The goal of this project has been to understand how the diversity of G protein alpha and betagamma subunits leads to specificity in transmembrane signaling. Our major effort has centered on the diversity of the betagamma subunits and has demonstrated that recombinant betagamma dimers of defined subunit composition have different activities in their ability to couple alpha subunits to receptors in the membrane or to directly activate effectors. These exciting results provide a potential explanation for signaling selectivity in the intact cell. During the next project period we plan to expand our study of the functional differences of recombinant betagamma dimers to better understand the specificity of cell signaling. The function of these betagamma dimers will be explored via three Specific Aims.
Aim 1 - To explore the functional role of betagamma dimers containing divergent beta subunits such as beta5. Our results with dimers composed of the beta5gamma2 subunits indicate selective coupling to the Gq alpha subunit and activation of a restricted set of effectors. We will examine the function of a panel of dimers containing the beta5 subunit combined with various gamma subunits for their ability to couple receptors to alpha subunits and to activate effectors. We will also examine the function of the variant (beta3S dimer, identified in hypertensive patients, which is missing one WD-40 repeat.
Aim 2 - To determine which domains of the gamma subunit interact with receptors and effectors. Using a series of chimeric gamma subunits containing switched and C-terminal domains from a pair of active and inactive gamma subunits, we will determine the role of these domains using assays of receptor interaction and effector function.
Aim 3 - To examine the possibility that the prenyl group of the gamma subunit participates in activating the betagamma dimer. Dimers containing gamma subunits modified with the C15 farnesyl group are usually less active than dimers containing gamma subunits modified with the C20 geranylgeranyl group. We will use the recent observation that the prenyl group interacts directly with the beta subunit to test the possibility that the two lipids cause different conformational changes in the beta subunit. The approach will be to make mutant beta subunits in which the prenyl group is not able to fold back into the beta subunit and test their activity in assays of receptor interaction and effector function.
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