G PROTEIN-DEPENDENT SIGNALING IN RENAL EPITHELIAL CELLS

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The long term goals of this research program is to understand the intracellular molecular mechanisms by which G protein-dependent signaling systems regulate renal epithelial cell function. G protein- coupled receptors (eg. for extracellular Ca, A11, and AVP) activate multiple parallel pathways involving distinct G proteins, kinases, and transcription factors to provide an integrated control network to regulate multiple cell functions. Despite the fact that the ligands for these receptors and their effects on second messengers and transport have been studied in considerable detail, the mechanisms by which information travels through the cell remain incompletely defined because: 1) many of the proteins are closely related in structure and function; 2) they are expressed at low levels; 3) chemica activators or inhibitors do not exist for many known proteins; and 4) many proteins probably remain to be identified. Signaling by the extracellular Ca receptor (Cao-R) will be examined using a model system that allows separation of the signaling process into components that can be analyzed with precision to test specific hypotheses. The components are: 1) the pattern of receptor - G protein coupling; 2) activation of intracellular kinases by individual G proteins; 3) activation of the Na/H exchanger promoter (NHE-1) by individual G proteins and kinases; and 4) identification of the NHE-1 promoter elements that respond to the G proteins and kinases. The hypotheses we will test are: 1) the Ca o -R activates of multiple G proteins (Gq~ G12' and a Gi): 2) the G proteins that couple to the Cao-R signal by different mechanisms, and activate different sets of MAP kinases; 3) the G proteins and kinase activate different elements of the NHE-1 promoter; and 4) the signals that regulate Na/H exchange also regulate other transporters in a coordinated manner. The studies will be performed in cells that allow simultaneous genetic and biochemical analyses, freshly isolated MTAL cells to verify the genetic and biochemical studies and cultured MTAL cells to test the effects of these signaling systems on regulation of transport. Establishing the mechanisms by which the Cao-R acts will improve our understanding of the genetic diseases linked to the Cao-R and the electrolyte abnormalities and interstitial nephritis associated with hypercalcemia.