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 [H]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.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. H. N. Yik, A. Saxena, J. A. Weigel, and P. H. Weigel Nonpalmitoylated Human Asialoglycoprotein Receptors Recycle Constitutively but Are Defective in Coated Pit-mediated Endocytosis, Dissociation, and Delivery of Ligand to Lysosomes J. Biol. Chem., October 18, 2002; 277(43): 40844 - 40852. [Abstract] [Full Text] [PDF]

				D. D. McAbee and X. Jiang Copper and Zinc Ions Differentially Block Asialoglycoprotein Receptor-mediated Endocytosis in Isolated Rat Hepatocytes J. Biol. Chem., May 21, 1999; 274(21): 14750 - 14758. [Abstract] [Full Text] [PDF]

				F.-Y. Zeng and P. H. Weigel Fatty Acylation of the Rat and Human Asialoglycoprotein Receptors. A CONSERVED CYTOPLASMIC CYSTEINE RESIDUE IS ACYLATED IN ALL RECEPTOR SUBUNITS J. Biol. Chem., December 13, 1996; 271(50): 32454 - 32460. [Abstract] [Full Text] [PDF]

				F.-Y. Zeng and P. H. Weigel Hydroxylamine Treatment Differentially Inactivates Purified Rat Hepatic Asialoglycoprotein Receptors and Distinguishes Two Receptor Populations J. Biol. Chem., September 8, 1995; 270(36): 21388 - 21395. [Abstract] [Full Text] [PDF]

				X. Liang, A. Nazarian, H. Erdjument-Bromage, W. Bornmann, P. Tempst, and M. D. Resh Heterogeneous Fatty Acylation of Src Family Kinases with Polyunsaturated Fatty Acids Regulates Raft Localization and Signal Transduction J. Biol. Chem., August 10, 2001; 276(33): 30987 - 30994. [Abstract] [Full Text] [PDF]