SURFACE AND INTERNAL GALACTOSYL RECEPTORS ARE HETEROOLIGOMERS AND RETAIN THIS STRUCTURE AFTER LIGAND INTERNALIZATION OR RECEPTOR MODULATION

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 °C express only active state 1 GalR on their surface. When these cells are equilibrated at 37 °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 °C with NHS-125I-AS0R, surface or internal GalR of state 1 cells or of state 1,2 cells incorporated 125I-AS0R 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 °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 2β detected cross-linked complexes of Mr ≃85K or ≃90K-115K, respectively, as well as un-cross-linked native subunits. In all four cases, the ratio of free to cross-linked subunits was ≥5:1 for RHL 1 and ≤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 to 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. © 1990, American Chemical Society. All rights reserved.