Protein tyrosine phosphorylation by protein tyrosine kinases (PTKases) plays a prominent role in the regulation of cell proliferation. For example, many receptor-linked PTKases upon binding of their ligands stimulate cell proliferation. The positive signals for cell proliferation transmitted by PTKases must, however, be counterbalanced by the action of protein tyrosine phosphatases (PTPases). Furthermore, given that hyperexpression or hyperactivity of PTKases causes tumors, then it can be expected that the inactivation of PTPases may also result in uncontrolled cell growth. As is the case with PTKases, there are both soluble cytoplasmic PTPases and receptor-linked PTPases. Whereas the biochemical study of the soluble PTPases is actively pursued by several groups, very little is known about the membrane-associated forms of PTPases. Recently we isolated several human and Drosophila cDNA clones (LCA, LAR, HPTPalpha, DLAR, and DPTP) that encode novel receptor-linked PTPases. Their structures suggest that the activities of the cytoplasmic phosphatase domains might be regulated by the binding of ligands to their extracellular receptor domains. The objective of this proposal is to fill the gap in our knowledge by studying the structure-function relationship of receptor-linked PTPases, mainly using the human LAR PTPase as a model system. In order to study the structural basis of the function and regulation of human receptor-linked PTPases, both cytoplasmic PTPase domains and extracellular receptor domains of LAR will be biochemically characterized. Functional topography and potential regulatory mechanisms of the cytoplasmic PTPase domain will be studied by isolating and biochemically characterizing various mutations, and by generating anti-PTPase monoclonal antibodies and mapping their epitopes. A method to detect the binding of the extracellular receptor domain to its ligand will be developed and used to identify and biochemically characterize the ligand. In order to examine the diversity of receptor-linked PTPases, cDNA clones encoding additional receptor-linked PTPases will be identified by hybridization with consensus oligonucleotides, by cross-hybridization to previously isolated PTPase cDNAs, and by polymerase chain reaction method. The genomic DNA clones for two human PTPases, LAR and HPTPalpha, will also be isolated and characterized. Finally, in order to understand the developmental and physiological role of receptor-linked PTPases, the mutations in the Drosophila PTPase gene DLAR will be identified and studied.
