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 receptor endocytosis and signal transduction and to Tsg101, a component of Endosomal Sorting Complex Required for Transport (ESCRT)-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 (RTKs) (e.b., epidermal growth factor receptor, hepatocyte growth factor receptor, etc.) and non-receptor tyrosine kinase, such as focal adhesion kinase (FAK), as well as substrates of tyrosine kinases, via preferential binding to the phosphopeptide motif pYXNX (where N is asparagines and X any residue) 
. 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. The sequence analysis of the isolated preys reported here revealed a fragment that contains the C-terminal half of Grb2 consisting of the entire C-SH3 domain and a short fragment of the SH2 domain, and the C-terminal end of GrpL containing the entire C-SH3 domain and a short peptide of the linker between the SH2 and C-SH3 domains. These data suggest that the C-terminal SH3 domains of both GrpL and Grb2 are sufficient for their interaction with HD-PTP in yeast two-hybrid assay. Moreover, this interaction seems to not require the phosphorylation by v-Src, since the colonies selected in conditions of v-Src expression (SD-Trp,-Leu,-His,-Met) were able to grow also under conditions in which v-Src expression was repressed (i.e., on SD-Trp,-Leu,-His). The interactions between Grb2 or GrpL and HD-PTP were verified in mammalian cells using coimmunoprecipitation, in vitro
pull-down and immunofluorescence assays. We found that Grb2 and GrpL were able to bind in vivo
and in vitro
to the full length HD-PTP, and that Grb2 and GrpL partially colocalized with HD-PTP to cytosolic vesicles. While this manuscript was in preparation Harkiolaki et al., 2009 
reported that HD-PTP could be pulled down with the C-terminal SH3 domain of GrpL. This result supports our findings. Since HD-PTP is a substrate for Src kinase 
and Grb2 and GrpL contain SH2 domains that bind peptide motifs containing phosphorylated tyrosines, it is conceivable that phosphorylated HD-PTP might interact also with the SH2 domains of these adapters. Further experiments are required to address this hypothesis and to determine whether HD-PTP could also bind to the N-terminal SH3 domain of the adapters.
Here we also show that the Histidine Domain (amino acid residues 705–1128) is indispensable for the interaction with Grb2 and GrpL in vitro
. Thus, by GST pull-down and Far-Western experiments we found that GST-Grb2 and GST-GrpL were able to bind to the Histidine Domain alone, but not to a construct lacking this domain. At the same time the results show that the C-terminal proline-rich domain is not involved in binding the Grb2 family adapters. The deletion of this domain (in EGFP-ΔPEST) did not prevent the interaction seen with the full length protein and its presence in the context of a protein lacking the HD (i.e., the ΔHD construct) did not result in an interaction with the adapters. Our results also show that three other domains of HD-PTP, namely the N-terminal Bro1 domain, the V-domain and the PTPc domain do not interact with Grb2 or GrpL under the conditions used in this study. However, it is plausible that any of these domains, which contain putative tyrosine phosphorylation sites, might interact with the SH2 domains of Grb2 adapters when phosphorylated. In summary, our data demonstrate that HD-PTP interacts with Grb2 and GrpL through the SH3 domain-binding motifs present in the Histidine Domain. The immunofluorescence data support this conclusion showing that when coexpressed with a mutant that contains only the Histidine Domain and the V-domain, GrpL and Grb2 relocate to cytoplasmic vesicles where they colocalize with the HD-PTP mutant. In contrast, when coexpressed with deletion mutants lacking the Histidine Domain, the two Grb2 adapters have a dispersed cytosolic and nuclear localization. The overexpressed Histidine Domain appeared diffusely located throughout the cytosol and the nucleus, similar with the two adapters. These results are supported by Harkiolaki et al., 2009 
where the interaction motif of HD-PTP with the C-SH3 domain of GrpL has been mapped to amino acids 717–725 (PPRPTAPKP) within the putative Histidine Domain. The Histidine Domain of HD-PTP has a unique sequence that contains 16 His and 2 Cys residues linked by proline-rich elements of 3 to 51 amino acid residues each. According to ELM prediction the Histidine Domain has 35 putative SH3 domain-binding motifs 
. It remains to investigate which of these motifs bind to Grb2. Within this domain there are binding motifs for Tsg101 and endophilin A1, two proteins involved in endocytosis 
. The putative Tsg101 binding motif, 720
(motif also found in Alix, another member of the Bro1 domain-containing proteins that has a domain structure similar to the N-terminal half of HD-PTP), overlaps with the GrpL binding motif. This suggests that the binding of Tsg101 and GrpL to HD-PTP are mutually exclusive.
Grb2 is a ubiquitously expressed adapter that acts as a critical downstream intermediary in several signaling pathways, including growth factor receptors pathways 
. In receptor tyrosine kinase (RTK)/Ras pathway Grb2 adapter mediates the activation of Ras by bringing the guanine nucleotide exchange factor Sos1 to the plasma membrane adjacent to the membrane-associated Ras. There activated Ras alters the activity of several effectors, such as Raf kinase, phosphatidylinositol-3 kinase, and Ral GTPase 
. Following the rapid and transient signaling initiated at the plasma membrane, RTKs are internalized and targeted to endosomal compartments where they can continue signaling 
. Grb2 and Ras accompany these receptors to the endosomes 
. At the endosomal membrane the activated RTKs are sorted and internalized in intralumenal vesicles, processes that end their signaling activity and generate multivesicular bodies. The internalized receptors are transported to the lysosomes for degradation. Cytosolic complexes such as ESCRTs and their associated proteins are involved in these highly dynamic and regulated processes. Grb2 seems to be involved in these processes as well, since mutations in either of its SH3 domains impede the epidermal growth factor receptor trafficking from the early to the late endosomes and the formation of the multivesicular bodies 
. Interestingly, a similar phenotype was observed when HD-PTP levels were knockdown in cell culture 
. The mechanisms by which Grb2 regulates endocytosis of RTKs is not fully understood, and we can hypothesize that HD-PTP and Grb2 work together, probably with other proteins as well, to assemble and/or coordinate the assembly of endosome-associated protein complexes essential for vesicle biogenesis and protein sorting.
GrpL, also known as Gads, Grap2, Mona and Grf40, is expressed only in hematopoietic tissues, including bone marrow, lymph node, and spleen 
. Both Grb2-family adapters found to bind to HD-PTP are important regulators in lymphocytes signaling and development 
. T-cell receptor (TCR) engagement with anti-CD3 antibodies or peptide MHC complexes induces a cascade of Tyr phosphorylations, which leads to the fast recruitment and subsequent activation of downstream effectors of the TCR/CD3 activated complex. Adapter proteins such as LAT become phosphorylated on multiple Tyr residues. Phosphorylation of LAT creates binding sites for SH2 domains of other proteins, including phospholipase C γ1 (PLC-γ1), Grb2, GrpL and Grap 
. Thus, SLP76, which is constitutively bound to GrpL is brought to the TCR signaling complex at the plasma membrane 
. In addition, Grb2 recruits Sos1 and E3 ubiquitin ligase c-Cbl, which are bound to its SH3 domains 
. These interactions are crucial for the regulation of calcium signaling in T cells 
and for coupling the TCR to Ras through a pathway involving PLC-γ1, Tec family kinases, and RasGRP 
. c-Cbl mediates the ubiquitination of TCRζ chain leading to TCR internalization into endosomal compartments and subsequent degradation of the receptor in activated T cells 
. c-Cbl also mediates the segregation of LAT/GrpL/SLP-76- containing microclusters from activated TCR/CD3 complexes and further induces their endocytosis 
. It is conceivable that these endocytozed microclusters contain other adapters and enzymes associated with activated LAT. Our results suggest that HD-PTP may be one of the adapters associated with LAT upon TCR activation and that it may modulate the endocytic trafficking of LAT/SLP-76 microclusters, thus downregulating the signaling output of the TCR. Further experiments are required to elucidate the molecular mechanisms controlled by HD-PTP in lymphocytes.
In conclusion, we have identified Grb2 and GrpL as binding partners of HD-PTP. These interactions with adapters, which are essential for numerous signaling pathways, suggest that HD-PTP might have a role in the regulation of downstream events of a plethora of receptors.