Abstract
Rat hepatic asialoglycoprotein receptors (ASGP-Rs) are hetero-oligomers composed of three homologous glycoprotein subunits, designated rat hepatic lectins (RHL) 1, 2, and 3. ASGP-Rs mediate the endocytosis and degradation of circulating glycoconjugates containing terminal N-acetylgalactosamine or galactose, including desialylated plasma glycoproteins. We have shown in permeable rat hepatocytes that the ligand binding activity of one subpopulation of receptors (designated State 2 ASGP-Rs) can be decreased or increased, respectively, by ATP and palmitoyl-CoA (Weigel, P. H., and Oka, J. A. (1993) J. Biol. Chem. 268, 27186-27190). We proposed that a reversible and cyclic acylation/deacylation process may regulate ASGP-R activity during endocytosis, receptor-ligand dissociation, and receptor recycling. In the accompanying paper (Zeng, F-Y., and Weigel, P. H. (1995) J. Biol. Chem. 270, 21388-21395), we show that the ligand binding activity of affinity-purified State 2 ASGP-Rs is decreased by treatment with hydroxylamine under mild conditions consistent with these ASGP-Rs being fatty acylated in vivo. In this study, we used a chemical method to determine the presence of covalently-bound fatty acids in individual ASGP-R subunits. The affinity-purified ASGP-R preparations were separated by SDS-polyacrylamide gel electrophoresis under nonreducing conditions, and the gel slices containing individual RHL subunits were treated with alkali to release covalently bound fatty acids, which were subsequently analyzed by gas chromatography and confirmed by gas chromatography-mass spectrometry. Both stearic and palmitic acids were detected in all three receptor subunits. Pretreatment of ASGP-Rs with hydroxylamine before SDS-polyacrylamide gel electrophoresis reduced the content of both fatty acids by 66-80%, indicating that most of these fatty acids are attached to cysteine residues via thioester linkages. Furthermore, when freshly isolated hepatocytes were cultured in the presence of [3H]palmitate, all three RHL subunits in affinity-purified ASGP-Rs were metabolically labeled. We conclude that RHL1, RHL2, and RHL3 are modified by fatty acylation in intact cells.
Highlights
Rat hepatic asialoglycoprotein receptors (ASGP-Rs) are hetero-oligomers composed of three homologous glycoprotein subunits, designated rat hepatic lectins (RHL) 1, 2, and 3
The affinity-purified ASGP-R preparations were separated by SDS-polyacrylamide gel electrophoresis under nonreducing conditions, and the gel slices containing individual RHL subunits were treated with alkali to release covalently bound fatty acids, which were subsequently analyzed by gas chromatography and confirmed by gas chromatography-mass spectrometry
These above results suggest that one or all ASGP-R subunits are modified by fatty acylation in vivo and that a reversible acylation/deacylation process may be involved in regulating the ligand-binding activity of ASGP-Rs as they function during receptor mediated endocytosis
Summary
THE THREE SUBUNITS FROM ACTIVE RECEPTORS CONTAIN COVALENTLY BOUND PALMITATE AND STEARATE*. In the accompanying paper [15], we demonstrate that the activity of one population of affinity-purified ASGP-Rs, the State 2 receptors, is selectively inactivated by treatment with hydroxylamine, a chemical frequently used to release thioester-linked fatty acids from proteins. These above results suggest that one or all ASGP-R subunits are modified by fatty acylation in vivo and that a reversible acylation/deacylation process may be involved in regulating the ligand-binding activity of ASGP-Rs as they function during receptor mediated endocytosis. Treatment of each RHL subunit with hydroxylamine under mild conditions released both fatty acids in relatively large amounts, indicating that most fatty acids are attached via thioester linkages
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