We previously showed that two subpopulations of asialoglycoprotein receptors (ASGP-Rs), designated State 1 and State 2 ASGP-Rs, are present in intact cells and that State 2 ASGP-Rs can be inactivated in permeable rat hepatocytes in a temperature- and ATP-dependent manner. These inactivated ASGP-Rs can be quantitatively reactivated by the addition of palmitoyl-CoA (Weigel, P. H., and Oka, J. A.(1993) J. Biol. Chem. 268, 27186-27190). Here we show that 50% of purified rat ASGP-Rs are inactivated by treatment with hydroxylamine under mild conditions. The activity of affinitypurified ASGP-Rs was assessed by measuring the specific binding of I-asialo-orosomucoid (ASOR) in a dot-blot assay after immobilization onto nitrocellulose. Treatment of ASGP-Rs in solution with 0.0125-1.0 M NHOH, pH 7.4, at 4 °C for 4 h resulted in a progressive loss of ASOR binding activity. ASGP-R inactivation with NHOH occurred more readily at basic pH or at room temperature. Similar treatment with Tris had no effect on ASGP-R activity. The kinetics of ASGP-R activity loss and the dose-response for this inactivation were both biphasic, indicating the presence of two equal populations of ASGP-Rs with different sensitivities to NHOH. The more sensitive population of ASGP-Rs (50%) was inactivated by treatment with 0.2 M NHOH (4 °C, 4 h) or with 1.0 M NHOH (4 °C, 1 h) without detectable peptide cleavage as assessed by SDS-polyacrylamide gel electrophoresis. State 1 ASGP-Rs, purified from chloroquine- or monensin-treated hepatocytes, showed significantly less sensitivity to NHOH treatment (both in kinetics and dose dependence). Furthermore, under mild conditions NHOH caused dissociation and inactivation of 50% of the total ASGP-Rs (State 1 and State 2) that were prebound to ASOR-Sepharose, whereas the same treatment caused dissociation of only <20% of State 1 ASGP-Rs from such preformed complexes. As shown in the accompanying paper (Zeng, F. Y., Kaphalia, B. S., Ansari, G. A. S., and Weigel, P. H.(1995) J. Biol. Chem. 270, 21382-21387) all three RHL subunits of active ASGP-Rs, in fact, contain covalently attached palmitate and stearate. In cultured cells, [H]palmitic acid is metabolically incorporated into all three subunits. These radiolabeled fatty acids are completely released from purified ASGP-Rs by mild NHOH treatment. We conclude that the NHOH-sensitive subpopulation of ASGP-Rs corresponds to the previously described State 2 ASGP-Rs and that these receptors require fatty acylation for their ligand binding activity.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. H. N. Yik and P. H. Weigel The Position of Cysteine Relative to the Transmembrane Domain Is Critical for Palmitoylation of H1, the Major Subunit of the Human Asialoglycoprotein Receptor J. Biol. Chem., November 27, 2002; 277(49): 47305 - 47312. [Abstract] [Full Text] [PDF]

				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]

				J. H. N. Yik, A. Saxena, and P. H. Weigel The Minor Subunit Splice Variants, H2b and H2c, of the Human Asialoglycoprotein Receptor Are Present with the Major Subunit H1 in Different Hetero-oligomeric Receptor Complexes J. Biol. Chem., June 14, 2002; 277(25): 23076 - 23083. [Abstract] [Full Text] [PDF]

				R. WESTERHUIS, G. VAN ZANDBERGEN, N. A. M. VERHAGEN, N. KLAR-MOHAMAD, M. R. DAHA, and C. VAN KOOTEN Human Mesangial Cells in Culture and in Kidney Sections Fail to Express Fc Alpha Receptor (CD89) J. Am. Soc. Nephrol., April 1, 1999; 10(4): 770 - 778. [Abstract] [Full Text]

				M. Manifava, J. Sugars, and N. T. Ktistakis Modification of Catalytically Active Phospholipase D1 with Fatty Acid in Vivo J. Biol. Chem., January 8, 1999; 274(2): 1072 - 1077. [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, B. S. Kaphalia, G. A. S. Ansari, and P. H. Weigel Fatty Acylation of the Rat Asialoglycoprotein Receptor J. Biol. Chem., September 8, 1995; 270(36): 21382 - 21387. [Abstract] [Full Text] [PDF]