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. In order to elucidate the spacial arrangement of the proteins that make up these signaling complexes a technique known as bioluminescence resonance energy transfer (BRET) is being used to investigate protein-protein interactions between the various signaling molecules in living cells. 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 the b2AR, the stimulatory heterotrimeric G protein (Gs), 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, G protein subunits and AC are tagged with GFP or RLuc these signaling molecules retain their biological activity. In HEK 293 cells co-expressing these tagged proteins BRET occurred between GFP-tagged G protein subunits and both the b2AR-RLuc and AC-RLuc. We have also shown that BRET occurs between the b2AR-GFP and AC-RLuc. These date indicate that the b2AR, Gs and AC form a complex in living cells. This complex is present in the absence of hormone stimulation, and BRET persists in the presence of hormone suggesting that these complexes exist in both the basal state and during signal transduction, thus 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 receptor dimers. 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 second and third intracellular loops 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 dimerization 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 second intracellular loop and one that is missing the third intracellular loop) 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.
