To identify which of the putative structural domains of WZ8040 HD-PTP interact with Grb2 and GrpL, in vitro pull-down assays were performed using affinity purified GST-Grb2 or GST-GrpL and lysates from cells transfected with EGFP-fusions of HD-PTP deletion mutants. As suggested by the presence of the proline-rich motifs, known to bind to SH3 domains, and by the interaction obtained in yeast two-hybrid of HD-PTP with the C-terminal SH3 domain of GrpL, we hypothesized that a region containing prolinerich motifs would be responsible for binding to the Grb2 family adapters. HD-PTP, a Bro1 domain-containing protein, essential for early embryo development, has very poorly understood functions. Being a large protein with 1636 amino acids and several putative structural domains it is likely that it can interact with numerous functional partners. However, only few partners have been identified so far. The center proline-rich Histidine Domain binds to endophilin A1, an SH3 protein involved in Trichostatin A 58880-19-6 receptor endocytosis and signal transduction and to Tsg101, a component of Endosomal Sorting Complex Required for Transport -I. The same Histidine Domain binds in a Ca2+ -dependent manner, to ALG-2, a protein important for apoptosis. The Bro1 domain interacts with CHMP4b, a component of ESCRT III. Furthermore, HD-PTP appears to interact in vivo with the focal adhesion kinase and to regulate both the kinase��s tyrosine phosphorylation state and its intracellular localization. These interactions indicate that HD-PTP might play roles in endosomal protein sorting and trafficking, apoptosis, and cell adhesion. In this study, we describe two novel interactions of HD-PTP. Using a yeast two-hybrid approach, we found that Grb2 and GrpL, two members of the Grb2 family adapters, are binding partners of HD-PTP. The Grb2 family consists of a central SH2 domain flanked by two SH3 domains. Through its SH2 domain, which is a conserved sequence of 90 amino acids, Grb2 can interact directly with receptor tyrosine kinases and non-receptor tyrosine kinase, such as focal adhesion kinase, as well as substrates of tyrosine kinases, via preferential binding to the phosphopeptide motif pYXNX. The C- and N-terminal SH3 domains, which have a conserved sequence of around 50 amino acids, bind proline-rich regions within the interacting proteins. In addition to these three domains, GrpL has a proline/ glutamine rich region of 135 amino acids between the SH2 domain and the C-terminal SH3 domain.