The mechanisms for the interactions of a family of guanine nucleotide binding proteins (G proteins) which act as intermediaries between hormone receptors on the cell surface and their intracellular effector systems will be investigated. There are at least four distinct members of this family, most with multiple isoforms, that serve to coordinate the activities of at least six different intracellular regulatory pathways. These proteins mediate important hormone or hormone-like effects in essentially all mammalian cells. They all have a similar subunit composition and share one common subunit. This application proposes to investigate how this common subunit reversibly associates with the other subunits of the proteins and provides a mechanism by which they affect each others function, thereby coordinating intracellular regulatory pathways. There are five components of this project. (Specific Aim 1) The study of subunit interactions in intact membranes from s49 mouse lymphoma cells grown in culture. These studies will indicate the role of subunit dissociation in the behavior of proteins which have not been solubilized from membranes with detergents and subsequently purified away from other proteins which may modulate their function. (Specific Aim 2) The study of subunit dissociation of G proteins purified from bovine brain or human erythrocytes, or analogous recombinant proteins, in detergent solution; which will determine what regulates the interactions of the different proteins with their shared subunit. (Specific Aim 3) The study of the functional consequences of these reversible subunit interactions of purified G proteins in detergent solution, determined by examining the kinetics of GTP binding and hydrolysis to the purified proteins. (Specific Aim 4) The study of how these reversible subunit interactions affect a specific intracellular regulatory system in intact membranes from s49 cells, the CAMP-adenylyl cyclase system. (Specific Aim 5) The identification of the subunit binding sites on the proteins, determined primarily by isolating chemically-modified fragments of the proteins responsible for blocking subunit interactions. These studies will provide important structural information about these proteins and how their subunits interact.
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