The elevated phosphorylation of key regulatory tyrosines on oncogenic signaling proteins that result from aberrant protein tyrosine kinases (PTKs) activity plays well-established roles in promoting tumorigenesis and in the high frequency with which resistance arises to existing therapeutic treatment. Protein tyrosine phosphatases (PTPs), by counterbalancing the activity of PTKs, play crucial tumor suppressive functions, and are thus considered as potential therapeutic targets. However, this therapeutic opportunity has yet to be fully met due to challenges in specifically targeting PTP catalytic domains, suggesting that alternative approaches to PTP targeting should be explored. Interestingly, both the PTK and PTP families include single-span transmembrane receptor-like enzymes, namely the receptor tyrosine kinases (RTKs) and the receptor-like PTPs (RPTPs), and these are some of the most important signaling regulatory proteins in oncogenic signaling. While RTK regulation has been studied in great detail, the mechanisms controlling RPTP activity and how they interact with their substrates have yet to be fully elucidated, although ther is some understanding of the roles that dimerization of RPTPs with themselves and with RTKs play. The investigation of these RPTP regulatory aspects beckons immediate attention because their elucidation could lead to useful new therapeutic opportunities, which will be explored in thi project. Thus, we propose to determine how dimerization regulates the activity of a RPTP (i.e., PTPRJ/DEP-1) that is critical for cell regulatory functions, how their interaction with the epidermal growth factor receptor (EGFR) affects EGFR tyrosine phosphorylation and downstream signaling in two different epithelial cell lines, and how these interactions can be leveraged for therapeutic purposes by designing novel peptide binders to target specific RPTP domains that will promote their activity. We hypothesize that: (1) PTPRJ form homodimers through its TM and/or JM domains, (2) PTPRJ associates with EGFR through specific TM and/or JM interactions, and (3) these interactions enable PTPRJ to regulate EGFR tyrosine phosphorylation and downstream signaling. A series of experiments has been outlined to address the integrated and diverse techniques and approaches needed to validate these hypotheses, culminating in the development of high affinity peptide binders of PTPRJ domains to promote its activity. Aim 1 will focus on identifying specific juxta- and trans-membrane domain interactions that participate in PTPRJ homodimerization and hetero-dimerization with EGFR, and on identifying dimerization-deficient PTPRJ point mutants. Aim 2 will demonstrate the effects of the PTPRJ point mutants on EGFR tyrosine phosphorylation and downstream signaling in epithelial cells. Aim 3 will lead to the design and testing of high affinity peptide binders in their ability to interfere with PTPRJ homo-dimerization and to attenuate EGFR-driven signaling in cancer. This project will thus result in important insights into cancer cell signaling and in new possibilities for targeting RPTPs for therapeutic modulation of EGFR in oncology.