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, recent evidence indicates that signaling molecules are corralled in microdomains such as caveolae on the cell surface suggesting that these systems are more well organized than previously thought. In order to determine if the organization of these systems extends to the molecular level, a technique know as bioluminescence resonance energy transfer (BRET) is being used to investigate protein-protein interactions between these 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 an enhanced variant of green fluorescent protein (EGFP). 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 EGFP 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) and the effector adenylyl cyclase (AC). Agonist stimulation of b2AR activates AC through Gs producing cyclic AMP. AC-RLuc and b2AR-EGFP were constructed and expressed in HEK 293 cells. Their functionality was confirmed by increased ligand binding and agonist-mediated cyclic AMP production. BRET was detected between b2AR-EGFP and AC-RLuc in the absence of agonist. These data suggest that in living cells, a receptor-effector complex exists even in the basal state, and provides support for the evolving view that G protein-mediated signaling systems exist as organized complexes.
