A specific antibody to the carbohydrate recognition domain of the asialoglycoprotein receptor RHL1 subunit does not react with RHL2/3 but blocks ligand binding.
The rat asialoglycoprotein receptor (ASGPR) is believed to be a hetero-oligomer composed of three subunits, designated rat hepatic lectin 1, 2, and 3 (RHL1, 2, and 3). The carbohydrate recognition domains (CRDs) of RHL1 and RHL2/3 are 56% identical. We developed a polyclonal antibody that specifically recognizes the CRD of RHL1 but not RHL2/3. When purified ASGPRs were bound to ligand-Sepharose, the CRD of RHL1, but not RHL2 or RHL3, was resistant to digestion with subtilisin. Antibody against purified RHL1 CRD recognized only RHL1 in Western blot analysis of crude cell extracts or purified receptors without detectable cross-reaction to RHL2/3. Although it does not recognize the CRD of RHL2 or RHL3, this antibody specifically inhibited 80-90% of the cell surface or total cellular 125I-ASOR binding to isolated rat hepatocytes and > 90% of ligand binding to purified rat ASGPRs. The antibody also immunoprecipitates active ASGPRs containing all three RHL subunits. The results indicate that homo-oligomeric RHL2/3 complexes, able to bind ASOR, do not form on hepatocytes by subunit rearrangement.
Department of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
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D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D037263:Asialoglycoprotein Receptor; D001665:Binding Sites / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D008024:Ligands; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000276:immunology*
D000906:Antibodies; D037263:Asialoglycoprotein Receptor; D000939:Epitopes; D008024:Ligands; D011956:Receptors, Cell Surface