The general aim of this research is to learn about the structure and functions of the membrane proteins of eukaryotic cells through analysis at the molecular level of the individual domains of such molecules. In the last year, methods have been developed to clone such genes, to insert them into expressing vectors, to reintroduce them into mammalian cells and to obtain expression of the authentic protein on the cell surface. In principle, these techniques can be applied to any gene coding for a cell-surface protein isolated from normal or malignant cells. The work in this proposal deals with one particular membrane protein - a virally-coded hemagglutinin.
The specific aims are: 1. to introduce mutations within the hydrophobic domains, the antigenic regions and the glycosylation sites of influenza virus hemagglutinin. 2. to construct chimeric genes in which the hydrophobic regions of influenza hemagglutinin are exchanged for those of other membrane proteins of both eukaryotic and bacterial origin. 3. to isolate large quantities of mutant and chimeric proteins and their wild-type homologs. The various biological functions of these proteins will be determined and correlated with their structures, which will be analyzed in detail if necessary by X-ray crystallography. 4. to construct mammalian vectors containing the signal and anchor hydrophobic regions of influenza hemagglutinin. These vectors will be used to express on the cell surface eukaryotic proteins that are normally found only in the interior of the cell. By these and other experiments, it is hoped to find out: how such proteins interact with the membranes of the cell; how such molecules become glycosylated and what function such glycosylation may serve; how membrane molecules are directed to particular surfaces of the cell and once there, how they carry out specific biological functions and serve as antigens and immunogens.
