Oligomeric structure of the human asialoglycoprotein receptor: nature and stoichiometry of mutual complexes containing H1 and H2 polypeptides assessed by fluorescence photobleaching recovery.
The interactions between H1 and H2, the two polypeptides comprising the human asialoglycoprotein receptor (ASGP-R), were investigated by immunofluorescence and lateral mobility measurements combined with antibody-mediated cross-linking and immobilization. Immunofluorescence microscopy revealed two ASGP-R populations on the cell surface, one homogeneously distributed and the other in micropatches. This was observed both in stably transfected NIH 3T3 lines expressing H1 and/or H2, and in the human hepatoma cell line HepG2. In transfected cells expressing both polypeptides (the 1-7-1 line), H1 and H2 were colocalized in the same micro aggregates. Moreover, enhancement of the patching of, e.g., H1 by IgG-mediated crosslinking was accompanied by copatching of H2. To quantify H1-H2 complex formation, the lateral diffusion of H1 and H2 was measured at 12 degrees C (to avoid internalization) by fluorescence photobleaching recovery. H1 (or H2) was immobilized by crosslinking with specific IgG molecules; the other chain was labeled with fluorescent monovalent Fab' fragments, and is lateral mobility was measured. In HepG2 cells, immobilization of either H1 or H2 led to an equal immobilization of the other, indicating that all the mobile H1 and H2 are in stable heterooligomers. In 1-7-1 cells, immobilization of H2 immobilized H1 to the same degree, but immobilization of H1 reduced the mobile fraction of H2 only by 2/3. Thus, in 1-7-1 cells all surface H1 molecules are associated with H2, but 1/3 of the H2 population is independent of H1. From these data and from measurements of the relative surface densities of H1 and H2, conclusions are drawn regarding the oligomeric structure and stoichiometry of the ASGP-R.
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel.
Y I
YI
Henis
Z
Z
Katzir
M A
MA
Shia
H F
HF
Lodish
D000818:Animals; D000906:Antibodies; D037263:Asialoglycoprotein Receptor; D002460:Cell Line; D003432:Cross-Linking Reagents; D005455:Fluorescent Antibody Technique; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection
D000906:Antibodies; D037263:Asialoglycoprotein Receptor; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011971:Receptors, Immunologic