The specific objectives are 1) to understand the molecular organization of membrane components involved in surface receptor patching and capping; and 2) to study the biogenesis of membrane proteins in normal and viral-transformed lymphoid cells. In the first part of the project, novel radioactive iodine and tritium labeling procedures will be used to analyze the biochemical composition of receptor patch and cap structures in order to further our understanding of """"""""lateral membrane protein interactions"""""""". Using the complementary methods of immunocytochemistry, enzyme-cytochemistry and autoradiography techniques, we will be able to analyze the relationships between receptor rearrangements and certain physiological responses monitored by membrane-associated enzymatic activities and hormone-receptor interactions. Furthermore, in order to test the """"""""transmembrane interaction"""""""" hypothesis, which suggests that certain regulatory molecule(s), (x protein(s)) may play an important role in the association of receptr patching/capping structures with contractile proteins, a new membrane fractionation procedure based on """"""""density perturbation theray"""""""" will be used to isolate receptor """"""""cap"""""""" structures. Analysis of the cap structure by a combination of chemical cross-linking and myosin affinity technique will allow us to gain a better understanding of the molecular linkages between certain membrane proteins (possibly, x protein(s)) and actin/myosin associated contractile proteins. In addition, we are going to determine the intracellular localization of the important calcium-binding protein, calmodullin, in patch/capped lymphoid cells and particularly its relationships with transmembrane processes and other contractile proteins using the double immuno-labeling (immunoperoxidase and immunoferritin) technique on frozen-thin sections. In the second part of this project, we propose to employ immunogenetics, immunobiochemistry and immunocytochemistry for the study of the biosynthesis pathways, turnover patterns and control mechanisms involved in the transport and assembly of three major membrane proteins such as Thy-1, HL-A (H-2) and viral-coded glycoprotein gp 69/71 into plasma membranes. In particular, the knowledge of the mechanisms involved in the incorporation of viral membrane glycoproteins into host plasma membranes may have significance for gaining a general understanding of viral-mediated cell transformation and a possible explanation for the behavior of malignant cells.
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