Heterotrimeric GTP binding proteins (G proteins) serve an essential role in transducing receptor-generated signals across the plasma membrane. One of the best characterized signal transduction pathways in the hormone-sensitive adenylyl cyclase system. The effector enzyme is under dual stimulatory and inhibitory control by two G proteins termed G and G. However, it is now known that each of these G proteins has roles in addition to regulation of adenylyl cyclase and, in the case of G there are three closely related gene products that can be expressed in a single type of cell. The function of each of the different forms of G is not known. An understanding of signal transduction mechanisms is fundamental to many aspect of medicine including hormone and drug action. Altered G protein activity has been observed in diseases such as heart failure, hypertension, and cancer. Knowledge of the location of specific G proteins may provide insight into the clinical relevance of such observation. Most information about the subcellular distribution of G proteins has come from relatively crude attempts at cellular fractionation, and there is little ultrastructural information on their important subject. G protein activity, although clearly present in the plasma membrane, has been reported to be associated with intracellular compartment, including the Golgiapparatus,mitochondria, endosomes, nucleus, and following activation, the cytoplasm. 1) The first specific aim is to utilize immunoelectron microscopy of ultrathin frozen sections to identify quantitatively the subcellular structures with which G protein subunits are associated in cells. Attempts will be made to identify specialized structural sites on the plasma membrane and intracellular membranes that are populated by G and each form of G. The hypothesis to be tested is hat differential distribution of individual G protein oligomers contributes to the specificity of their participation in signal transduction pathways and that knowledge of G protein localization will shed light on their functional roles. 2) The second specific aim involves examination of the role of defined structural features of alpha subunits in membrane localization. The distribution of endogenous G proteins will be examined using quantitative morphological and biochemical techniques on whole cells and plasma membranes exposed to agents known to alter G protein function. Based in part on the results of these experiments, mutant G protein alpha subunits in the active and inactive states. 3) The third specific aim is to determine the relationship between the localization of receptors and their affiliated G proteins. Receptors and G proteins will be examined for differential distribution to basolateral or apical plasma membranes of epithelial cells. In addition, the fate of G during desensitization may provide insight into additional functions of G proteins as well as mechanisms to explain G protein specificity in signal transduction.
