G-protein coupled receptors in thymocyte migration and development

T cells, like all hematopoietic cells, must be continually replaced throughout life. Common lymphoid progenitors migrate from the bone marrow compartment to the thymus where they undergo a coordinated process of development marked by distinct patterns of localization within the organ. The directional migration, therefore, of both progenitors and thymocytes is essential to T cell differentiation. Their migration is thought to be mediated through G-protein coupled receptors (GPCRs) and integrins, however the receptors and signaling pathways remain unclear.; My research focuses on developing and applying novel genetic technologies that inhibit GPCR signaling pathways. I have developed a novel genetic system, Degrakine, which specifically and stably inactivates chemokine receptors (CKR) by localizing to the ER, trapping its target CKR, and attracting the host proteosome. I have established that SDF-1, MDC, and RANTES based Degrakines specifically inactivate their respective receptor functions and that CXCR4 is required for homing of hematopoietic progenitor cells to the bone marrow immediately following transplantation. The Degrakine , therefore, provides an effective tool to dissect receptor functions in a number of biological systems.; GPCR-mediated G-protein (Galpha) signaling pathways play important roles in T cell development and activation. However the Galpha12 family, which consists of Galpha12 and Galpha13, has not been well studied in either process. To investigate whether Galpha12/Galpha13 signaling is involved in thymopoiesis, we constructed a retrovirus expressing only the RGS and PH domains of p115RhoGEF. The p115RGS acts as a dominant negative mutant, binding activated Galpha12/Galpha13 to turn off their signaling cascade through RhoA. We reconstituted fetal thymic lobes with progenitor cells expressing either vector or p115RGS and showed that inhibition of Galpha12/13 signaling resulted in a developmental block by the DN3 pre-thymocyte stage. The expression of mini-gene constructs, which specifically inhibit either Galpha12 or Galpha13, demonstrated that Galpha13 signaling, but not Galpha12, is required for early thymopoiesis. Bcl2 expression rescued the defect in DN3, but only partially rescued T cell development. The discovery that Galpha13 is a critical mediator of a novel signaling cascade necessary for pre-thymocyte survival not only increases our understanding of T cell development, but also opens novel avenues for modulating thymopoiesis.