Harvey F. Lodish

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.

Intracellular transport of secretory and membrane proteins in hepatoma cells infected by vesicular stomatitis virus.

Abstract Rat hepatoma cells which secrete several serum proteins have been infected with a temperature sensitive mutant of Vesicular Stomatitis Virus, which is defective in RNA replication and transcription. These infected cells, grown at 39.5°C, continue normal secretion of albumin and transferrin, so that synthesis and maturation of secretory and membrane proteins, including the VSV G protein, can be studied in the same cells. Serum proteins are not secreted from the cells at the same rate; half of the newly synthesized albumin appears in the medium after 23 minutes, while half of the glycoprotein transferrin is secreted only after about 40 minutes. By contrast, two membrane proteins—the VSV G glycoprotein and a high molecular weight hepatoma surface protein—are detected at the cell surface approximately 23 minutes after their synthesis. The differences in the rate of externalization among the secretory and membrane glycoproteins are due to differences in the rate of transport from the rough endoplasmic reticulum (ER) into or through the Golgi region, as judged by the time required for maturation of the asn-linked oligosaccharides. Unglycosylated VSV G does not reach the cell surface, while unglycosylated transferrin is secreted at the same rate as the glycosylated species. Thus neither the synthesis nor the presence of the asn-linked oligosaccharide chain are determinants in the rate of transferrin secretion. The monovalent ionophore monensin inhibits both secretion of serum proteins and migration of membrane proteins to the cell surface. However, the intracellular transport of transferrin is blocked in a stage where the molecule is still sensitive to endo-β-N-acetylglucosaminidase H, while VSV G reaches a stage in which it is resistant to the enzyme. These results indicate that at least part of the intracellular pathway may be different for membrane and secretory proteins and glycoproteins.

Expression cloning of the TGF-β type II receptor, a functional transmembrane serine/threonine kinase

A cDNA encoding the TGF-beta type II receptor protein has been isolated by an expression cloning strategy. The cloned cDNA, when transfected into COS cells, leads to overexpression of an approximately 80 kd protein that specifically binds radioiodinated TGF-beta 1. Excess TGF-beta 1 competes for binding of radioiodinated TGF-beta 1 in a dose-dependent manner and is more effective than TGF-beta 2. The predicted receptor structure includes a cysteine-rich extracellular domain, a single hydrophobic transmembrane domain, and a predicted cytoplasmic serine/threonine kinase domain. A chimeric protein containing the intracellular domain of the type II receptor and expressed in E. coli can phosphorylate itself on serine and threonine residues in vitro, indicating that the cytoplasmic domain of the type II receptor is a functional kinase. This result implicates serine/threonine phosphorylation as an important mechanism of TGF-beta receptor-mediated signaling.

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.

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.

Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain: acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation.

gACRP30, the globular subunit of adipocyte complement-related protein of 30 kDa (ACRP30), improves insulin sensitivity and increases fatty acid oxidation. The mechanism by which gACRP30 exerts these effects is unknown. Here, we examined if gACRP30 activates AMP-activated protein kinase (AMPK), an enzyme that has been shown to increase muscle fatty acid oxidation and insulin sensitivity. Incubation of rat extensor digitorum longus (EDL), a predominantly fast twitch muscle, with gACRP30 (2.5 μg/ml) for 30 min led to 2-fold increases in AMPK activity and phosphorylation of both AMPK on Thr-172 and acetyl CoA carboxylase (ACC) on Ser-79. Accordingly, concentration of malonyl CoA was diminished by 30%. In addition, gACRP30 caused a 1.5-fold increase in 2-deoxyglucose uptake. Similar changes in malonyl CoA and ACC were observed in soleus muscle incubated with gACRP30 (2.5 μg/ml), although no significant changes in AMPK activity or 2-deoxyglucose uptake were detected. When EDL was incubated with full-length hexameric ACRP30 (10 μg/ml), AMPK activity and ACC phosphorylation were not altered. Administration of gACRP30 (75 μg) to C57 BL/6J mice in vivo led to increased AMPK activity and ACC phosphorylation and decreased malonyl CoA concentration in gastrocnemius muscle within 15–30 min. Both in vivo and in vitro, activation of AMPK was the first effect of gACRP30 and was transient, whereas alterations in malonyl CoA and ACC occurred later and were more sustained. Thus, gACRP30 most likely exerts its actions on muscle fatty acid oxidation by inactivating ACC via activation of AMPK and perhaps other signal transduction proteins.

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.

Fetal Anemia and Apoptosis of Red Cell Progenitors in Stat5a−/−5b−/− Mice: A Direct Role for Stat5 in Bcl-XL Induction

The erythropoietin receptor (EpoR) is essential for production of red blood cells; a principal function of EpoR is to rescue committed erythroid progenitors from apoptosis. Stat5 is rapidly activated following EpoR stimulation, but its function in erythropoiesis has been unclear since adult Stat5a-/-5b-/- mice have normal steady-state hematocrit. Here we show that Stat5 is essential for the high erythropoietic rate during fetal development. Stat5a-/-5b-/- embryos are severely anemic; erythroid progenitors are present in low numbers, show higher levels of apoptosis, and are less responsive to Epo. These findings are explained by a crucial role for Stat5 in EpoRs antiapoptotic signaling: it mediates the immediate-early induction of Bcl-X(L) in erythroid cells through direct binding to the Bcl-X promoter.

Intracellular site of asialoglycoprotein receptor-ligand uncoupling: Double-label immunoelectron microscopy during receptor-mediated endocytosis

In rats infused with asialoglycoprotein for 60 min, receptor-mediated endocytosis of the ligand occurred exclusively in hepatic parenchymal cells. We have used double-label immunoelectron microscopy on ultrathin cryosections of rat liver to identify the site at which the asialoglycoprotein receptor and its ligand dissociate following their common endocytosis. Asialoglycoprotein receptor, ligand and clathrin were identified and quantitated by the use of monospecific antibodies followed by gold-protein A complexes. Both receptor and ligand were found associated with the membrane of clathrin-coated vesicles close to the cell surface. We identified other vesicles that contained ligand accumulated within the lumen. The membranes of these latter vesicles contained little receptor, but receptor was concentrated in tubular extensions that were largely free of ligand. We call this organelle CURL (compartment of uncoupling of receptor and ligand). CURL vesicles appear to transform into secondary lysosomes, wherein the ligand is degraded. The tubular vesicles are, we propose, an intermediate in recycling the receptor to the cell surface.

Cloning and functional expression in bacteria of a novel glucose transporter present in liver, intestine, kidney, and β-pancreatic islet cells

Abstract The well-characterized erythrocyte glucose transporter is also expressed in brain, adipocytes, kidney, muscle, and certain transformed cells, but not in liver, intestine, or the islets of Langerhans. Using as probe a cDNA encoding the rat brain glucose transporter, we isolated from a rat liver cDNA library a clone encoding a protein 55% identical in sequence to the rat brain transporter, and with a superimposible hydropathy plot. We expressed this protein in an E. coli mutant defective in glucose uptake; the protein was incorporated into the bacterial membrane and functioned as a glucose transporter. This new transporter is expressed in liver, intestine, kidney, and the islets of Langerhans; immunofluorescence analysis showed that it is present in the plasma membrane of the insulin-producing β cells. Insulinoma cells express, inappropriately, the erythrocyte glucose transporter, and we suggest that this may be related to their inability to secrete insulin in response to elevations in glucose.