The four members of the ErbB family of mammalian receptor tyrosine kinases are essential for the development and maintenance of a variety of tissues.  Aberrant activation or over-expression of these receptors leads to the progression of epithelial and brain tumors.  An understanding of the mechanistic and structural changes that occur to activate the intracellular receptor tyrosine kinase domain via extracellular ligand binding and receptor dimerization is desirable for identifying specific and efficient methods to inhibit signal transduction.  Previous attempts to study the mechanism of ErbB activation attained limited success due to low protein yields, poor water solubility of the membrane protein, and the large size of receptor.  Here, we report progress on overcoming these technical hurdles by forming the ErbB family members in nanolipoprotein particles (NLPs).  NLPs are ~20 nm disc-shaped cell membrane analogs.  NLPs containing the ErbB1 receptor, also known as EGFR, can be produced using an optimized cell-based method or a novel cell-free.  The cell-produced ErbB1-NLPs retain kinase activity and are assembled at higher yields compared to cell-free produced ErbB1-NLPs.  However, the cell-free produced receptors can be produced in about four hours and contain regions of correct folding.  In addition, the cell-free produced constructs can be labeled conveniently with unnatural amino acids to enable characterization of dimerization interactions.  These two receptor production systems can be utilized synergistically to enable new studies of conformational changes associated with ligand binding or protein mutations, as well as to identify small molecule inhibitors of these receptors.

Functional and structural characterization of EGFR-containing nanolipoprotein particles (NLP) utilizing cell-based and cell-free assembly methods

246th National Meeting (2013)

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