SUMMARY - Protein tyrosine phosphorylation and dephosphorylation, which are balanced by counteracting
protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), are essential for molecular
communications in signal transduction cascades. The receptor-like PTPs (RPTPs) are a family of cell surface
PTPs containing a usually large extracellular domain, a single-pass transmembrane domain, and either a single
or tandem cytoplasmic phosphatase domain. The functions of RPTPs are attributable to both catalytic activities
and extracellular interactions, resembling that of receptor tyrosine kinases (RTKs). However, our molecular
understanding of RPTP activity regulation is far from complete compared with that of the structurally and
functionally well-characterized RTKs. Structural studies focused on phosphatase domains failed to define
generalizable and conclusive mechanisms. We have been engaged in structural and functional analysis of an
important RPTP family member, namely CD148/PTPRJ, which is the most abundant RPTP in platelets and
megakaryocytes and has an established positive role in platelet aggregation, an essential process for hemostasis
and thrombosis. Our preliminary biochemical and structural studies of CD148 have yielded many intriguing
observations that begin to define the structural basis of CD148 activity regulation, supporting our hypothesis that
dimerization of RPTPs is largely attributable to extracellular and transmembrane domains that govern dimer
formation of the phosphatase domain and regulation of catalytic activity. Using innovative construct designs and
a novel human megakaryocyte progenitor cell line derived from induced pluripotent stem (iPS) cells, we will
examine how the CD148 activity is regulated by dimerization and ligand binding, and how these regulations
affect the function of human megakaryocytes and platelets. Using the recently developed proximity-dependent
labeling method, we will perform proteomic profiling of CD148 substrates in both resting and activated human
megakaryocytes and platelets. Using a combination of crystallographic, electron microscopy, nuclear magnetic
resonance, and other multifaceted biochemical and biophysical approaches, we will define the structural basis
of CD148 activity regulation through the size of ectodomain and the dimerization mediated by the extracellular
and transmembrane domains, and the structural basis of ligand binding. The effect of individual domains, N-
linked glycosylation, specific disease-associated polymorphisms, and ligand binding on the structure,
dimerization, and activity of CD148 will be examined. These complementary Specific Aims will advance our
understanding of the molecular basis for the regulation of CD148 PTP activity and will facilitate the development
of novel strategies for modulating CD148 function by selectively targeting either dimerization or ligand binding in
the treatment of diseases such as thrombosis and cancer. Approaches established in this study will be readily
applicable to other members of the RPTP family, which will improve our family-wide understanding of the
molecular mechanisms of RPTP activity regulation.
