Most of our projects focus on the function of segments of the two polypeptides that constitute the human hepatic asialoglycoprotein receptor. They are very similar yet exhibit different properties in transfected cells. We will determine the cell-surface distribution of the receptor polypeptides with respect to coated pits, and determine whether ligand or antibody triggers receptor accumulation in coated pits and endocytosis. We showed that when one receptor polypeptide, H2, is expressed without the other, H1, in 3T3 cells, it is synthesized but rapidly degraded. We want to determine whether H2 is degraded in lysosomes, and whether it gets there from the ER without traversing the Golgi. We will make several deletion mutants of H1 and H2, and also chimeras of H1 and H2: by expressing these in 3T3 dells by themselves, or together with normal H1 or H2, we should define segments important in ligand binding, low pH-induced ligand dissociation, endocytosis, transport from ER to Golgi, and rapid degradation. Then we hope to use peptides corresponding to segments of the wild type and mutant proteins to search for cell proteins that bind specifically to that functional segment, and that might be a """"""""transport receptor"""""""" or """"""""sorting protein."""""""" Other studies focus on regulated transport or newly made hepatic secretory proteins from the ER to the Golgi, emphasizing retinol binding protein and alpha1 antitrypsin. Using 3T3 cells expressing wild type or mutant proteins we want to define the requirements for exit from the ER. We want to identify the putative ER-to-Golgi transport receptor by virtue of its specific binding to the holo, not the apo- form of RBP, and to the wild type and not mutant forms of these proteins. By making and expressing chimeras of RBP and alpha1-antitrypsin, and studying the requirements for their maturation from the ER, we hope to determine whether retention of unfolded proteins in the ER, or active export of folded proteins to the Golgi, is employed.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035012-09
Application #
2177704
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1984-07-01
Project End
1994-12-31
Budget Start
1993-01-01
Budget End
1994-12-31
Support Year
9
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Whitehead Institute for Biomedical Research
Department
Type
DUNS #
076580745
City
Cambridge
State
MA
Country
United States
Zip Code
02142
Tolchinsky, S; Yuk, M H; Ayalon, M et al. (1996) Membrane-bound versus secreted forms of human asialoglycoprotein receptor subunits. Role of a juxtamembrane pentapeptide. J Biol Chem 271:14496-503
Yuk, M H; Lodish, H F (1993) Two pathways for the degradation of the H2 subunit of the asialoglycoprotein receptor in the endoplasmic reticulum. J Cell Biol 123:1735-49
Lodish, H F; Kong, N (1993) The secretory pathway is normal in dithiothreitol-treated cells, but disulfide-bonded proteins are reduced and reversibly retained in the endoplasmic reticulum. J Biol Chem 268:20598-605
Wikstrom, L; Lodish, H F (1993) Unfolded H2b asialoglycoprotein receptor subunit polypeptides are selectively degraded within the endoplasmic reticulum. J Biol Chem 268:14412-6
Lodish, H F; Kong, N; Wikstrom, L (1992) Calcium is required for folding of newly made subunits of the asialoglycoprotein receptor within the endoplasmic reticulum. J Biol Chem 267:12753-60
Wikstrom, L; Lodish, H F (1992) Endoplasmic reticulum degradation of a subunit of the asialoglycoprotein receptor in vitro. Vesicular transport from endoplasmic reticulum is unnecessary. J Biol Chem 267:5-8
Lederkremer, G Z; Lodish, H F (1991) An alternatively spliced miniexon alters the subcellular fate of the human asialoglycoprotein receptor H2 subunit. Endoplasmic reticulum retention and degradation or cell surface expression. J Biol Chem 266:1237-44
Lodish, H F; Kong, N (1991) Cyclosporin A inhibits an initial step in folding of transferrin within the endoplasmic reticulum. J Biol Chem 266:14835-8
Lodish, H F (1991) Recognition of complex oligosaccharides by the multi-subunit asialoglycoprotein receptor. Trends Biochem Sci 16:374-7
Wikstrom, L; Lodish, H F (1991) Nonlysosomal, pre-Golgi degradation of unassembled asialoglycoprotein receptor subunits: a TLCK- and TPCK-sensitive cleavage within the ER. J Cell Biol 113:997-1007

Showing the most recent 10 out of 28 publications