Mast Cell Activation And The Allergic Response: Quantifying Proteins Involved In The FcєRI Signaling Pathway

Mast cells play an important role in the body’s immune response to pathogens, but can also trigger allergic reactions that are responsible for diseases such as allergic rhinitis and asthma. Understanding the pathways involved in mast cell activation could suggest ways to treat these diseases.  In mast cells, allergens interact with multiple immunoglobulin E (IgE) molecules each bound to a high affinity IgE receptor (FcεRI) causing receptor clustering. This clustering initiates a signaling cascade that leads to de-granulation (release of pro-inflammatory factors) and initiation of the allergic response. The concentration of available signaling proteins may influence its response to FcεRI activation.  Determining the absolute quantities of downstream molecules in this pathway will provide information for the development of computational models. We are using quantitative western blot analysis with known quantities of recombinant protein as standards, to estimate the number of protein molecules per cell in rat basophilic leukemia (RBL) cells.  Using this method, we have quantified two downstream signaling proteins in the FcεR1 pathway, Grb2 and LAT.  The amount of Grb2 was estimated to be 128,000 molecules/cell +/- 21,000 and the amount of LAT was estimated to be 370,000 molecules/cell +/- 36,000.  We are currently quantifying cytoplasmic proteins that regulate FcεRI activity focusing on Lyn and Fyn, kinases responsible for phosphorylating FcεRI, and Csk, a negative regulator of these kinases.  Estimates of protein numbers will be incorporated into mathematical models to identify rate-limiting interactions in the pathway, which could potentially aid in the development of novel treatments for allergic diseases.