G protein-coupled receptors transduce diverse extracellular signals, such as neurotransmitters, hormones, chemokines, and sensory stimuli, into intracellular responses through activation of heterotrimeric G proteins. G proteins play critical roles in determining specificity and kinetics of subsequent biological responses by modulation of effector proteins. We have developed a fluorescence resonance energy transfer (FRET)-based assay to directly measure mammalian G protein activation in intact cells and found that Gi proteins activate within 1-2 s, which is considerably slower than activation kinetics of the receptors themselves. More importantly, FRET measurements demonstrated that G␣i-and G␤␥-subunits do not dissociate during activation, as has been previously postulated. Based on FRET measurements between G␣i-yellow fluorescent protein and G␤␥-subunits that were fused to cyan fluorescent protein at various positions, we conclude that, instead, G protein subunits undergo a molecular rearrangement during activation. The detection of a persistent heterotrimeric composition during G protein activation will impact the understanding of how G proteins achieve subtype-selective coupling to effectors. This finding will be of particular interest for unraveling G␤␥-induced signaling pathways. Avariety of physiological signals such as neurotransmitters, hormones, and light are detected by members of the seven transmembrane domain receptor family. These G protein-coupled receptors (GPCRs) activate G proteins by promoting binding of GTP in exchange for GDP. Both, G␣ and G␤␥-subunits of activated G proteins can regulate downstream effectors such as adenylyl cyclases, phospholipases, or ion channels. Based on biochemical experiments and structural studies, it is known that conformational rearrangements in the ''switch regions'' of the ␣-subunits on GTP binding weaken the interaction with G␤␥-subunits (1-3). It is generally assumed that the reduced affinity of G␣-GTP for G␤␥ causes G proteins to dissociate into G␣-GTP and a G␤␥ complex. Reassociation is then assumed to occur on hydrolysis of the GTP bound to G␣, which can be accelerated by RGS proteins (4-6). However, this model fails to explain well established phenomena in G protein-mediated signaling. Most importantly, a major open question is how G␤␥ effectors, such as G proteinactivated inwardly rectifying K ϩ (GIRK) channel, are selectively regulated through specific G␣ subtypes despite the lack of subtype selectivity for G␤␥ subtypes (7-10). In attempts to answer this central question, it has been hypothesized that selectivity may be caused by scaffolding of G proteins and effectors, either by direct binding of G␣ to its effector (11), or by temporally and spatially restricting G protein activity (12, 13).The possibility to investigate protein-protein interactions in living cells by using fluorescence resonance energy transfer (FRET) between recombinant fluorescent proteins (14), has recently led to new insights in temporal signaling properties of G protein effectors (1...

The interaction of activated G protein‐coupled receptors with G proteins is a key event in signal transduction. Here, using a fluorescence resonance energy transfer (FRET)‐based assay, we measure directly and in living cells the interaction of YFP‐labeled α2A‐adrenergic receptors with CFP‐labeled G proteins. Upon agonist stimulation, a small, concentration‐dependent increase in FRET was observed. No specific basal FRET was detected in the absence of agonist. Kinetics of the onset of receptor/G protein interaction were <100 ms and depended on expression levels of Gα. Simultaneously recorded G protein‐regulated inwardly rectifying K+ channel currents revealed a maximal current response already at agonist concentrations producing submaximal FRET amplitudes. By analyzing FRET signals in the presence of a Gα mutant, which dissociates more slowly from activated receptors, it was demonstrated that only a fraction of wild‐type G proteins interacts with the activated receptor at any time. Our data suggest that α2A‐adrenergic receptors and G proteins interact by rapid collision coupling and indicate that there is no significant precoupling between these receptors and G proteins.

G proteins transmit a variety of extracellular signals into intracellular responses. The G␣ and G␤␥ subunits are both known to regulate effectors. Interestingly, the G␣ subunit also determines subtype specificity of G␤␥ effector interactions. However, in light of the common paradigm that G␣ and G␤␥ subunits dissociate during activation, a plausible mechanism of how this subtype specificity is generated was lacking. Using a fluorescence resonance energy transfer (FRET)-based assay developed to directly measure mammalian G protein activation in intact cells, we demonstrate that fluorescent G␣ i1,2,3 , G␣ z , and G␤ 1 ␥ 2 subunits do not dissociate during activation but rather undergo subunit rearrangement as indicated by an activation-induced increase in FRET. In contrast, fluorescent G␣ o subunits exhibited an activation-induced decrease in FRET, reflecting subunit dissociation or, alternatively, a distinct subunit rearrangement. The ␣ B/C -region within the ␣-helical domain, which is much more conserved within G␣ i1,2,3 and G␣ z as compared with that in G␣ o , was found to be required for exhibition of an activation-induced increase in FRET between fluorescent G␣ and G␤␥ subunits. However, the ␣ B/C -region of G␣ i1 alone was not sufficient to transfer the activation pattern of G␣ i to the G␣ o subunit. Either residues in the first 91 amino acids or in the C-terminal remainder (amino acids 93-354) of G␣ i1 together with the ␣ B/Chelical region of G␣ i1 were needed to transform the G␣ o -activation pattern into a G␣ i1 -type of activation. The discovery of subtype-selective mechanisms of G protein activation illustrates that G protein subfamilies have specific mechanisms of activation that may provide a previously unknown basis for G protein signaling specificity.Heterotrimeric G proteins transduce and modulate many biological signals such as neurotransmissions, hormone-mediated signals, chemotaxis signals, cell growth signals, and also visual and olfactory signals. Common to all heterotrimeric G proteins is the fact that their activation by G protein-coupled receptors involves binding of GTP in exchange for GDP. Both G␣ and G␤␥ subunits of activated G proteins can regulate downstream effectors such as enzymes or ion channels. To achieve proper regulation of cellular responses, it is critically important that G proteins interact with effectors in a subtypespecific manner. However, it is unknown how this specificity of G protein subtypes for effectors is achieved. The principle of generating G protein subtype specificity for G␣ effector interaction may be based on the existence of a direct contact between G␣ and its effectors, allowing discrimination between different G␣ subtypes. Much more puzzling is the question of how effectors for G␤␥ subunits are able to discriminate between G␤␥ subunits derived from different G␣ subtypes. The common notion over the past two decades has been that, upon activation, G proteins will dissociate, and each of the subunits can interact independently with and modulate their effector...

Zusammenfassung. Anhand einer Stichprobe von N = 1252 Patienten, die in einer von drei Ambulanzen der Christoph-Dornier-Stiftung für Klinische Psychologie behandelt wurden und hauptsächlich unter Angststörungen litten, wurde die Reliabilität und Validität des Brief Symptom Inventory (BSI; Derogatis, 1993 ) untersucht. Vergleiche mit Daten der Symptom-Check-Liste (SCL-90-R) zeigten ähnliche psychometrische Qualitäten für beide Instrumente. Für die Primärdimensionen des BSI ergaben sich befriedigende interne Konsistenzen von .70 bis .89 mit einem α = .96 für den Global Severity Index (GSI). Die konvergente Validität zeigte hohe Korrelationen zu inhaltsnahen klinischen Selbstbeurteilungsskalen. Mit Hilfe der Prä-Post-Daten von N = 617 Patienten, die kognitiv-verhaltenstherapeutisch behandelt wurden, konnten vergleichbare Effektstärken für die SCL-90-R und das BSI ermittelt werden. Die Generalisierbarkeit der Befunde ist durch das Vorherrschen von Angstpatienten in dieser Stichprobe eingeschränkt.

After stimulation with agonist, G protein-coupled receptors (GPCRs) activate G proteins and become phosphorylated by G protein-coupled receptor kinases (GRKs), and most of them translocate cytosolic arrestin proteins to the cytoplasmic membrane. Agonist-activated GPCRs are specifically phosphorylated by GRKs and are targeted for endocytosis by arrestin proteins, suggesting a connection between GPCR conformational changes and interaction with GRKs and arrestins. Previously, we showed that by substitution of histidine for residues at the cytoplasmic side of helix 3 (H3) and helix 6 (H6) of the parathyroid hormone (PTH) receptor (PTHR), a zinc metal ion-binding site is engineered that prevents PTH-stimulated G s activation (Sheikh, S. P., Vilardaga, J.-P., Baranski, T. J., Lichtarge, O., Iiri, T., Meng, E. C., Nissenson, R. A., and Bourne, H. R. (1999) J. Biol. Chem. 274, 17033-17041). These data suggest that relative movements between H3 and H6 are critical for G s activation. Does this molecular event play a similar role in activation of GRK and arrestin and in PTHRmediated G q activation? To answer this question, we utilized the two previously described mutant forms of PTHR, H401 and H402, which contain a naturally present histidine residue at position 301 in H3 and a second substituted histidine residue at positions 401 and 402 in H6, respectively. Both mutant receptors showed inhibition of PTH-stimulated inositol phosphate and cAMP generation in the presence of increasing concentrations of Zn(II). However, the mutants showed no Zn(II)-dependent impairment of phosphorylation by GRK-2. Likewise, the mutants were indistinguishable from wild-type PTHR in the ability to translocate ␤-arrestins/green fluorescent protein to the cell membrane and were also not affected by sensitivity to Zn(II). These results suggest that agonist-mediated phosphorylation and internalization of PTHR require conformational switches of the receptor distinct from the cAMP and inositol phosphate signaling state. Furthermore, PTHR sequestration does not appear to require G protein activation.Transduction and regulation of signals by G protein-coupled receptors (GPCRs) 1 require the interaction of the receptors with three families of proteins: G proteins, G protein-coupled receptor kinases (GRKs), and arrestins (1). Agonist binding to GPCRs allows G proteins to bind to the cytoplasmic parts of the receptor and to become activated by accelerating GDP/GTP exchange of the G protein ␣-subunits of the heterotrimer (2). This event promotes activation of effector enzymes and ion channels by the activated G␣⅐GTP as well as the G␤␥ complex (3). Signaling by most agonist-activated GPCRs is regulated by a two-step process (also referred to as the rapid form of GPCR desensitization) in which the activated receptors become a target for phosphorylation by GRKs and then interact with cytosolic arrestin proteins (1, 4). Binding of arrestin to the agonist-activated receptor has at least two consequences: uncoupling of receptors from G proteins, resulting in...