Abstract. The human asialoglycoprotein receptor is a heterooligomer of the two homologous subunits H1 and H2. As occurs for other oligomeric receptors, not all of the newly made subunits are assembled in the RER into oligomers and some of each chain is degraded. We studied the degradation of the unassembled H2 subunit in fibroblasts that only express H2 (45,000 mol wt) and degrade all of it. After a 30 min lag, H2 is degraded with a half-life of 30 min. We identified a 35-kD intermediate in H2 degradation; it is the COOH-terminal, exoplasmic domain of H2. After a 90-min chase, all remaining intact H2 and the 35-kD fragment were endoglycosidase H sensitive, suggesting that the cleavage generating the 35-kD intermediate occurs without translocation to the medial Golgi compartment. Treatment of cells with leupeptin, chloroquine, or NI-LCI did not affect H2 degradation. Monensin slowed but did not block degradation. Incubation at 18-20°C slowed the degradation dramatically and caused an increase in intracellular H2, suggesting that a membrane tratficking event occurs before H2 is degraded. Immunofluorescence microscopy of cells with or without an 18°C preincubation showed a colocalization of H2 with the ER and not with the Golgi complex. We conclude that H2 is not degraded in lysosomes and never reaches the medial Golgi compartment in an intact form, but rather degradation is initiated in a pre-Golgi compartment, possibly part of the ER. The 35-kD fragment of H2 may define an initial proteolytic cleavage in the ER. MANY cell surface receptors and secreted proteins are composed of multiple subunits. The assembly of the individually synthesized subunits into complexes is not a perfectly efficient process; in many instances, a fraction of the subunits made is never assembled or routed to the cell surface (e.g., Dulis et al., 1982;Merlie et al., 1982;Plant and Grieninger, 1986;Corless et al., 1987;Minami et al., 1987). The human hepatic asialoglycoprotein (ASGP) ~ receptor is an attractive system in which to study this process. This receptor is composed of two strongly homologous subunits, H1 and H2. Studies using chemical cross-linking and antibody-induced degradation showed that, in the human hepatoma cell line HepG2, the receptor is a heterooligomer (Bischoff et al., 1988). The functional complex is at least a trimer, containing at least one H2 and two (or three) HI polypeptides. HI is synthesized as a 40-kD core-glycosylated precursor, oriented in the ER membrane with its NH2 terminus in the cytoplasm and its COOH terminus in the ER lumen. During transit through the Golgi complex, the modification of its two N-linked oligosaccharide chains causes a size increase to ~46 kD. Similarly, H2J. E Amara's present address is Procept lnc,, 840 Memorial Drive, Cambridge, MA 02139. : ASGE asiatoglycoprotein; endo, endoglycosidase. is synthesized as a 43-kD transmembrane protein with three high-mannose oligosaccharide chains and is then modified in the Golgi complex to the mature form of ~50 kD. In HepG2 cells, about one thi...

Abstract. We have identified a vesicle fraction that contains arantitrypsin and other human HepG2 hepatoma secretory proteins en route from the rough endoplasmic reticulum (RER) to the cis face of the Golgi complex.[35S]Methionine pulse-labeled cells were chased for various periods of time, and then a postnuclear supernatant fraction was resolved on a shallow sucrose-D20 gradient. This intermediate fraction has a density lighter than RER or Golgi vesicles. Most chantitrypsin in this fraction (P1) bears N-linked oligosaccharides of composition similar to that of chantitrypsin within the RER; mainly MansGlcNac2 with lesser amounts of ManTGlcNac2 and MangGlcNac2; this suggests that the protein has not yet reacted with ct-mannosidase-I on the cis face of the Golgi complex. This light vesicle species is the first post-ER fraction to be filled by labeled ¢tl-antitrypsin after a short chase, and newly made secretory proteins enter this compartment in proportion to their rate of exit from the RER and their rate of secretion from the cells: al-antitrypsin and albumin faster than preC3 and ctrantichymotrypsin, faster, in turn, then transferrin. Deoxynojirimycin, a drug that blocks removal of glucose residues from ¢tl-antitrypsin in the RER and blocks its intracellular maturation, also blocks its appearance in this intermediate compartment. Upon further chase of the cells, we detect sequential maturation of ctrantitrypsin to two other intracellular forms: first, P2, a form that has the same gel mobility as P1 but that bears an endoglycosidase H-resistant oligosaccharide and is found in a compartment-probably the medial Golgi complex-of density higher than that of the intermediate that contains P1; and second, the mature sialylated form of ctrantitrypsin.

Abstract. Antibody-induced degradation and chemical cross-linking experiments have been carried out to assess the nature of the interaction between the two asialoglycoprotein-receptor polypeptides, H1 and H2, synthesized in HepG2 cells. Incubation of HepG2 cell monolayers with anti-H1 antibody caused a specific and equal loss of both H1 and H2 polypeptides. The same result was obtained with anti-H2 antibody. Control serum did not affect the level of H1 or H2 nor did anti-H1 or anti-H2 antibodies affect the level of the transferrin receptor. The chemical cross-linking reagent, difluorodinitrobenzene, has been used to demonstrate that H1 can be cross-linked to H2 in HepG2 cell microsomal membranes. Dimer and trimer species with apparent molecular masses of 93 and 148 kD, respectively, were readily observed upon chemical cross-linking and some dimers and trimers were immunoreactive with both anti-H1 and anti-H2 antibodies. The putative trimer, possibly two H1 and one H2 molecules, is a minimum estimate of the true size of the asialoglycoprotein receptor in intact HepG2 cell, and it is possible that larger hetero-oligomeric forms of the receptor exist. The results of both types of experiments indicate that H1 and H2 form an oligomeric complex in HepG2 cells and thus, both polypeptides constitute the human asialoglycoprotein receptor. THE asialoglycoprotein receptor (ASGP-R) ~ is a liverspecific membrane glycoprotein that binds to terminal galactose and N-acetylgalactosamine residues on serum glycoproteins (for review, 2, 6). This receptor has been studied extensively in rabbit liver, rat liver, and human liver, as well as the human hepatoma cell line HepG2. In each of these species, the receptor activity appears to consist of multiple polypeptide chains. In rabbit liver, 40-and 48-kD proteins have been observed (12, 13). In rat liver, three polypeptide species of 41.5, 49, and 54 kD have been characterized (7,22,28). In human liver and in HepG2 cells (3, 23), a single polypeptide of 46 kD has been observed; more recently, a 50-kD protein has also been characterized (5). In all cases, the higher molecular mass species are less abundant than the lower molecular mass polypeptides. Protein sequencing (7) and cDNA cloning (11,18, 26,27) of the rat and human receptors has revealed that the polypeptides are distinct from each other. The human cDNAs and their protein products are referred to as H1 and H2 (26) while the rat hepatic lectin (RHL) cDNAs are referred to as RHL-1 and RHL-2/3 (7). RHL-1 is more homologous to H1 than to RHL-2/3, and RHL-2/3 is more homologous to H2 than to 26). RHL-2/3 is so named because it consists of two polypeptide species of 49 and 54 kD, respectively; the increment in ap-1, Abbreviations used in this paper: ASGP-R, asialoglycoprotein receptor; DFDNB, 1,5-difluoro-2,4 dinitrobenzene; DTSP, dithiobis (succinimidyl) propionate; RHL, rat hepatic lectin. parent molecular mass has been attributed to differential oligosaccharide modifications (10).The fact that the proteins are distinct molecules r...

MicroRNAs are small non‐coding RNAs that are important regulators of gene expression. MicroRNA profiling studies have determined a number of tissue‐specific microRNAs. miR‐1 and miR‐133 are examples of tissue‐specific microRNAs and are exclusively muscle‐specific being expressed only in the heart and skeletal muscle. miR‐206 is preferentially expressed in the skeletal muscle. We have determined the expression pattern of these tissue‐specific microRNAs in C2C12 cells in an effort to see whether we can use them to study their regulation in an in vitro cell culture system. We describe the induction of miR‐1 and miR‐133 expression specifically during the myogenic conversion of C2C12 myoblasts and in primary human myoblasts. Furthermore, we have used genomic location analyses to determine the occupancy of the myogenic factor MyoD and Myogenin in upstream regions of these microRNAs. Our data suggests that these myogenic factors are important in regulating the expression of miR‐1 and miR‐133 in C2C12 cells, and by extension, in normal skeletal muscle.

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