Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently crosslink to galactosyl receptors on isolated rat hepatocytes

Herzig, Maryanne C.; Weigel, Paul H.

doi:10.1021/bi00428a028

Cited by 17 publications

(10 citation statements)

References 53 publications

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“…Our previous immunoprecipitation results are consistent with this prediction (11). In contrast to our results, Herzig and Weigel (28) have recently proposed that RHL-1 interacts with RHL-2/3 in the ratio 2:1. The critical difference is their model assumes all RHL-1 polypeptides to be present in the heterooligomeric complex.…”

Section: Discussioncontrasting

confidence: 99%

Assembly of a heterooligomeric asialoglycoprotein receptor complex during cell-free translation.

Sawyer¹,

Doyle²

1990

Proc. Natl. Acad. Sci. U.S.A.

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We have translated RNAs for the two rat asialoglycoprotein receptor polypeptides together in a cell-free system containing dog pancreatic microsomes and immunoprecipitated the products with antibodies that distinguish the two proteins. In this system the proteins oligomerize, as judged by their coprecipitation with either of the subunit-specific antisera. Oligomerization does not occur between subunits synthesized without microsomes or between subunits synthesized on separate microsomes mixed during detergent solubilization. Thus, oligomerization occurs within the microsomal membrane. We calculate that oligomerization proceeds with an efficiency of -85%. The receptor complex appears to represent a specific oligomer because it excludes a third membrane glycoprotein synthesized in the same reaction. Oligomerization of the asialoglycoprotein receptor in vitro should provide a useful system to study the assembly of a membrane-protein complex.The hepatic asialoglycoprotein receptor of rats, rat hepatic lectin (RHL), consists of three different subunits (1-3). Two of these (RHL-2 and RHL-3) have the same polypeptide chain (RHL-2/3) but differ in carbohydrate modifications, whereas the remaining subunit (RHL-1) is a distinct, but related, polypeptide (4). The receptor binds serum glycoproteins that have carbohydrate chains terminating in galactose, and each subunit has a galactose-binding site (4-6). As in rat, the receptor exists as more than one polypeptide in the mouse, rabbit, and human (7-9). Sequence analysis shows that the two receptor polypeptide subtypes identified in human hepatoma cells are quite similar in their primary structure to those of rat (4, 10).We have previously reported that all three rat receptor subunits can be immunoprecipitated with antisera reacting specifically with the carboxyl terminus of either the RHL-1 or RHL-2/3 polypeptide (11). Thus, the various rat receptor subunits can form a heterotypic oligomer that may be the principal form found on the cell surface. A heterooligomer of the human polypeptides has also been proposed on the observation that antibodies specific for one of the molecules accelerate the turnover of both (12). In addition, the same antibodies will precipitate a complex of both human subunits after chemical cross-linking (12).The physical evidence for the heterooligomeric structure of the receptor is corroborated by cDNA transfection experiments. Synthesis in various cell lines of transfected asialoglycoprotein receptor with properties similar to that of the endogenous receptor of hepatocytes occurs only when both polypeptides are expressed in the same cell (13)(14)(15). Though each subunit has a galactose-binding site (4-6), the heterooligomeric complex apparently has to be assembled to achieve high-affinity uptake of serum glycoproteins.Despite the above information, the RHL complex has yet to be well characterized. The carboxyl-terminal antibodies we have generated have been of limited usefulness in this regard because these react with only a fraction ofthe ...

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Section: Discussioncontrasting

confidence: 99%

Assembly of a heterooligomeric asialoglycoprotein receptor complex during cell-free translation.

Sawyer¹,

Doyle²

1990

Proc. Natl. Acad. Sci. U.S.A.

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“…We do not know if there is a structural basis for the two functionally distinct Gal receptor populations. However, we recently proposed a new structural model for the receptor, in which three different heterohexamers can be formed from the three rat Gal receptor polypeptides [38].…”

Section: Discussionmentioning

confidence: 99%

Differential effects of leupeptin, monensin and colchicine on ligand degradation mediated by the two asialoglycoprotein receptor pathways in isolated rat hepatocytes

Clarke

Weigel

1989

Biochemical Journal

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We have shown that degradation of asialo-orosomucoid (ASOR) in isolated rat hepatocytes occurs by two different intracellular pathways [Clarke, Oka & Weigel (1987) J. Biol. Chem. 262, 17384-17392] mediated by two subpopulations of cell surface galactosyl (Gal) receptors, designated State 1 or State 2 receptors. In the present study, several inhibitors were tested for their effects on ligand degradation by the State 1 or State 2 pathway. Leupeptin, monensin and chloroquine completely inhibited degradation of 125I-labelled ASOR in both pathways. Dose-response studies showed, however, that the State 2 pathway was more sensitive to leupeptin or monensin than the State 1 pathway. No differences were observed with chloroquine. For example, the onset of inhibition in the State 2 and State 1 pathways occurred at about 0.05 and 0.3 microM-leupeptin respectively, a 6-fold difference. At 3.5 microM-monensin, 125I-ASOR degradation in the State 2 pathway was completely blocked, whereas degradation in the State 1 pathway was essentially unaffected. Colchicine was observed to give the largest differential sensitivity between the two pathways. The State 2 degradation pathway was about 30-fold more sensitive to colchicine than the State 1 pathway. Lumicolchicine had no affect. The onset of inhibition of the rate of 125I-ASOR degradation in the State 2 and State 1 pathways occurred at approximately 0.1 and 3.0 microM-colchicine respectively. At very high concentrations (greater than 0.1 mM), the State 1 pathway could be completely inhibited. We conclude that intracellular 125I-ASOR processing or delivery to degradative compartments in both the State 1 and State 2 Gal receptor pathways requires low pH. Ligand delivery to the degradative compartment does not require microtubules in the State 1 pathway, consistent with the very rapid onset of degradation in this pathway. The State 2 degradation pathway does require microtubules.

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“…Heterooligomerization appears to create a productive arrangement of the subunit binding sites. All subunits of the complex are involved in ligand binding as judged by affinity labeling and cross-linking of ligand and receptor (Lee & Lee, 1986 Herzig & Weigel, 1989).…”

Section: A Mammalian Hepatic Lectinmentioning

confidence: 99%

The asialoglycoprotein receptor: a model for endocytic transport receptors

1990

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Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently crosslink to galactosyl receptors on isolated rat hepatocytes

Cited by 17 publications

References 53 publications

Assembly of a heterooligomeric asialoglycoprotein receptor complex during cell-free translation.

Assembly of a heterooligomeric asialoglycoprotein receptor complex during cell-free translation.

Differential effects of leupeptin, monensin and colchicine on ligand degradation mediated by the two asialoglycoprotein receptor pathways in isolated rat hepatocytes

The asialoglycoprotein receptor: a model for endocytic transport receptors

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