to increases in endothelin in the lungs of young rats and that endothelin receptor blockade ameliorates the hypoxia-induced increases in lung water found in these animals.     

Transient reexpression of an embryonic autonomous growth phenotype by adult carotid artery smooth muscle cells after vascular injury

High rates of vascular smooth muscle cell (SMC) replication are observed, at least transiently, after injury to the arterial wall and contribute to the formation of a neointima. Neutralizing antibodies designed to inhibit growth of SMC have only been variably successful in inhibiting neointima formation, raising the possibility that neointimal cell proliferation involves unique growth mechanisms. This study examined the possibility that SMC isolated from injured rat carotid arteries would express an autonomous, mitogen-independent growth phenotype similar to that utilized by embryonic vascular SMC during periods of rapid growth. We found that primary cultures of SMC isolated 7 and 14 days after injury, times at which high in vivo replication rates were observed, demonstrated high intrinsic DNA synthetic rates compared to SMC isolated from uninjured arteries or at 2, 4, 21, and 28 days after injury where in vivo replication rates were far less. Subcultured SMC isolated from 7-day injured vessels (Neo7 SMC) exhibited a stable, autonomous growth phenotype, did not secrete detectable mitogenic activity, and had decreased alpha-actin and myosin expression compared to mitogen-dependent SMC. Heterokaryons constructed between autonomous Neo7 SMC and mitogen-dependent SMC exhibited a mitogen-dependent growth phenotype suggesting that nonautonomous SMC produce factors that actively inhibit autonomous growth. In contrast, heterokaryons constructed between Neo7 SMC and autonomous embryonic SMC retained an autonomous growth phenotype. We examined the expression of known tumor suppressors to determine if any of these factors played a role in inhibiting SMC autonomous growth. p27, p53, pRb, and PTEN were abundantly expressed by Neo7 SMC and e17 SMC under both basal and serum stimulated conditions. The data suggest that the mechanisms driving SMC replication during neointimal formation are self-driven and self-regulated, and that at specific times after injury, SMC escape normal growth suppressive mechanisms through the loss of intracellular growth suppressor activity.     

The structure of human collapsin response mediator protein 2, a regulator of axonal growth

Axonal growth cone guidance is a central process in nervous system development and repair. Collapsin response mediator protein 2 (CRMP-2) is a neurite extension-promoting neuronal cytosolic molecule involved in the signalling of growth inhibitory cues from external stimuli, such as semaphorin 3A and the myelin-associated glycoprotein. We have determined the crystal structure of human tetrameric CRMP-2, which is structurally related to the dihydropyriminidases; however, the active site is not conserved. The wealth of earlier functional mapping data for CRMP-2 are discussed in light of the three-dimensional structure of the protein. The differences in oligomerisation interfaces between CRMP-1 and CRMP-2 are used to model CRMP-1/2 heterotetramers.