Role of guanosine triphosphatase regulators in fibroblast transformation and lymphocyte development

Guanosine triphosphatases (GTPases) are signaling mediators involved in regulation of diverse cellular processes including regulation of the actin cytoskeleton, gene transcription, cell cycle regulation, apoptosis and transformation. Regulatory, proteins including G protein coupled receptors (GPCR), GTPase activating proteins (GAP) and guanine nucleotide exchange factors (GEF) influence the activity of GTPases. Balanced regulation of GTPase activity is critical in coordinating normal cellular responses. This thesis addresses the contributions of GTPase regulators in cellular growth control, differentiation and transformation. Over-expression of G2A or PAR-1, two GPCRs, in NIH 3T3 fibroblasts induced a full range of phenotypes characteristic of oncogenic transformation. Co-expression of dominant negative Rho or LscRGS (Lbc's second cousin regulator of G protein signaling domain), a negative regulator of Gan and Gai3 GTPases, suppressed transformation via these GPCRs. Activation of Gai2, Gai3 and Rho GTPases are thus required for transformation via these GPCRs. Gai2 and Gai3 are unique in that they are activated upstream of Rho. Moreover, Rho GTPase activity is regulated via GEFs. Gct-mediated activation of GEFs and downstream smaller molecular weight GTPases appears to be an important mechanism of linking divergent GTPases downstream of GPCR activation. To elucidate the role of Gai2 and Ga i3 GTPases in lymphocyte development, transgenic mice expressing LscRGS were generated. Analyses of lymphocytes from these mice revealed that LscRGS expression did not overtly affect lymphocyte development. These results indicate that Gai2 and Ga i3 are not required for lymphocyte development. Rho and Cdc42 are two GTPases involved in lymphocyte development. Previous studies by others demonstrated that loss of Rho function partially blocked differentiation and survival of CD47CD8double negative (DN) thymocytes and expression of another Rho family GTPase, Cdc42, enhanced the proliferative capacity of DN thymocytes. In addition, results from other studies revealed that expression of activated Rho augments positive selection and induces CD4 +/CD8and CD47CD8single positive (SP) thymocyte hypersensitivity to TCR-induced proliferation in vitro. Dbs is a Rhoand Cdc42-activating GEF normally expressed in thymus. To determine how Dbs influences lymphocyte development, transgenic mice were generated expressing an activated form of Dbs. Expression of activated Dbs in lymphocytes promoted the accumulation of early thymocytes and restricted the production of mature thymocytes. Activated Dbs expression also led to increased proliferation of DN thymocytes. The Dbs transgene caused reduced numbers of SP thymocytes and mature splenic T lymphocytes. In addition, transgenic CD4 + /CD8 + double positive (DP) thymocytes expressed higher levels of T cell receptor (TCR) and were hypersensitive to apoptosis induced by injection of anti-CD3. Moreover, Dbs transgenic thymocytes displayed impaired positive selection. Thymocyte culture experiments revealed that proliferation in response to anti-CD3 was reduced in SP thymocytes from Dbs transgenic mice. Expression of activated Dbs, a positive regulator of Rho and Cdc42, promoted the accumulation of DN thymocytes; this is the opposite of the DN phenotype observed in thymocytes lacking Rho function and similar to the phenotype displayed by Cdc42 transgenic mice. Thus the accumulation of DN thymocytes is likely to occur via Rho and/or Cdc42 activation. Activated Dbs expression also caused reduced in vitro SP thymocyte proliferation in response to TCR cross-linking and impaired thymocyte positive selection. These results are contrary to previous reports describing transgenic mice expressing activated Rho; thus, impaired thymocyte proliferation and positive selection in Dbs transgenic mice is likely to involve a pathway independent of Rho. Results presented in this thesis provide insights into the contributions of GTPase regulators in regulation of cellular growth control, differentiation and transformation.