Heterotrimeric guanine nucleotide binding (G) proteins composed of individual alpha, beta, and gamma subunits play pivotal roles in linking receptors to effector proteins at the plasma membrane and are involved in the physiology of an increasing variety of cellular processes. These proteins play an equally important role in pathways that control cell differentiation, growth, and oncogenesis. Consistent with its central role in signaling cascades required for differentiation, Gai-2 is a protooncogene in renal cells. Temporal repression of Gai-2 expression in vivo is associated with renal morphologic abnormalities in transgenic mice. The applicant has utilized the porcine LLC-PK1 kidney cell as a model to study G protein coupled processes during their programmed differentiation in culture. Polar LLC-PK1 cells have geographically segregated Gai-2 and Gai-3 proteins that allow ordered signaling for second messenger generation, ion channel regulation and protein transport. In mitotically active pre-differentiated LLC-PKI cells, Gi protein function in growth and differentiation cascades link to p42/44 and/or JNK/SAPK mitogen-activated protein kinases. The applicant proposes to continue studies on the mechanism of G protein ai subunit gene expression and their interaction with effectors in LLC-PK1 cells. The rationale for such studies is based on findings that polar LLC-PK1 cells: 1) undergo a highly regulated genetic program for ai-2 and ai-3 subunit expression during culture; and 2) these subunits are differentially expressed and targeted to differing membrane sites for interaction with and modulation of their respective effectors. For targeting and effector coupling studies, the applicant has established methods to express or produce chimeric ai subunits, localize them, monitor their effector interactions, and clone novel effectors. For gene expression studies, the applicant has characterized the porcine ai-2 and ai-3 gene promoters and several transactivating factors. By utilizing these resources, the applicant has demonstrated a molecular switching paradigm for regulation of the ai-2 gene, in polarized cells by activating a """"""""CCAAT"""""""" box family of DNA binding proteins and in differentiating LLC-PK1 cells by the coordinated activating and repressing activities of homologous zinc finger transcription factors Egr-1 and WT1 (Wilms' tumor suppressor). The information gained from these studies should provide fundamental knowledge concerning the mechanisms of Gi protein expression and effector coupling which influence kidney cellular physiology in health and disease.
