The adapter protein CrkL associates with CD34

Abstract

CD34 is a cell-surface transmembrane protein expressed specifically at the stem/ progenitor stage of lymphohematopoietic development that appears to regulate adhesion. To elucidate intracellular signals modified by CD34, we designed and constructed glutathione-S-transferase (GST)-fusion proteins of the intracellular domain of full-length CD34 (GST-CD34i full ). Precipitation of cell lysates using GSTCD34i full identified proteins of molecular mass 39, 36, and 33 kd that constitutively associated with CD34 and a 45-kd protein that associated with CD34 after adhesion. By Western analysis, we identified the 39-kd protein as CrkL. In vivo, CrkL was coimmunoprecipitated with CD34 using CD34 antibodies, confirming the association between CrkL and CD34. CD34 peptide inhibition assays demonstrated that CrkL interacts at a membrane-proximal region of the CD34 tail. To identify the CrkL domain responsible for interaction with CD34, we generated GST-fusion constructs of adapter proteins including GSTCrkL3 (C-terminal SH3) and GST-CrkL5 (N-terminal SH2SH3). Of these fusion proteins, only GST-CrkL3 could precipitate endogenously expressed CD34, suggesting that CD34 binds the C-terminal SH3 domain of CrkL. Interestingly, there appears to be differential specificity be-

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