Abstract
Rat hepatocytes, freshly isolated by a collagenase perfusion technique, bound [3H]asialo-orosomucoid in a sugar-specific and calcium-dependent manner as expected for the hepatic asialoglycoprotein receptor. At least 90% of the total cell surface-bound [3H]asialo-orosomucoid represented specific binding and could be removed by washing with EDTA. Freshly isolated cells had about 7 x 10(4) surface receptors per cell. However, when cells were incubated at 37 degrees C, the number of surface receptors per cell rapidly increased 2- to 3-fold to about 2.2 x 10(5). This increase in receptor number occurred in the absence of serum and began within minutes, depending on the particular conditions used to keep the cells in suspension. (The maximal rate of appearance of new receptors at 37 degrees C was about 70 receptors per cell per s.) When cells were first exposed to a brief EDTA treatment at 4 degrees C, before measuring the binding of [3H]asialo-orosomucoid, the number of surface receptors per cell was found to increase by about 45%. Therefore, about 30% of the surface receptors on freshly isolated cells have already bound endogenous asialoglycoproteins or are present in the membrane in a cryptic form. At 4 degrees C the binding of [3H]asialo-orosomucoid was rapid (kon greater than or equal to 1.8 x 10(4) M-1s-1), whereas the dissociation of bound [3H]asialo-orosomucoid, measured in the presence of excess nonradioactive glycoprotein, was extremely slow (koff less than or equal to 0.9 x 10(-5) s-1). The association constant calculated from these data (Ka = 2.0 x 10(9) M-1) agreed well with that obtained from equilibrium binding experiments (Ka = 2.4 x 10(9) M-1) using untreated cells or cells which had first been treated with EDTA or incubated at 37 degrees C. In all cases, when the concentration of [3H]asialo-orosomucoid was higher than about 600 ng/ml, the Scatchard plots were curvilinear. The data are, however, consistent with the conclusion that there is a single high affinity receptor on the hepatocyte surface. The additional receptors that appear on the surface when cells are incubated at 37 degrees C or exposed to EDTA are identical with those on untreated cells,
Highlights
To assessing the binding of [3H]asialo-orosomucoid, there was If there are,two separate classes of asialoglycoprotein a virtually identical increase in the number of apparent high receptors, they are presentin identical ratios on freshly and low affinity receptors per cell. This is evident when the isolated cells and in thereceptorpopulations exposed by fraction of total receptors that arehigh affinity is calculated treatment with EDTA or by incubation a t 37°C
Hepatocytes howed a calcium-dependen(tFig. 3) and could suggest the possibility that there is a second class of sugar-specific (Fig. 2) binding of asialo-orosomucoid consist- lower affinity sites, we believe it is more likely that there is ent with the properties of the membrane-bound and solubi- really one typoef receptor
When freshly isolated cells are state conditions the totalcellular pool of functioning receptors placed at 37°C (under which conditions rapid endocytosis of would be present in particular amounts at each stage of the asialoglycoproteins occurs [14]), EDTA-exposable sites can recycling process
Summary
Tanabe et al purified the receptorfrom rat liver and examined its topological distribution in subcellular membrane fractions [6] They found viable receptor activity on the cytosolic surface of membranes in the lysosomal fraction. Secered and characterized by Morel et al more than 12 years ond, the asialoglycoprotein receptor appears tobe involved in ago [1].This receptor functionisn uiuo to effect the rapid and efficient clearance of desialylated serum glycoproteins It was the first mammalian protein identified as a lectin-like molethe specific adhesion of freshlyisolated rat hepatocytes to syntheticcarbohydrate surfaces [15]. For both of the studies mentioned above, iwt as necessary was assessed by including in the binding medium an excess of nontocharacterizethe asialoglycoprotein receptorson freshly radioactive asialo-orosomucoid (at least 50-fold). Steer and Ashwell subsequently reported on the receptor in isolated hepatocytes and provided additional evidence that the receptor isrecycled during the catabolism of asialo-orosomucoid [18].A preliminaryreport of our results has appeared [19]
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