The goal of this research is the isolation and biochemical and genetic characterization of mammalian cells carrying mutations in the structural and regulatory genes that participate in receptor-mediated endocytosis. Receptor-mediated endocytosis is a process by which animal cells bind and internalize physiologically active extracellular macromolecules including serum transport proteins such as low density lipoprotein (LDL) and peptide hormones. Defects in endocytosis can result in major human disease. For example, familial hypercholesterolemia (FH), a relatively common genetic disease, is the result of the defective endocytosis of LDL, the major cholesterol-transporter in human plasma. We have developed a Chinese hamster ovary (CHO) cell culture system for the genetic and biochemical analysis of endocytosis and we have described four genes which are required for the expression of LDL receptor activity (ldlA-ldlD). Additional types of mutants in endocytosis will be isolated using previously established and newly developed selection techniques. CHO cells carrying conditional-lethal mutations and specific types of dominant mutations will be isolated using modifications of our previously described reconstituted LDL and MeLoCo-Amphotericin B methods. Genetic analysis of the mutants will include complementation and reversion studies. The phenotypes of the mutants will be characterized and a thorough biochemical analysis will be conducted. We will use cloned LDL receptor probes to compare the LDL receptor gene structure and expression in wild type cells to that in the mutants. Recombinant DNA methods will be used when appropriate. Our long term goal is to define the molecular mechanisms by which extracellular macromolecules such as LDL regulate cellular metabolism in order 1) to expand our general understanding of fundamental biological processes and 2) to build a firm foundation for the rational diagnosis and treatment of human disease, such as atherosclerosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030243-05
Application #
3277888
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1982-07-01
Project End
1988-07-31
Budget Start
1986-08-01
Budget End
1987-07-31
Support Year
5
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Type
Other Specialized Schools
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139