The objective of the proposed research is to utilize a genetic in combination with a biochemical, a cell biological, and a cell-engineering approach to examine the mechanisms involved in the regulation of plasma membrane biogenesis, primarily in normal but also in malignant cells. The system that is being studied consists of two membrane glycoproteins, denoted D and K, whose structures are specified by the major histocompatibility locus of the mouse, the histocompatibility-2 region on chromosome 17. Lactoperoxidase-catalyzed iodination and sodium borotritide reduction of cells treated in situ with glactose oxidase are used to label the D and K proteins accessible on the plasma membrane of macrophages, lymphocytes, and hepatocytes of different inbred mouse strain. Metabolic labeling with fucose, mannose or methionine are used to label the total cellular complement of these proteins in the differnt strains. Cell fractionation methods, in combination with the differential labeling techniques, are used to localize and quantitate the D and K glycoproteins among the different membrane systems of the mouse cells. The D and K glycoproteins are being isolated from these membrane systems, using specific allo and monoclonal antibodies; they are analyzed and quantitated by two-dimensional polyacrylamide gel electrophoretic techniques. Using these types of analyses, we have shown differential expression of these two H-2-encoded polypeptides in (1) different tissues of the same mouse strain and (2) in the same tissue of different mouse strains. The genetic and biochemical basis for these differences will be examined. Attempts will also be made to differentially modulate the D and K polypeptides in order to trace the intracellular localization and route of biogenesis of the perturbed glycoprotein. As part of these studies, attempts are being made to alter the expression of the D and K polypeptides and to change the histocompatibility-2 phenotype of mouse cells by (1) transferring specificities from one cell to another, using membrane reconstitution and fusion methods and using gene transfer techniques. The expectation is that the above approaches will yield important insight into the genetic control of expression of these membrane glycoproteins, their mode of biogenesis and turnover, and their metabolic function within the cell.
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