Elucidating The Structural Organization Of G-protein Cou

Neurological Disorders And Stroke

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Abstract

G protein-mediated signal transduction pathways 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 ion channel kinetics. G protein-mediated signal transduction occurs when an agonist binds selectively to its heptahelical receptor leading to the activation of a heterotrimeric G protein. These G proteins are composed of alpha, beta (Gb) and gamma (Gg) subunits, and when activated they are able to regulate the activity of specific effectors. Two fluorescence-based techniques are being used to resolve the question of whether or not receptors, G proteins, and effectors are present as protein complexes in the living cell. These techniques, known as bioluminescent resonance energy transfer (BRET), and bimolecular fluorescence complementation (BiFC), can provide both spacial and temporal information about the formation and dissolution of protein complexes. BRET involves the exogenous expression of fusion proteins tagged with either luciferase (Luc) or a fluorescent protein (eg. GFP or YFP). BRET occurs when the bioluminescent energy of the Luc tag is transferred to the fluorescent tag causing it to fluoresce. This only occurs if the tags are juxtaposed (less than 100 angstroms apart) because the fusion proteins associate to form a complex. BiFC is based on the fact that peptide fragments of YFP consisting of amino acids 1-158 or 159-238 are not fluorescent when co-expressed, but will reconstitute a fluorescent YFP if they are brought together by fusing them to proteins that associate to form a complex. The heptahelical beta2-adrenergic receptor (b2AR) triggers the activation of G proteins leading to the regulation of effectors including adenylyl cyclase (AC) and G protein-coupled inwardly rectifying K+ (Kir3) channels. When these proteins were tagged for BRET and BiFC experiments they retained their biological activity. BRET was used to show that the b2AR forms a complex with AC and with the Kir3 channel subunit, Kir3.1. These complexes exist in the absence of signal transduction and persist during signal transduction. BRET was also used to show that G protein subunits form complexes with the b2AR, AC and Kir3.1. BRET between the G protein subunits and these signaling proteins was affected by a receptor agonist. Experiments designed to probe the nature of the agonist-induced effects indicated that they were caused by altered conformations within a protein complex that remains intact. Experiments also indicate that these agonist sensitive complexes are formed before they reach the plasma membrane. BiFC was combined with BRET to determine if the simultaneous presence of three signaling proteins within the same complex could be detected. As a result we have identified complexes of either b2AR or effectors with both Gb and Gg subunits. The technique of combining BiFC and BRET is now being used to show that b2AR, G protein subunits and effectors are all part of the same complex in living cells. In summary our data support the hypothesis that the b2AR, G proteins and effectors are assembled into functional complexes before being transported to the plasma membrane, and that these complexes exist regardless of whether or not the signal transduction pathway is activated by an agonist. This arrangement may explain the specificity and efficacy that is often observed during G protein-mediated signal transduction.