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H F Lodish

Publications and source records attributed to H F Lodish.

At least 109 records · Page 6Linked to original sources
PubMed1995

Regulated secretion of prolactin by the mouse insulinoma cell line beta TC-3.

Our aim is to use cultured cells capable of regulated protein secretion for the production of recombinant proteins that require particular types of post-translational modifications. Here we have generated a stable transfected beta TC-3 cell line, beta TC-IPR9, that secretes high levels of recombinant prolactin. Transfected cells synthesize both the 27 kDa glycosylated and a 23 kDa nonglycosylated prolactin; the 23 kDa nonglycosylated species was secreted preferentially when cells were placed in secretion medium containing isobutylmethylxanthine (IBMX) and high concentrations of glucose, K+, and Ca2+. When the cells were cultured in medium containing low concentrations of glucose, K+, and Ca2+, most of the prolactin and insulin were not secreted; much of the prolactin was proteolytically converted to a 16 kDa form. Within the first 30 minutes after transferring the cells to medium containing secretagogues there was a 20-fold increase in the rate of secretion of prolactin; all of the 16 kDa species was secreted. The recombinant cells could be cycled several times between medium in which prolactin was biosynthesized and medium in which it was secreted. Preferential secretion of proteolytically processed prolactin in a medium without contaminating proteins offers an example of the advantage of this technology for production of other recombinant proteins.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Signal transduction of a G protein-coupled receptor in caveolae: colocalization of endothelin and its receptor with caveolin.

Caveolae are small invaginations of the plasma membrane 50-100 nm in diameter. Since calcium channels, inositol 1,4,5-trisphosphate receptors, and heterotrimeric GTP-binding proteins (G proteins) are localized in caveolae, they may participate in signal transduction by G protein-coupled receptors. Here we show that the G protein-coupled endothelin receptor subtype A (ETA) and its bound endothelin ligand are found in plasma membrane caveolae. ETA and its bound ligand coimmunoprecipitate with caveolin, a structural component of caveolae, in extracts of cells expressing transfected ETA receptors. Confocal fluorescence microscopy shows colocalization of ETA receptors and caveolin in micropatches at or near the plasma membrane, in the absence of endothelin ligands. These observations demonstrate a functional role for plasma membrane caveolae in signal transduction by this G protein-coupled receptor.

Amino Acid Sequence↗
PubMed1994

Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein.

Long chain fatty acids (LCFAs) are an important energy substrate used by cardiac myocytes and other cells, but the mechanism whereby these molecules cross the plasma membrane is poorly understood. We used an expression cloning strategy and a cDNA library from 3T3-L1 adipocytes to identify a cDNA that, when expressed in cultured cells, augments uptake of LCFAs. This cDNA encodes a novel 646 amino acid fatty acid transport protein (FATP) with six predicted membrane-spanning regions and that is integrally associated with membranes. Immunocytochemistry and subcellular fractionation of 3T3-L1 adipocytes show that FATP is localized to the plasma membrane. We propose that FATP is a plasma membrane transporter for LCFAs.

3T3 Cells↗
PubMed1994

Expression cloning of SR-BI, a CD36-related class B scavenger receptor.

Scavenger receptors are integral membrane proteins that mediate the endocytosis of modified lipoproteins. The first of these to be purified and cloned were the type I and II macrophage scavenger receptors (SR-AI and SR-AII; class A scavenger receptors). Subsequently, the cell surface protein CD36 was shown to bind oxidized low density lipoprotein (oxidized LDL). From a Chinese hamster ovary (CHO) cell variant we have cloned by expression the cDNA for a new member of the CD36 family of membrane proteins, SR-BI, whose predicted protein sequence of 509 amino acids is approximately 30% identical to those of the four previously identified family members. Both SR-BI and CD36 displayed high affinity binding for acetylated LDL with an apparent dissociation constant on the order of approximately 5 micrograms of protein/ml. The ligand binding specificities of CD36 and SR-BI, determined by direct binding or competition assays, were similar, but not identical; both bind modified proteins (acetylated LDL, oxidized LDL, maleylated bovine serum albumin), but not the broad array of other polyanions (e.g. fucoidin, polyguanosinic acid, carrageenan) which are ligands of the class A receptors. Thus, SR-BI and CD36 define a second class of scavenger receptors, designated class B. Native LDL, which does not bind to either class A receptors or CD36, unexpectedly bound with high affinity to SR-BI. Northern blot analysis of murine tissues showed that SR-BI was most abundantly expressed in fat and was present at moderate levels in lung and liver. Furthermore, SR-BI mRNA expression was induced upon differentiation of 3T3-L1 cells into adipocytes. Thus, the tissue distribution of expression and ligand binding properties of SR-BI raise the possibility that this cell surface receptor may play an important role in lipid metabolism.

Amino Acid Sequence↗
PubMed1994

The pancreatic beta-cell glucose sensor.

Pancreatic beta cells secrete insulin in response to an increase in the level of blood glucose above 5 mM, which is characteristic of the fasting state. Glucose metabolism is essential for glucose sensing, and both the high-Km glucose transporter GLUT2 and the high-Km glucose phosphorylating enzyme glucokinase have been implicated in coupling insulin secretion to extracellular glucose levels. Experiments in isolated islets, immortalized beta-cell lines and transgenic animals, together with findings in humans with maturity-onset diabetes of the young, indicate that the primary beta-cell glucose sensor is glucokinase. Although the level of GLUT2 is frequently reduced in animal models of type II diabetes, GLUT2 does not limit glucose metabolism in beta cells and does not appear to regulate glucose induction of insulin secretion.

Animals↗
PubMed1994

The types II and III transforming growth factor-beta receptors form homo-oligomers.

Affinity-labeling experiments have detected hetero-oligomers of the types I, II, and III transforming growth factor beta (TGF-beta) receptors which mediate intracellular signaling by TGF-beta, but the oligomeric state of the individual receptor types remains unknown. Here we use two types of experiments to show that a major portion of the receptor types II and III forms homo-oligomers both in the absence and presence of TGF-beta. Both experiments used COS-7 cells co-transfected with combinations of these receptors carrying different epitope tags at their extracellular termini. In immunoprecipitation experiments, radiolabeled TGF-beta was bound and cross-linked to cells co-expressing two differently tagged type II receptors. Sequential immunoprecipitations using anti-epitope monoclonal antibodies showed that type II TGF-beta receptors form homo-oligomers. In cells co-expressing epitope-tagged types II and III receptors, a low level of co-precipitation of the ligand-labeled receptors was observed, indicating that some hetero-oligomers of the types II and III receptors exist in the presence of ligand. Antibody-mediated cross-linking studies based on double-labeling immunofluorescence explored co-patching of the receptors at the cell surface on live cells. In cells co-expressing two differently tagged type II receptors or two differently tagged type III receptors, forcing one receptor into micropatches by IgG induced co-patching of the receptor carrying the other tag, labeled by noncross-linking monovalent Fab'. These studies showed that homo-oligomers of the types II and III receptors exist on the cell surface in the absence or presence of TGF-beta 1 or -beta 2. In cells co-expressing types II and III receptors, the amount of heterocomplexes at the cell surface was too low to be detected in the immunofluorescence co-patching experiments, confirming that hetero-oligomers of the types II and III receptors are minor and probably transient species.

Affinity Labels↗
PubMed1994

Induction of caveolin during adipogenesis and association of GLUT4 with caveolin-rich vesicles.

Caveolae, also termed plasmalemmal vesicles, are small, flask-shaped, non-clathrin-coated invaginations of the plasma membrane. Caveolin is a principal component of the filaments that make up the striated coat of caveolae. Using caveolin as a marker protein for the organelle, we found that adipose tissue is the single most abundant source of caveolae identified thus far. Caveolin mRNA and protein are strongly induced during differentiation of 3T3-L1 fibroblasts to adipocytes; during adipogenesis there is also a dramatic increase in the complexity of the protein composition of caveolin-rich membrane domains. About 10-15% of the insulin-responsive glucose transporter GLUT4 is found in this caveolin-rich fraction, and immuno-isolated vesicles containing GLUT4 also contain caveolin. However, in non-stimulated adipocytes the majority of caveolin fractionates with the plasma membrane, while most GLUT4 associates with low-density microsomes. Upon addition of insulin to 3T3-L1 adipocytes, there is a significant increase in the amount of GLUT4 associated with caveolin-rich membrane domains, an increase in the amount of caveolin associated with the plasma membrane, and a decrease in the amount of caveolin associated with low-density microsomes. Caveolin does not undergo a change in phosphorylation upon stimulation of 3T3-L1 adipocytes with insulin. However, after treatment with insulin it is associated with a 32-kD phosphorylated protein. Caveolae thus may play an important role in the vesicular transport of GLUT4 to or from the plasma membrane. 3T3-L1 adipocytes offer an attractive system to study the function of caveolae in several cellular trafficking and signaling events.

3T3 Cells↗
PubMed1994

A constitutively activated erythropoietin receptor stimulates proliferation and contributes to transformation of multipotent, committed nonerythroid and erythroid progenitor cells.

If the env gene of spleen focus-forming virus (SFFV) is replaced by a cDNA encoding a constitutively active form of the erythropoietin receptor, EPO-R(R129C), the resultant recombinant virus, SFFVcEPO-R, induces transient thrombocytosis and erythrocytosis in infected mice. Clonogenic progenitor cell assays of cells from the bone marrow and spleens of these infected mice suggest that EPO-R(R129C) can stimulate proliferation of committed megakaryocytic and erythroid progenitors as well as nonerythroid multipotent progenitors. From the spleens of SFFVcEPO-R-infected mice, eight multiphenotypic immortal cell lines were isolated and characterized. These included primitive erythroid, lymphoid, and monocytic cells. Some expressed proteins characteristic of more than one lineage. All cell lines resulting from SFFVcEPO-R infection contained a mutant form of the p53 gene. However, in contrast to infection by SFFV, activation of PU.1 gene expression, by retroviral integration, was not observed. One cell line had integrated a provirus upstream of the fli-1 gene, in a location typically seen in erythroleukemic cells generated by Friend murine leukemia virus infection. This event led to increased expression of fli-1 in this cell line. Thus, infection by SFFVcEPO-R can induce proliferation and lead to transformation of nonerythroid as well as very immature erythroid progenitor cells. The sites of proviral integration in clonal cell lines are distinct from those in SFFV-derived lines.

Animals↗
PubMed1994

Activation and inhibition of erythropoietin receptor function: role of receptor dimerization.

Members of the cytokine receptor superfamily have structurally similar extracellular ligand-binding domains yet diverse cytoplasmic regions lacking any obvious catalytic domains. Many of these receptors form ligand-induced oligomers which are likely to participate in transmembrane signaling. A constitutively active (factor-independent) mutant of the erythropoietin receptor (EPO-R), R129C in the exoplasmic domain, forms disulfide-linked homodimers, suggesting that the wild-type EPO-R is activated by ligand-induced homodimerization. Here, we have taken two approaches to probe the role EPO-R dimerization plays in signal transduction. First, on the basis of the crystal structure of the ligand-bound, homodimeric growth hormone receptor (GH-R) and sequence alignment between the GH-R and EPO-R, we identified residues of the EPO-R which may be involved in intersubunit contacts in an EPO-R homodimer. Residue 129 of the EPO-R corresponds to a residue localized to the GH-R dimer interface region. Alanine or cysteine substitutions were introduced at four other residues of the EPO-R predicted to be in the dimer interface region. Substitution of residue E-132 or E-133 with cysteine renders the EPO-R constitutively active. Like the arginine-to-cysteine mutation at position 129 in the exoplasmic domain (R129C), E132C and E133C form disulfide-linked homodimers, suggesting that constitutive activity is due to covalent dimerization. In the second approach, we have coexpressed the wild-type EPO-R with inactive mutants of the receptor missing all or part of the cytosolic domain. These truncated receptors have a dominant inhibitory effect on the proliferative action of the wild-type receptor. Taken together, these results strengthen the hypothesis that an initial step in EPO- and EPO-R-mediated signal transduction is ligand-induced receptor dimerization.

Amino Acid Sequence↗
PubMed1994

The transforming growth factor beta type II receptor can replace the activin type II receptor in inducing mesoderm.

The type II receptors for the polypeptide growth factors transforming growth factor beta (TGF-beta) and activin belong to a new family of predicted serine/threonine protein kinases. In Xenopus embryos, the biological effects of activin and TGF-beta 1 are strikingly different; activin induces a full range of mesodermal cell types in the animal cap assay, while TGF-beta 1 has no effects, presumably because of the lack of functional TGF-beta receptors. In order to assess the biological activities of exogenously added TGF-beta 1, RNA encoding the TGF-beta type II receptor was introduced into Xenopus embryos. In animal caps from these embryos, TGF-beta 1 and activin show similar potencies for induction of mesoderm-specific mRNAs, and both elicit the same types of mesodermal tissues. In addition, the response of animal caps to TGF-beta 1, as well as to activin, is blocked by a dominant inhibitory ras mutant, p21(Asn-17)Ha-ras. These results indicate that the activin and TGF-beta type II receptors can couple to similar signalling pathways and that the biological specificities of these growth factors lie in their different ligand-binding domains and in different competences of the responding cells.

Activin Receptors↗
PubMed1993

The transforming growth factor beta receptors types I, II, and III form hetero-oligomeric complexes in the presence of ligand.

Transforming growth factors beta (TGF-beta s) are disulfide-linked dimers. In Rat-1 cells both radioiodinated TGF-beta 1 and -beta 2 bind to and can be chemically cross-linked to type I and II receptors (which are thought to mediate effects of cell growth suppression and gene activation), to type III proteoglycan receptors, and to a novel approximately 50-kDa protein. After detergent solubilization of cells that were cross-linked with radioiodinated TGF-beta, antibodies specific for the type II receptor precipitated labeled receptor types I and III as well as type II. In these cells, the type III receptor is the predominant TGF-beta-binding protein, and antibodies specific for it precipitate mainly this cross-linked receptor. Thus, in the presence of TGF-beta ligand, receptor types II and III and types II and I form heteromeric complexes. The majority of the type III receptor does not associate with receptor types I and II, probably reflecting the relative amounts of the three receptors on the surface of Rat-1 cells. Since TGF-beta 1 but not TGF-beta 2 binds to the exoplasmic domain of the type II receptor in the absence of the type III receptor, and since both TGF-beta 1 and -beta 2 bind with high affinity to the type III receptor, we suggest that TGF-beta 2, and possibly TGF-beta 1, bind initially to the type III receptor. The TGF-beta 2-type III receptor complex would then interact with a type II receptor, thus modulating the affinity of the type II receptor for TGF-beta 2.

Amino Acid Sequence↗
PubMed1993

Both megakaryocytopoiesis and erythropoiesis are induced in mice infected with a retrovirus expressing an oncogenic erythropoietin receptor.

Increasing direct and indirect evidence suggests that erythropoietin (Epo) promotes both erythropoiesis and megakaryocytopoiesis. Here we report that, in mice infected with a recombinant spleen focus-forming retrovirus (SFFV) expressing an oncogenic erythropoietin receptor (EpoR), there was an increase in platelet count preceding the ensuing erythrocytosis. Concurrently, there was a substantial increase in splenic megakaryocytes. Culture of the bone marrow and spleen cells from infected mice showed enhanced numbers of multipotent megakaryocytic progenitors. DNA polymerase chain reaction analysis of individual megakaryocyte-containing colonies showed recombinant SFFV (SFFVcEpoR) proviral integration. Immunofluorescence of spleen sections showed overexpression of EpoR protein in the megakaryocytes. Mice infected with a strain of SFFV also developed splenic megakaryocytosis without activating overexpression of the EpoR in megakaryocytes. This in vivo system shows that a relationship between erythropoiesis and thrombopoiesis can exist at the level of the Epo-EpoR signaling pathway. Also, SFFV-based vectors may be excellent vehicles for the introduction of genes into multipotent, hematopoietic progenitors, in vitro.

Animals↗
PubMed1993

Unfolding of newly made retinol-binding protein by dithiothreitol. Sensitivity to retinoids.

In cultured hepatoma HepG2 cells, serum retinol-binding protein (RBP) is secreted more rapidly in the presence of retinol than in its absence (Tosetti, F., Ferrari, N., Pfeffer, U., Brigati, C., and Vidali, G. (1992) Exp. Cell Res. 200, 467-472). In the presence of millimolar concentration of DTT, HepG2 cells synthesize fully reduced RBP within the endoplasmic reticulum (ER) which, upon removal of DTT, forms disulfide bonds post-translationally. Secretion of this post-translationally folded RBP is also dependent on the presence of retinol. Using nonreducing gel electrophoresis, we resolved disulfide-bonded RBP folding intermediates. In addition, two other intracellular folding intermediates, compact I and II, which co-migrate with mature RBP were resolved by their different sensitivity to DTT-induced unfolding. Retinol, as well as retinoic acid, stabilized both compact I and II RBP intermediates to DTT-induced unfolding, suggesting that RBP assumes different conformations in the ER in the presence and absence of a ligand. However, only RBP synthesized in the presence of retinol is rapidly secreted, indicating that the ER export quality control system recognizes RBP containing retinol, but not retinoic acid, as fully folded and competent for export. Folding of RBP so that it is stabilized to DTT reduction is not a sufficient condition for ER exit.

Disulfides↗
PubMed1993

In vitro unfolding of retinol-binding protein by dithiothreitol. Endoplasmic reticulum-associated factors.

HepG2 cells in the presence of DTT synthesize fully reduced serum retinol-binding protein (RBP) within the endoplasmic reticulum (ER). Upon removal of DTT, RBP forms disulfide bonds and a folding intermediate, compact II, accumulates within the ER. Compact II RBP co-migrates on nonreducing gel electrophoresis with the secreted form of RBP but is differentiated from secreted RBP by its sensitivity to DTT-induced unfolding (see accompanying article; Kaji, E. H., and Lodish, H. F. (1993) J. Biol. Chem. 268, 22188-22194). Here, we have reconstituted DTT-induced unfolding of compact II RBP in a broken cell system and demonstrate that ER-associated factors enhance the unfolding of RBP by DTT. Protein disulfide isomerase is likely to be one such factor since it enhances the rate of RBP unfolding by DTT in vitro; protein disulfide isomerase-induced unfolding requires the absence of retinoids, similar to the DTT-induced unfolding in vivo. ATP enhances the unfolding of RBP in the absence but not in the presence of retinol, both in intact and broken cells. Thus, protein disulfide isomerase and other ATP-dependent factors can unfold partly folded (or misfolded) RBP in the ER, suggesting how improperly folded proteins might be correctly refolded in vivo.

Adenosine Triphosphate↗
PubMed1993

The secretory pathway is normal in dithiothreitol-treated cells, but disulfide-bonded proteins are reduced and reversibly retained in the endoplasmic reticulum.

Addition of 2 mM dithiothreitol (DTT) to HepG2 human hepatoma cells blocks secretion of newly made albumin and causes it to accumulate in the endoplasmic reticulum in the reduced state. Subsequent incubation of the cells in the absence of DTT allows the reduced albumin to form apparently normal disulfide bonds and to be secreted normally. Similarly, DTT treatment causes all newly made subunits of the H1 subunit of the asialoglycoprotein receptor to be retained in the endoplasmic reticulum in the reduced state; following removal of DTT H1 subunits form disulfide bonds via a normal endoplasmic reticulum folding intermediate and mature to the Golgi. Thus, apparently normal formation of disulfide bonds on these two proteins can occur post-translationally, as has been shown previously for the influenza HA hemagglutinin (Braakman, I., Helenius, J., and Helenius, A. (1992) EMBO J. 11, 1717-1722; Braakman, I., Helenius, J., and Helenius, A. (1992) Nature 356, 260-262). alpha 1-Antitrypsin contains no disulfide bonds; in the continuous presence of DTT it acquires a normal complement of complex oligosaccharides and is secreted at an only slightly reduced rate. Thus, the secretory pathway functions efficiently even when it is reduced by DTT. Endoplasmic reticulum retention of albumin and H1 during treatment with DTT presumably occurs because, without disulfide bonds, these proteins cannot fold properly, not because of any defect in the overall processes of vesicular transport and protein secretion.

Asialoglycoprotein Receptor↗