The long term goals of this laboratory are to understand the signaling systems regulating hepatic function. This project period focuses on the heterotrimeric G proteins which couple receptors to effectors. New insights into the complexity of this system show that; (a) there is a significant diversity among the proteins that comprise the alpha and beta gamma subunits of the heterotrimeric G proteins; (b) the betagamma subunits are able to directly activate important effectors such as PLC-beta, adenyl cyclase or ion channels; (c) there is significant diversity among the combination of betagamma subunits expressed in cells. The experiments proposed will explore the hypothesis that the composition of the betagamma subunit plays a role in determining the selectivity of the cellular signalling mechanisms according to the following Specific Aims. (1) To understand the interaction of the betagamma subunits with the alpha subunit and receptors: Using recombinant G protein coupled receptors expressed in the membranes of Sf9 cells reconstituted with various pure, recombinant G protein alpha and betagamma subunits we will examine which of the many possible combinations of beta gamma subunits provides productive interaction between alpha subunits and receptors. We will assay high affinity ligand binding and the rate of receptor activated GDP/GTP exchange ont eh alpha subunit, both early steps in the interaction of receptors and alpha subunits that are critically dependent on betagamma. (2) To understand the role of the lipid modification on the gamma subunit in the interactions between receptors an the alphabetagamma complex. We will explore the function of recombinant gamma subunits with farnesyl or geranylgeranyl modifications on their C-terminus in the assays of receptor coupling developed in Aim 1. (3) To understand athe interaction of the betagamma subunits with PLC-beta; Using pure, recombinant G protein betagamma subunits and PLC-beta reconstituted into lipid vesicles, we will examine the activation of PLC-beta by the betagamma dimers. We will examine the ability of a panel of bet gamma subunits to activate PLC-beta and will also examine the role of the gamma subunit's prenyl group in the activation of the phospholipase. To identify the domains responsible for the interaction between betagamma and PLC-beta, we will test the activity of peptides known to mimic or block beta gamma function in other systems int eh PLC assay. Finally, we will use chemical crosslinking followed by protein sequencing to directly identify the domains of betagamma that interact with PLC-beta.
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