Platelet derived growth factor (PDGF)--the product of c-sis proto-oncogene (p28sis), initiates cellular replication and transformation by binding to a high affinity cell surface receptor. Recently I purified the human PDGF-receptor and demonstrated for the first time the presence of PDGF-stimulatable tyrosine kinase activity in the solubilized and purified receptor preparation. One of the objectives of this proposal is to purify the receptor to homogeneity and to test rigorously whether the tyrosine kinase is intrinsic to the receptor.
Other aims relate to studies on biosynthesis, processing, subcellular localization and endocytic fate of the receptor. My specific objectives are as follows:
Aim 1. Further purification of human and murine PDGF-receptor to homogeneity and structural/functional characterization of the purified receptor. The purified receptor will be tested for its ability to be convalently labeled by ATP analogues. The domain sub-structure of the receptor will be studied by limited proteolysis. The receptor fragment representing the putative tyrosine kinase domain will be identified by autophosphorylation assay and by affinity labeling with a radiolabeled ATP analogue. The fragment representing the growth factor binding domain will be identified by 125I-PDGF binding and by chemical cross-linking to 125I-PDGF.
Aim 2. Generation of polyclonal and monoclonal antibodies to different functional sites of the receptor. The antibodies will be classified according to their specificity for various sites, such as PDGF-binding site, tyrosine kinase site, etc. All the antibodies will be tested for PDGF-like mitogenic activity.
Aim 3. Use the antibodies to investigate biosynthesis, turnover, glycosylation and maturation of the PDGF-receptor.
Aim 4. Studies on endocytic fate and recycling behavior of the PDGF-receptor in response to PDGF. These studies will involve indirect immunofluorescent staining and immunogold electron microscopy of cells using appropriate anti-receptor antibodies. Recycling behaviour of the internalized receptor will be examined by both immunocythchemical techniques and by 125I-PDGF binding assay. In addition, I plan to study localization of the receptor in simian sarcoma virus (SSV)-transformed cells.
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