G protein-mediated signal transduction systems are involved in the responses of organisms and their constituent cells to a wide variety of stimuli including light, gustants, odorants, hormones, and neurotransmitters. The nature of the response can be equally diverse varying from changes in gene transcription to altered transmembrane ion permeability. The three core components of this system are the heptahelical receptors, heterotrimeric G proteins and effector molecules which must interact in order to convey information from one component to the next. The prevailing view has been that these interactions are the result of random collisions between signaling molecules that move about freely in the plasma membrane. However, accumulating data has provided evidence that signaling molecules are organized into macromolecular complexes on the cell surface. To elucidate the spatial arrangement of the proteins that make up these signaling complexes in living cells, I have used bioluminescence resonance energy transfer (BRET). The signaling molecules are expressed in transfected mammalian cells as fusion proteins tagged with either the bioluminescent protein luciferase (RLuc) or green fluorescent protein (GFP). If the tags are brought into juxtaposition by a stable protein-protein interaction between two signaling molecules, BRET occurs because light emitted by the RLuc tag will be absorbed by the GFP tag which then fluoresces. For these studies, we are using the prototypical beta2-adrenergic receptor (b2AR) signaling system that consists of b2AR, the stimulatory heterotrimeric G protein (Gs) which is made up of an alpha (G-alpha), a beta (G-beta) and a gamma subunit, and the effector adenylyl cyclase (AC). Agonist stimulation of the b2AR results in the Gs-mediated stimulation of AC leading to the production of cyclic AMP. When the b2AR is tagged with GFP or AC with RLuc these signaling molecules retain their biological activity. BRET occurred when the b2AR-GFP and AC-RLuc were co-expressed in HEK 293 cells indicating that they form a complex in living cells. BRET experiments indicate that this complex is present in the absence of hormone stimulation suggesting that the it exists in the basal state. Currently, G-alpha-GFP and G-beta-RLuc are being used to investigate the interaction of G protein subunits with each other, and with receptor (ie. b2AR-GFP and b2AR-RLuc) and effector (ie. AC-RLuc) proteins. These data are providing support for the evolving view that G protein-mediated signaling systems exist as organized complexes that contribute significantly to the specificity and efficacy of the signal transduction process. In addition to forming complexes with down stream signaling molecules, heptahelical receptors interact with each other to form homomultimers (most probably homodimers). As the receptor's polypeptide chain passes back and forth through the plasma membrane it creates loops that extend into the extra- and intracellular spaces and deposits the C-terminus within the cytoplasmic milieu. The third intracellular loop and the C-terminus are both critical for interaction with downstream signaling components, and the lack of either domain renders the receptor unable to mediate signal transduction despite retention of the ability to bind ligand. It has been hypothesized that receptor oligomerization results in intermolecular interactions in which a portion of one receptor is associated with the complementary part of the other, thus forming a domain that interacts with the heterotrimeric G proteins. To test this hypothesis two signaling deficient b2ARs (one that is missing the third intracellular loop and one that is missing the C-terminal amino acid sequence) are being co-expressed in HEK 293 cells in order to determine if a fully functional b2AR can be reconstitute from the two mutated receptors
