The B chain of PDGF is closely related to the protein encoded by the oncogene v-sis, isolated from simian sarcoma virus. Experiments are proposed to examine the structural and functional properties of PDGF-related proteins. In addition, experiments will be conducted to further investigate the interaction between the v- sis protein and the PDGF receptor which results in autocrine stimulation and transformation. This proposal thus represents a comprehensive approach to the study of PDGF and related proteins. It is anticipated that these experiments will provide new insights into the structure and function of an important growth factor which is involved not only in normal physiology but also in oncogenesis. The following areas of research are proposed: 1) Structure- function of the v-sis protein: The disulfide bonds of the v-sis B:B homodimer will be characterized by peptide mapping, using mutant proteins lacking each of the cysteine residues. The reported interaction between PDGF and alpha-macroglobulin will also be characterized. 2) Interactions of v-sis protein with the A chain and with the PDGF receptor. The biological activity and physical structure of the A:A and B:B homodimers will be compared. An N-linked oligosaccharide site in the A chain will be removed and a corresponding N-linked oligosaccharide site will be introduced into the B chain. The effects of these mutations will be determined. Linker insertion mutants will also be constructed to identify regions of the v-sis protein required for receptor recognition and binding. 3) In vivo folding of v-sis protein: Experimental conditions will be investigated for the in vitro folding of v-sis protein into a biologically active protein. The effects of protein disulfide isomerase and also proline cis/trans isomerization for in vitro folding will be determined. 4) Nuclear targeting of growth factors: The nuclear form of the v-sis protein which we have identified will be further characterized as to whether it mediates any known biological effects of PDGF. Other growth factors will be examined for the existence of a nuclear targeting sequence, including the A chain of PDGF, NGF, EGF and insulin. 5) Studies of a membrane-anchored v-sis protein: The autocrine interaction between v-sis protein and the PDGF receptor will be characterized using a cell line in which synthesis of v- sis protein is expressed under control of the heat-inducible hsp70 promoter. Various cell lines will be constructed with the v-sis protein fused to the transmembrane anchor domain of the VSV glycoprotein and expressed from the heat-inducible hsp70 promoter. 6) Vesicle sorting of v-sis will then be examined so as to identify the vesicle sorting signal within the protein. The sorting signal of v-sis will also be studied in gene fusions to see if it can confer regulated secretion upon a protein which normally follows the constitutive pathway. 7) Chemotactic signals within the v-sis protein: The v-sis protein will be examined for chemotactic activity for cells such as fibroblasts, smooth muscle cells and monocytes. Truncated forms of the v-sis protein as well as synthetic peptides will be studied to identify the chemotactic signal.
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