Expression, Purification Of The PTB Domain Of Human APPL2 And Screen For Its Peptide Ligands

APPL proteins, including APPL1 and APPL2, are important messengers between cell membrane and nucleus and indispensable to cell proliferation. APPL proteins consist of BAR domain (bin-amphysin-rvs), PH domain (pleckstrin homology) and PTB domain (phosphotyrosine binding). All these domains are able to interact with a series of different signal molecules, and play roles of signal transduction. Till now, two subunits, APPL1 and APPL2, have been discovered and confirmed to exist in eukaryotic only. In addition, APPL1 and APPL2 share 54% sequence homology. APPL1 was first discovered in the interaction with serine/threonine kinase, AKT2, in the yeast two hybrid screening.Along with the development of research on APPL proteins, more and more new theories and discoveries are reported all the time. For instance, APPL1 was formerly found to interact with the non-active form of AKT2. However, AKT in this form mainly exists in cytoplasm. Therefore, it is not as possible as before that AKT2 could locates in endosomes with APPL proteins. What is more, DCC (deleted in colorectal carcinoma) is another interacting partner of APPL1. With the absence of ligands, DCC could induce the cell apoptosis through activating caspase-3 and caspase-9, in which APPL1 is involved. With the help of APPL1, DCC could also activate p53, which is a unit of NuRD/MeCP1 complex. The activation of p53 could bring cells stopping growing or apoptosis, although this depends on activation of downstream transcriptional signals. Besides, another role of DCC is a messenger in signal pathways of neurocytes.APPL1 and APPL2 are substantiated to interact with Rab5 through the PTB domain. Recently, Nechamen et al reported that APPL2 could not only interact with FSHR, but also form a dimer with APPL1 through the BAR domain.As PTB domain could binds with phosphotyrosine in different proteins, it plays an important role in different signal pathways. Nonetheless, this binding could be phosphotyrosine independent as well. Similar to the construction of GLUT2 (glucose transporter 2), PTB domain of APPL proteins consist of two anti-parallelβsheets and threeαhelices. Although PTB domains of APPL1 and APPL2 share higher sequence homology, some proteins are confirmed to bind APPL1 only. This suggests that these two proteins play different roles possibly, even in an antagonistic manner. Therefore, identifying the interacting molecules is of great importance.In this study, we expressed and purified the PTB domain of human APPL2 (hAPPL2) in Escherichia coli. Phage display technology was then used to screen for the interacting peptides with this domain. After 3 rounds of panning, 48 single phage plaques were randomly picked and ELISA analysis was performed. Two 7-mer peptides, ERLPFFY and YLTSPKH were found to bind to the PTB domain of hAPPL2 in a specific manner. Both wild type and mutant forms of P3-GST fusion protein, with each amino acid substituted by alanine, were prepared and evaluated for binding capacity with hAPPL2 PTB by ELISA. Results showed that each residue was indispensible in the binding between this peptide to hAPPL2 PTB domain. This study shed light on structural interactions between the PTB domain of APPL2 and the binding proteins.