Reversible G protein subunit interactions will be studied to determine the properties of these proteins important for mediating hormone action. G proteins are heterotrimeric GTP binding proteins composed of alpha, beta and gamma subunits. They mediate signalling across the plasma membranes of cells for many hormones and neurotransmitters, as well as for numerous other regulatory substances. External signals activate G proteins inside cells by stimulating GTP-dependent dissociation of their alpha subunit from their associated betagamma dimer. These parts of the G protein then independently control signalling pathways inside cells so that the cells respond appropriately to signals in their environment. Errors in such signalling systems are likely contributors to disease processes such as diabetes, essential hypertension and cancer. Based upon the fact that some hormones require very specific G protein isoform combinations for their effects, it is hypothesized that G protein subunit dissociation and reassociation during hormone signalling provides a key mechanism for coordinating cell responses. For example, if G protein subunits form transient or dynamic heterotrimeric complexes, hormones will only be able to stimulate cells when G proteins containing the correct subunits are present. To test this hypothesis the regions of betagamma dimers that bind G protein alpha subunits will be identified and this information will be used to develop sensitive and general assays for G protein activation by receptors in intact membranes and cells.
The Specific Aims of this application are: 1). To determine the structure and diversity of naturally occurring gamma subunits in G protein heterotrimers. (2). To determine the post-translational modifications of the beta subunits of the G proteins. 3). To use the information from 1) and 2) to define regions in G protein subunits involved in the binding of betagamma dimers to alpha subunits. 4). To determine if G protein heterotrimers are formed of specific subunit combinations in cells and whether reversible subunit dissociation produces heterotrimers of changing subunit composition. 5). To determine the occurrence, regulation and function of G protein subunit dissociation in intact membranes using fluorescently-labeled peptides based upon the work in Specific Aims 1-3. These studies will test the hypothesis that changing G protein subunit composition alters hormonal responses of cells. Regardless of the validity of this hypothesis, these studies will define the properties of the G proteins important for the action of the hormones that regulate them.
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