Our goal is to show the extent to which MHC antigens influence ligand binding by peptide hormone receptors and in turn are modified as a consequence of ligand binding to specific receptors. We have shown as association between HLA (genotype and phenotype) and the affinity of insulin receptors on B lymphoblasts. We will now further analyze the genetic associations between HLA and insulin receptor affinity and extend the analysis to the physical relationships between the antigens and receptor molecules. The genetic analysis will proceed through examination of mutant lymphoblasts, deleted for expression of some HLA antigens, which have been transfected with genes for the missing antigens. We will also attempt to rescue expression of HLA antigens in Daudi cells, to examine the effects of such rescue on insulin binding by Daudi. The physical analysis will take two approaches: 1. an attempt, based on precedents in the literature, to co-precipitate insulin receptors, detected by their endogenous tyrosine kinase activity, with class I MHC antigens, using specific anti-receptor and anti-class I monoclonal antibodies and 2. determination of the proximity of class I MHC antigens and insulin receptors in intact cells, using resonance energy transfer between endogenously (Beta-2-m) labeled class I antigens, and insulin receptors which we have shown can be specifically labeled with biotinyl insulin and avidin phycobilliproteins. Energy transfer will be measured in the flow cytometer in terms of donor quenching sensitized emission of acceptor. We plan to extend our work on the metabolic effects of epidermal growth factor binding on the display and phosphorylation state of wild-type and mutant class I MHC antigens, using flow cytometry to quantitate the display and standard immunochemical and biochemical approaches to determine the phosphorylation state of the antigens associated with changes in the display.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI019814-06
Application #
3129246
Study Section
Allergy and Immunology Study Section (ALY)
Project Start
1983-07-01
Project End
1990-05-31
Budget Start
1988-12-01
Budget End
1990-05-31
Support Year
6
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
Schools of Arts and Sciences
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218