Mutational analysis of interdomain binding interface of Grb7

Abstract

Grb7 is a member of the Grb7 adaptor protein family (Grb7, Grb10 and Grb14) and is a suspected promoter of tumor progression. The multi‐domain Grb7 protein is composed of the Proline‐Rich (PR), Ras‐associating (RA), Pleckstrin Homology (PH), and Src Homology 2 (SH2) domain[Office1] s. Grb7 is observed over‐expressed, along with the receptor tyrosine kinase ErbB2, in 20–30% of breast cancer cells, and is involved in increased cell migration and formation of metastases [1]. It is believed the RA, PH and SH2 domains interact with various downstream signaling partners to potentiate Grb7's role in cell migration [1]. Grb7 undergoes intermolecular homo‐dimerization through its SH2 domain [2] while also undergoing an intramolecular domain interaction via its SH2 and RA‐PH domains [3]. These interactions are believed to affect the Grb7 protein functional state. Based upon our previous results substantiating the Grb7 intramolecular domain interaction, we now seek to characterize the binding interface between the Grb7 RA‐PH, and Grb7 SH2 domains. Our approach is to use mutational analysis to target the RA‐PH and SH2 domain interfaces we hypothesize may be involved in the intramolecular interaction. At present we have successfully produced the SH2 domain mutants V45A, K46R, D47E and S48A by overexpression in a bacterial system and GST fusion‐protein based purification. We have also expressed and purified the Grb7 RA‐PH two‐domain construct by a method previously developed in our laboratory. With these protein domains in hand we will perform biophysical studies to probe the interaction between the Grb7 RA‐PH, and SH2 domains. Proposed experimental approaches for this study include isothermal titration calorimetry, surface plasmon resonance, nuclear magnetic resonance, circular dichroism, and dynamic light scattering. These studies will help us to better understand how Grb7 functions in tumor progression, and further our ultimate goal of designing Grb7‐based therapeutics for treating aggressive breast cancers.[Office1]You can't use acronyms in an abstract without first defining them. Your reader will get lost.Support or Funding InformationNIH5‐SC3‐GM116728This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.