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H F Lodish

Publications and source records attributed to H F Lodish.

At least 91 records · Page 5Linked to original sources
PubMed1996

Regulation of the murine adipocyte fatty acid transporter gene by insulin.

A cDNA encoding a novel fatty acid transport protein (FATP) was identified recently using expression cloning methodologies. We have studied the expression of FATP in differentiating 3T3-L1 cells and adipose tissue in vivo. When 3T3-L1 preadipocytes are treated with a combination of methylisobutylxanthine, dexamethasone, and insulin to induce differentiation, the abundance of FATP mRNA decreases within 24 h to less than one-third that of preadipocytes and increases subsequently, such that mature adipocytes have 5-7 times more FATP mRNA than fibroblastic precursors. In fully differentiated 3T3-L1 adipocytes, insulin alone is sufficient to down-regulate FATP mRNA levels 10-fold. The concentration of insulin necessary for half-maximal repression (I0.5) is approximately 1 nM and is specific for insulin; insulin-like growth factor I (IGF-I) has little effect at similar concentrations. Kinetic analysis indicates that the reduction in expression of FATP mRNA by insulin is rapid (t1/2 = approximately 4 h) and reversible upon withdrawal of insulin. The half-lives of FATP mRNA are 2.9 h and 4.4 h in the absence and presence of insulin, respectively. The insulin-mediated decrease in FATP steady state mRNA level correlates with a decrease in its transcription rate as measured by nuclear run-on transcription assay. To determine whether physiological conditions that alter insulin concentration in vivo affect adipose FATP levels, feeding/fasting studies are employed. Fasting of C57BL/6J mice for 48 h results in an 11-fold up-regulation of FATP mRNA expression in adipose tissue. Refeeding of fasted animals for 72 h results in a return of FATP mRNA to basal levels. In sum, these results indicate that the expression of FATP gene is negatively regulated by insulin at the transcriptional level in cultured adipocytes and that transporter mRNA expression in murine adipose tissue is altered in a manner consistent with insulin being a negative regulator of gene activity.

1-Methyl-3-isobutylxanthine↗
PubMed1995

The specificity of the transforming growth factor beta receptor kinases determined by a spatially addressable peptide library.

Type I and II receptors for the transforming growth factor beta (TGF-beta) are transmembrane serine/threonine kinases that are essential for TGF-beta signaling. However, little is known about their in vivo substrates or signal transduction pathways. To determine the substrate specificity of these kinases, we developed combinatorial peptide libraries synthesized on a hydrophilic matrix that is easily accessible to proteins in aqueous solutions. When we subjected these libraries to phosphorylation by the cAMP-dependent protein kinase, we obtained the optimal peptide sequence RRXS (I/L/V), in perfect agreement with the substrate sequence deduced from mutagenesis and crystal structure analyses. By using the same libraries, we showed that the optimal substrate peptide for both the type I and II TGF-beta receptors was KKKKKK(S/T)XXX. Since the two kinases are thought to play different roles in intracellular signal transduction, it was a surprise to find that they have almost identical substrate specificity. Our method is direct, sensitive, and simple and provides information about the kinase specificity for all the amino acid residues at each position.

Activin Receptors, Type I↗
PubMed1995

A novel serum protein similar to C1q, produced exclusively in adipocytes.

We describe a novel 30-kDa secretory protein, Acrp30 (adipocyte complement-related protein of 30 kDa), that is made exclusively in adipocytes and whose mRNA is induced over 100-fold during adipocyte differentiation. Acrp30 is structurally similar to complement factor C1q and to a hibernation-specific protein isolated from the plasma of Siberian chipmunks; it forms large homo-oligomers that undergo a series of post-translational modifications. Like adipsin, secretion of Acrp30 is enhanced by insulin, and Acrp30 is an abundant serum protein. Acrp30 may be a factor that participates in the delicately balanced system of energy homeostasis involving food intake and carbohydrate and lipid catabolism. Our experiments also further corroborate the existence of an insulin-regulated secretory pathway in adipocytes.

3T3 Cells↗
PubMed1995

Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor.

Erythropoietin (EPO) is the principal growth factor regulating the production of circulating erythrocytes. We introduced null mutations into both Epo and the EPO receptor (EpoR) gene. Both heterozygotes appeared normal. Homozygous animals exhibited reduced primitive erythropoiesis and died around embryonic day 13, owing to failure of definitive fetal liver erythropoiesis. Both types of mutations exhibited identical phenotypes, indicating that EPO and the EPOR are crucial for definitive erythropoiesis in vivo and that no other ligands or receptors can replace them. Committed erythroid BFU-E and CFU-E progenitors were present in both homozygous fetal livers. Thus, neither EPO nor the EPOR is required for erythroid lineage commitment or for the proliferation and differentiation of BFU-E to CFU-E progenitors. EPO and the EPOR are crucial in vivo for the proliferation and survival of CFU-E progenitors and their irreversible terminal differentiation.

Animals↗
PubMed1995

Interaction of the erythropoietin and stem-cell-factor receptors.

Mutations in the KIT transmembrane protein-tyrosine kinase receptor affect erythropoiesis, resulting in fewer committed late progenitors (colony-forming unit erythroid, CFU-E) in the fetal liver. As the survival and proliferation of CFU-Es depend absolutely on erythropoietin (EPO), these results suggest that CFU-Es cannot proliferate or mature further unless both the KIT and EPO receptor signalling pathways are functional. How KIT affects proliferation or differentiation of CFU-Es is not clear. Here we show that the KIT ligand SCF (for stem-cell factor) can replace EPO in supporting the growth and survival of HCD57 cells, an EPO-dependent erythroid-progenitor cell line expressing high levels of KIT. SCF supports the proliferation of 32D cells that express KIT only if they also express the EPO receptor. In HCD57 cells, SCF rapidly induces tyrosine phosphorylation of the EPO receptor, and KIT physically associates with the extended box 2 region in the cytoplasmic domain of the EPO receptor. Our results indicate that KIT may activate the EPO receptor by tyrosine phosphorylation to induce further proliferation and maturation of CFU-Es.

Animals↗
PubMed1995

Enhanced folding and processing of a disulfide mutant of the human asialoglycoprotein receptor H2b subunit.

Unfolded forms of the H2b subunit of the human asialoglycoprotein receptor, a galactose-specific C-type lectin, are degraded in the endoplasmic reticulum (ER), whereas folded forms of the protein can mature to the cell surface (Wikström, L., and Lodish, H. F. (1993) J. Biol. Chem. 268, 14412-14416). There are eight cysteines in the exoplasmic domain of the protein, forming four disulfide bonds in the folded protein. We have constructed double cysteine to alanine mutants for each of the four disulfide bonds and examined the folding and metabolic fate of each of the mutants in transfected 3T3 fibroblasts. We find that mutation of the two cysteines nearest to the transmembrane region (C1) does not prevent proper folding of the protein, whereas mutations of the other three disulfides prevent proper folding of the protein and all of the mutant proteins are degraded in the ER. A normal (approximately 20%) fraction of the C1 mutant protein exists the endoplasmic reticulum and is processed in the Golgi complex, and it does so at a faster rate compared to the wild-type. Furthermore, the folded form of this mutant protein is more resistant to unfolding by dithiothreitol than the wild-type. The C1 mutant protein is expressed on the cell surface and can form a functional receptor with the H1 subunit with similar binding affinities for natural ligands as that of the wild-type receptor. The same fraction of newly made mutant and wild-type proteins (approximately 80%) remain in the ER, but the mutant protein is degraded more quickly. Thus, the presence of the C1 disulfide bond in the wild-type receptor both reduces the rate of protein folding and exit to the Golgi and slows the rate of ER degradation of the portion (approximately 80%) of the receptor that never folds properly.

3T3 Cells↗
PubMed1995

Epitope tagging of the human endoplasmic reticulum HSP70 protein, BiP, to facilitate analysis of BiP--substrate interactions.

We modified BiP, the resident endoplasmic reticulum (ER) heat shock protein 70, to contain an epitopetag sequence close to the C-terminus (BiP-tag); the epitope is derived from an influenza hemagglutinin (HA) subtype and is recognized by the monoclonal antibody 12CA5. This antibody both immunoprecipitates BiP-tag and detects it on Western blots. Using transient expression of cDNAs in COS cells, we studied the interaction of BiP-tag with several membrane proteins. Consistent with previous work on BiP, BiP-tag bound poorly and transiently to newly made wild-type influenza HA glycoprotein and strongly and irreversibly to an HA mutant that misfolds and is retained in the ER. Most newly made erythropoietin receptor (EPO-R) polypeptides are retained in the ER and degraded there; we show here that, in cotransfected COS cells, newly made EPO-R is bound to BiP-tag prior to its degradation. Thus, by several criteria the BiP-tag molecule is fully functional in binding newly made proteins. Because it can be immunoprecipitated by a readily available antibody, it offers several advantages to the study of protein folding in the ER and the role of chaperones in this process.

Amino Acid Sequence↗
PubMed1995

Diffuse vesicular distribution of Rab3D in the polarized neuroendocrine cell line AtT-20.

The neuroendocrine cell line AtT-20 has two types of storage vesicles: dense core granules and synaptic vesicles, both sequestered at the tip of the processes. Here we show that Rab3D protein, which is abundant in fat cells, is also expressed in AtT-20 cells. Differently from Rab3A, which is localized in secretory vesicles accumulated at the tips, Rab3D has a diffuse vesicular distribution in the cytoplasm of the cell body, the processes and the tips. In AtT-20 cells, Rab3D may define a regulated secretory pathway which functions independently from cell polarity.

Amino Acid Sequence↗
PubMed1995

Cooperative binding of transforming growth factor (TGF)-beta 2 to the types I and II TGF-beta receptors.

TGF-beta 1 binds with high affinity (KD = 25-50 pM) directly to the TGF-beta type II receptor serine-threonine kinase (T beta-RII) in the absence of expression of the TGF-beta type I or III receptors (T beta-RI and T beta-RIII). The serine-threonine kinase T beta-RI is essential for TGF-beta 1 signaling but not for binding to T beta-RII. TGF-beta 2, in contrast, does not bind directly to T beta-RII, although coexpression of T beta-RIII does allow binding and cross-linking of TGF-beta 2 to T beta-RII. Here we show that in transfected COS cells binding and cross-linking of 125I-TGF-beta 2 to T beta-RI or T beta-RII requires expression of both receptors. In cells transfected with the c-myc-tagged human T beta-RII cDNA, only low amounts of 125I-TGF-beta 2 cross-linked to T beta-RI and T beta-RII were detected even with high concentrations (700 pM) of ligand. Cotransfection of the influenza-hemagglutinin-tagged human T beta-RI cDNA dramatically increased the binding of TGF-beta 2 to T beta-RII; the concentration of 125I-TGF-beta 2 required for half-maximal binding and cross-linking to T beta-RI and T beta-RII was approximately 40 pM. Coimmunoprecipitation studies showed that the high affinity receptor for TGF-beta 2 is composed of a hetero-oligomer of T beta-RI and T beta-RII. Thus TGF-beta 1 and -beta 2 bind to TGF-beta receptors in different ways; TGF-beta 1 binds directly to T beta-RII, while binding of TGF-beta 2 to T beta-RII requires coexpression of T beta-RI or T beta-RIII.

Activin Receptors, Type I↗
PubMed1995

Caveolin isoforms differ in their N-terminal protein sequence and subcellular distribution. Identification and epitope mapping of an isoform-specific monoclonal antibody probe.

Caveolin, an integral membrane protein, is a principal component of caveolae membranes in vivo. Two isoforms of caveolin have been identified: a slower migrating 24-kDa species (alpha-isoform) and a faster migrating 21-kDa species (beta-isoform). Little is known about how these isoforms differ, either structurally or functionally. Here we have begun to study the differences between these two isoforms. Microsequencing of caveolin reveals that both isoforms contain internal caveolin residues 47-77. In a second independent approach, we recombinantly expressed caveolin in a caveolin-negative cell line (FRT cells). Stable transfection of FRT cells with the full-length caveolin cDNA resulted in the expression of both caveolin isoforms, indicating that they can be derived from a single cDNA. Using extracts from caveolin-expressing FRT cells, we fortuitously identified a monoclonal antibody that recognizes only the alpha-isoform of caveolin. Epitope mapping of this monoclonal antibody reveals that it recognizes an epitope within the extreme N terminus of caveolin, specifically residues 1-21. These results suggest that alpha- and beta-isoforms of caveolin differ in their N-terminal protein sequences. To independently evaluate this possibility, we placed an epitope tag at either the extreme N or C terminus of full-length caveolin. Results of these "tagging" experiments clearly demonstrate that (i) both isoforms of caveolin contain a complete C terminus and (ii) that the alpha-isoform contains a complete N terminus while the beta-isoform lacks N-terminal-specific protein sequences. Mutational analysis reveals that these two isoforms apparently derive from the use of two alternate start sites: methionine at position 1 and an internal methionine at position 32. This would explain the approximately 3-kDa difference in their apparent migration in SDS-polyacrylamide electrophoresis gels. In addition, using isoform-specific antibody probes we show that caveolin isoforms may assume a distinct but overlapping subcellular distribution by confocal immunofluorescence microscopy. We discuss the possible implications of these differences between alpha- and beta-caveolin.

Amino Acid Sequence↗
PubMed1995

Nonneuronal expression of Rab3A: induction during adipogenesis and association with different intracellular membranes than Rab3D.

Rab3A is a small GTP-binding protein expressed predominantly in brain and neuroendocrine cells, in which it is associated with synaptic and synaptic-like vesicles, respectively. Here we report that adult mouse fat cells and 3T3-L1 adipocytes also express Rab3A mRNA and protein. They do not express synaptophysin, an abundant protein in synaptic vesicles or synaptic-like vesicles. The amount of Rab3A mRNA and protein, like that of the highly homologous isoform Rab3D, increases severalfold during differentiation of 3T3-L1 fibroblasts into mature adipocytes. In fat cells, most Rab3D and Rab3A protein is bound to membrane, irrespective of insulin addition. Rab3A and Rab3D are localized in different subcellular compartments, since about half of the Rab3A, but none of the Rab3D, is associated with a low-density organelle(s). Rab3D and Rab3A may be involved in different pathways of regulated exocytosis in adipocytes. Moreover, in adipocytes Rab3A may define an exocytic organelle that is different from synaptic vesicles or synaptic-like microvesicles found in neuronal and endocrine cells.

3T3 Cells↗
PubMed1995

Endothelin-induced endocytosis of cell surface ETA receptors. Endothelin remains intact and bound to the ETA receptor.

We demonstrate unusual features of the intracellular processing of endothelin-1 (ET-1) and its receptor ETA, the receptor subtype that mediates contraction of vascular smooth muscle cells. First, we show that in stably transfected CHO cells expressing ETA, binding of an ET-1 ligand induces rapid endocytosis of cell surface ETA. Receptor endocytosis was measured both by immunofluorescence and by radioiodinated antibodies specific for ETA. Second, we demonstrate that ET-1 remains intact for up to 2 h after endocytosis and, as judged by co-immunoprecipitation, internalized 125I-ET-1 remains bound to ETA receptors. We hypothesize that internalized ET-1, bound to ETA receptors, continues to activate a signal-transducing G protein, thus accounting for the prolonged period of contraction induced in smooth muscle cells by a single administration of ET-1.

Animals↗
PubMed1995

Specific recruitment of SH-PTP1 to the erythropoietin receptor causes inactivation of JAK2 and termination of proliferative signals.

The binding of erythropoietin (EPO) to its receptor (EPO-R) activates the protein tyrosine kinase JAK2. The mechanism of JAK2 inactivation has been unclear. We show that the hematopoietic protein tyrosine phosphatase SH-PTP1 (also called HCP and PTP1C) associates via its SH2 domains with the tyrosine-phosphorylated EPO-R. In vitro binding studies suggest that Y429 in the cytoplasmic domain of the EPO-R is the binding site for SH-PTP1. Mutant EPO-Rs lacking Y429 are unable to bind SH-PTP1; cells expressing such mutants are hypersensitive to EPO and display prolonged EPO-induced autophosphorylation of JAK2. Our results suggest that activation of SH-PTP1 by binding to the EPO-R plays a major role in terminating proliferative signals.

Animals↗
PubMed1995

The soluble exoplasmic domain of the type II transforming growth factor (TGF)-beta receptor. A heterogeneously glycosylated protein with high affinity and selectivity for TGF-beta ligands.

The transforming growth factor (TGF)-beta type II receptor is a transmembrane serine/threonine kinase which is essential for all TGF-beta-induced signals. In several cell types TGF-beta 2 is as potent as TGF-beta or TGF-beta 3 in inducing cellular responses, yet TGF-beta 2 does not bind to the majority of expressed type II receptors. Here we characterized the properties of the soluble extracellular domain of the human TGF-beta type II receptor synthesized in COS-7 cells. Like the membrane-attached type II receptor, the soluble receptor contains complex N-linked oligosaccharides as well as additional sialic acid residues that cause it to migrate heterogenously upon SDS-polyacrylamide gel electrophoresis. 125I-TGF-beta 1 binds to and is chemically cross-linked to this protein. Unlabeled TGF-beta 1 inhibits the binding of 125I-TGF-beta 1 with an apparent dissociation constant (Kd) of approximately 200 pM, similar to the apparent Kd (approximately 50 pM) of the cell-surface type II receptor. TGF-beta 3 inhibits the binding of 125I-TGF-beta 1 to the soluble type II receptor with a similar dissociation constant, approximately 500 pM. In contrast, 125I-TGF-beta 2 cannot bind and be chemically cross-linked to the soluble type II receptor, nor does as much as a 125-fold excess of unlabeled TGF-beta 2 inhibit the binding of 125I-TGF-beta 1 to the soluble receptor. This is the first demonstration of the binding affinities of the type II receptor in the absence of the other cell-surface molecules known to bind TGF-beta. Expressed alone in COS-7 cells the type II receptor also cannot bind TGF-beta 2; co-expression of type III receptor enables the type II receptor to bind TGF-beta 2. Thus, the type III receptor or some other component is required for transmission of TGF-beta 2-induced signals by the type II receptor.

Animals↗
PubMed1995

GH3 pituitary tumor cells contain heteromeric type I and type II receptor complexes for transforming growth factor beta and activin-A.

Transforming growth factors beta (TGF-beta s) and activins induce and inhibins block secretion of follicle-stimulating hormone by rat GH3 pituitary tumor cells. Cheifetz et al. (Cheifetz, S., Ling, N., Guillemin, R., and Massagué, J. (1988) J. Biol. Chem. 263, 17225-17228) reported that GH3 cells express a approximately 50-kDa surface protein, termed the type IV TGF-beta receptor, that directly binds all of these peptide hormones. Here we show that GH3 cells express the previously identified type I and type II receptors for TGF-beta and activin-A. Immunoprecipitation of affinity-labeled surface binding proteins with antisera specific to known receptors demonstrated independent heteromeric complexes of TGF-beta types I and II receptors and of activin types I and II receptors. As judged by ligand-binding and cross-linking analysis, TGF-beta binding to the TGF-beta receptors is not inhibited by activin-A and activin-A binding to its receptors is not inhibited by TGF-beta. Screening of a cDNA library from GH3 cells for potential receptor serine-threonine kinases yielded the known types I and II TGF-beta and activin receptors. The presumed common intracellular signaling pathway for TGF-beta and activin in GH3 cells appears to be mediated by distinct cell-surface receptors.

Activin Receptors↗
PubMed1995

Increased cell surface expression and enhanced folding in the endoplasmic reticulum of a mutant erythropoietin receptor.

In both transfected and normal hematopoietic cells, the majority of newly made erythropoietin receptor (EPO-R) subunits are retained in the endoplasmic reticulum (ER), destined for degradation. Only a small fraction exit the ER and are competent to bind EPO, suggesting that the EPO-R folds inefficiently. The EPO-R contains a 5-amino acid motif, WSXWS, in the extracellular domain that is conserved among members of the cytokine receptor family. We describe a mutant EPO-R with a change in the middle residue of this motif, A234E, that is transported from the ER more efficiently than the wild-type (wt) receptor and is expressed in elevated numbers at the cell surface. This mutant polypeptide is processed more efficiently in the ER than its wt counterpart, suggesting that it folds better than the wt EPO-R. Inefficient folding and processing of the wt EPO-R in the ER may be one mechanism for controlling the number of plasma membrane receptors.

Amino Acid Sequence↗
PubMed1995

Molecular characterization of a type I serine-threonine kinase receptor for TGF-beta and activin in the rat pituitary tumor cell line GH3.

GH3 pituitary tumor cells have surface receptors for transforming growth factors-beta (TGF-beta s) and activins/inhibins. GH3 cell mRNA was screened by a novel reverse transcriptase-polymerase chain reaction technique with primers for receptor serine-threonine kinases. We isolated rat homologs of previously identified clones for type I (ALK-2 and ALK-5) and type II (ActRII, TGF-beta RII) activin and TGF-beta receptors, together with a novel clone, whose full-length version was isolated from a GH3 cell cDNA library. Named B1, it encodes a 505-amino-acid protein belonging to the family of type I receptor serine/threonine kinases. The kinase domain of B1 exhibits 90% identity to that of the TGF-beta type I receptor. B1 mRNA is expressed not only in pituitary cells but also in all other cells and tissues examined. B1 protein can be expressed on the cell surface, but cannot bind ligand unless a type II receptor is also present. When coexpressed with the type II receptors specific for TGF-beta or activin, B1 can be efficiently cross-linked to either ligand, suggesting that it can form heteromeric complexes with both type II receptor subunits.

Activin Receptors↗