By application of a dual selection strategy using two distinct membrane receptors to simultaneously internalize different glycoprotein toxin conjugates, mutant hepatoma (HuH-7) cell lines termed Trafficking 1 and 2 (Trfl, TrfZ) were isolated. The pleiotropic phenotype of the Trfl cell line is distinct from that of all other endocytosis mutants previously isolated. The mutation harbored by Trfl cells reduces the cell surface expression of several unrelated membrane-proteins and provides the first genetic evidence for the existence of different subpopulations of cell surface receptors. By expression cloning of a cDNA that complements the trfl gene we identified a novel subunit of Casein Kinase 2 alpha, CK2 alpha. The long-term objective of this application is to identify the biochemical mechanisms that govern the subcellular trafficking of membrane-proteins in hepatocytes.
The Specific Aims of this project are designed to define the molecular basis of differential membrane-protein trafficking.
In Aim 1, real time confocal microscopy of a green fluorescent-asialoglycoprotein receptor fusion protein coupled with mutational analysis will provide the means to determine whether an acid cluster of amino acids localized in the cytoplasmic domain of the receptor is responsible for regulated traffic between the plasma membrane and the sorting/recycling endosomal compartment.
In Aim 2, a construct encoding the cytoplasmic domain of the receptor will be utilized to isolate and identify proteins that selectively interact with the acid-cluster sorting motif that are responsible for maintaining the fidelity of vesicular traffic between intercellular compartments along the microtubular network.
In Aim 3, by expression cloning, we will define the genetic and molecular basis of the Trf2 phenotype. Initially, somatic cell hybridization will be used to determine whether the TrfZ phenotype is recessive or dominant. Based on these findings, a cloning strategy will be designed. Once a complementary cDNA is obtained and sequenced, antibodies will be raised to characterize and localize the trf2 gene product in both the Trf2 mutant and parental HuH-7 cells. In addition, using improved selection agents, attempts to isolate new HuH-7 mutants in the endocytic pathway will be undertaken.
Showing the most recent 10 out of 122 publications
