Dr. Clarks laboratory is interested in the mechanisms by which the B cell antigen receptor initiates specific signals and their biological consequences. Within the confines of this overall goal we have three active areas of research. Previous work by several laboratories, including our own, has demonstrated that the resting B cell antigen receptor is assembled with tyrosine kinases and that these kinases become activated following receptor ligation. Recently, using different approaches, we have elucidated two mechanisms by which receptor mediated tyrosine kinase activation is coupled to distal signaling pathways. First, using a chimeric system developed in our laboratory, we demonstrated that the non-ITAM tyrosines in the cytoplasmic tail are involved in coupling the BCR to PLC? activation. In a second project, we have recently demonstrated that another linker, GrpL is involved in coupling the BCR to global tryosine phosphorylation. In collaboration with Celeste Simon (U. Pennsylviana) we have been characterizing the signaling defects in mice deficient in the Ets transcription factors PU.1 and SpiB. These mice have diminished B cells in the periphery. When these B cells were analyzed it became apparent they had a very proximal defect in signaling. Cross-linking the BCR induced the phosphorylation of Iga/Ig and the recruitment of Syk. However, subsequent phosphorylation of cytosolic substrates was diminished. RNA subtractive hybridization demonstrated that the linker GrpL was not expressed. The GrpL promoter was shown to be a target of PU.1 and SpiB. Finally, transgenic reconstitution of PU.1-/+/SpiB-/- mice rescued global tyrosine phosphorylation. Thus, using both genetic and biochemical approaches we have begun to elucidate how the BCR couples to distal signaling pathways.

It has been thought that the BCR acted primarily as an efficient handle to deliver antigen to endosomal compartments where it could be processed. We have recently demonstrated that specific signals from the BCR regulate the physical and biochemical characteristics of the endosomal compartment in which antigen is processed. These observations are novel in that it has never been demonstrated before that signals from a receptor regulate the endocytic pathway. We demonstrated that both Iga and Ig??and the recruitment of Syk, were required for sorting through early endosomes to the antigen processing compartment. Most recently, we have shown that the BLNK recruitment to Iga is required for entry into late endosomes. These data provide a model in which the recruitment of specific signaling molecules facilitate receptor trafficking through at least two independent checkpoints.

We have developed a system in which retroviruses are used to reconstitute the bone marrow from Igb deficient animals. In our first experiments, we have been able to demonstrate that the cytoplasmic tail of Ig is required for B cell development. Subsequent experiments have indicated that substitution of the Iga for Igb only partially rescues B cell development. We are now using this system to elucidate the specific differences between Iga and Ig.