Phage display based cloning of proteins interacting with the cytoplasmic tail of membrane immunoglobulins.

摘要

The reduced quantity and quality of serum immunoglobulins (sIgs) in mutant mice expressing truncated cytoplasmic tails of IgE and IgG1 indicate an active role for the cytoplasmic domains of mIgG1 and mIgE. We used phage display technology to identify candidate proteins able to interact with the cytoplasmic tail of mIgE. Using a murine cDNA B cell library displayed on the surface of phage as prey and the 28 amino acid long cytoplasmic tail of IgE as bait, we isolated phage encoding the murine hematopoietic progenitor kinase 1 (HPK1). Surface plasmon resonance analysis measurements confirmed affinity of HPK1 to the mIgE cytoplasmic tail and revealed association to other immunoglobulin isotypes as well. Immunoprecipitation experiments, using lysates from two B cell lines expressing nitrophenyl (NP) specific mIgE molecules showed co-precipitation of IgE and HPK1. The interaction of HPK1 with the cytoplasmic domains of membrane immunoglobulins indicate an active role of the tails as part of an isotype specific signal transduction, independent from the Igalpha/Igbeta heterodimers, and may represent a missing link to upstream regulatory elements of HPK1 activation.