Surface and internal galactosyl receptors are heterooligomers and retain this structure after ligand internalization or receptor modulation

• Published in: Biochemistry (Easton) Vol. 29; no. 27; pp. 6437 - 6447
• Main Authors: Herzig, Maryanne C. S, Weigel, Paul H
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 10.07.1990
• Subjects: 550201 - Biochemistry- Tracer Techniques; Affinity Labels; Animals; Asialoglycoprotein Receptor; Asialoglycoproteins; ATP; BASIC BIOLOGICAL SCIENCES; Biological and medical sciences; Cell receptors; Cell structures and functions; CHEMICAL REACTIONS; CROSS-LINKING; Cross-Linking Reagents; DERIVATIZATION; ELECTROPHORESIS; Endocytosis; ESTERS; Fundamental and applied biological sciences. Psychology; Galactose - metabolism; GLYCOPROTEINS; Glycoproteins - metabolism; hepatocytes; Kinetics; LIGANDS; Liver - metabolism; Male; MEMBRANE PROTEINS; Miscellaneous; Molecular and cellular biology; MOLECULAR STRUCTURE; NUCLEOTIDES; ORGANIC COMPOUNDS; Orosomucoid - analogs & derivatives; POLYMERIZATION; PROTEINS; Rats; Rats, Inbred Strains; RECEPTORS; Receptors, Cell Surface - classification; Receptors, Cell Surface - metabolism; Receptors, Cell Surface - ultrastructure; Receptors, Immunologic - classification; Receptors, Immunologic - metabolism; Receptors, Immunologic - ultrastructure; Purification; Rat; Liver; Rodentia; Crosslink agent; Subunit; Spatial structure; Characterization; Vertebrata; Mammalia; Substrate specificity; Immunoblotting assay; Chemical synthesis; Biological receptor
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00479a015

We have developed a specific chemical affinity reagent for the hepatic galactosyl receptor (GalR) by derivatizing asialoorosomucoid (ASOR) with the homobifunctional N-hydroxysuccinimide (NHS) ester cross-linker disuccinimidyl suberate [Herzig, M. C. S., & Weigel, P. H. (1989) Biochemistry 28, 600]. NHS-ASOR cross-links with 30-50% efficiency to the three GalR subunits, designated rat hepatic lectins (RHL) 1, 2, and 3. Here, we examined the subunit structure of both surface and internal receptors of two functionally distinct GalR subpopulations, designated state 1 or state 2 GalR. Freshly isolated cells, referred to as state 1 cells, kept at 4 degrees C express only active state 1 GalR on their surface. When these cells are equilibrated at 37 degrees C, they then express both state 1 GalR and state 2 GalR on their surface. These cells are referred to as state 1,2 cells. After incubation at 4 degrees C with NHS-125I-ASOR, surface or internal GalR of state 1 cells or of state 1,2 cells incorporated 125I-ASOR into all three RHL subunits. As analyzed by autoradiography of SDS-PAGE, radiolabeling was identical for all conditions and was in a ratio of 1:1:1 for RHL 1:2:3. Native GalR structure was also examined by first cross-linking nonradiolabeled NHS-ASOR at 4 degrees C to surface or internal receptors of state 1 or state 1,2 hepatocytes. These cells were then washed with EGTA, extracted with Triton X-100, immunoprecipitated with anti-orosomucoid antibody, and subjected to Western blot analysis. Antisera specific for RHL 1 or RHL 2/3 detected cross-linked complexes of Mr congruent to 85K or congruent to 90K-115K, respectively, as well as un-cross-linked native subunits. In all four cases, the ratio of free to cross-linked subunits was greater than or equal to 5:1 for RHL 1 and less than or equal to 0.5:1 for RHL 2/3. Internalized GalR had the same ratio of free to cross-linked subunits as noninternalized GalR. Depletion of ATP either before or after cross-linking GalR to NHS/ASOR also did not alter the ratio of free cross-linked RHL subunits. We conclude that the surface and internal GalR of the two functionally distinct GalR populations have the same heterooligomeric subunit composition and that this GalR structure persists following endocytosis or ATP depletion.