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Gβγ dimers get specific at the synapse: In contrast to how much is known about the specificity of G protein α subunits for their cognate GPCRs and their downstream effectors, comparatively little is known about Gβγ dimers, which regulate synaptic transmission by inhibiting the release of neurotransmitter-containing vesicles from presynaptic neurons. Yim et al. applied mass spectrometry to epinephrine-treated and untreated synaptosomes from mouse brain to determine the identities of the Gβ and Gγ subunits that interacted with the SNARE complex, which mediates vesicle exocytosis. This analysis, which revealed neuron specificity and GPCR-dependent changes in the Gβγ dimers that interacted with SNARE proteins, provides molecular details about the regulation of synaptic transmission. Ligand binding to G protein–coupled receptors (GPCRs), such as the α2a-adrenergic receptor (α2aAR), results in the activation of heterotrimeric G proteins, which consist of functionally distinct Gα subunits and Gβγ dimers. α2aAR-dependent inhibition of synaptic transmission regulates functions such as spontaneous locomotor activity, anesthetic sparing, and working memory enhancement and requires the soluble NSF attachment protein receptor (SNARE) complex, a Gβγ effector. To understand how the Gβγ-SNARE complex underlies the α2aAR-dependent inhibition of synaptic transmission, we examined the specificity of Gβγ subunits for the SNARE complex in adrenergic neurons, in which auto-α2aARs respond to epinephrine released from these neurons, and nonadrenergic neurons, in which hetero-α2aARs respond to epinephrine released from other neurons. We performed a quantitative, targeted multiple reaction monitoring proteomic analysis of Gβ and Gγ subunits bound to the SNARE complex in synaptosomes from mouse brains. In the absence of stimulation of auto-α2aARs, Gβ1 and Gγ3 interacted with the SNARE complex. However, Gβ1, Gβ2, and Gγ3 were found in the complex when auto-α2aARs were activated by epinephrine. Further understanding of the specific usage of distinct Gβγ subunits in vivo may provide insights into the homeostatic regulation of synaptic transmission and the mechanisms of dysfunction that occur in neurological diseases. [ABSTRACT FROM AUTHOR]

Agonist-activated G protein-coupled receptors (GPCRs) must correctly select from hundreds of potential downstream signaling cascades and effectors. To accomplish this, GPCRs first bind to an intermediary signaling protein, such as G protein or arrestin. These intermediaries initiate signaling cascades that promote the activity of different effectors, including several protein kinases. The relative roles of G proteins versus arrestins in initiating and directing signaling is hotly debated, and it remains unclear how the correct final signaling pathway is chosen given the ready availability of protein partners. Here, we begin to deconvolute the process of signal bias from the dopamine D1 receptor (D1R) by exploring factors that promote the activation of ERK1/2 or Src, the kinases that lead to cell growth and proliferation. We found that ERK1/2 activation involves both arrestin and Gαs, while Src activation depends solely on arrestin. Interestingly, we found that the phosphorylation pattern influences both arrestin and Gas coupling, suggesting an additional way the cells regulate G protein signaling. The phosphorylation sites in the D1R intracellular loop 3 are particularly important for directing the binding of G protein versus arrestin and for selecting between the activation of ERK1/2 and Src. Collectively, these studies correlate functional outcomes with a physical basis for signaling bias and provide fundamental information on how GPCR signaling is directed. [ABSTRACT FROM AUTHOR]

We present a model of interaction of Gi protein with the activated receptor (R*) rhodopsin, which pinpoints energetic contributions to activation and reconciles the β2 adrenergic receptor-Gs crystal structure with new and previously published experimental data. In silico analysis demonstrated energetic changes when the Gα C-terminal helix (α5) interacts with the R* cytoplasmic pocket, thus leading to displacement of the helical domain and GDP release. The model features a less dramatic domain opening compared with the crystal structure. The α5 helix undergoes a 63° rotation, accompanied by a 5.7-Å translation, that reorganizes interfaces between α5 and α1 helices and between α5 and β6-α5. Changes in the β6-α5 loop displace αG. All of these movements lead to opening of the GDP-binding pocket. The model creates a roadmap for experimental studies of receptor-mediated G-protein activation. [ABSTRACT FROM AUTHOR]

Abstract: Local movements of receptors in the plasma membrane have been extensively studied, as it is generally believed that the dynamics of membrane distribution of receptors regulate their functions. However, the properties of large-scale (>5μm) receptor movements in the membrane are relatively obscure. In the present study, we addressed the question as to whether the large-scale movement of receptor in the plasma membrane at the whole cell level can be explained quantitatively by its local diffusive properties. We used HEK 293 cells transfected with human β2-adrenoceptor fused to photoconvertible fluorescent protein dendra2 as a model system; and found that 1) functional integrity of the dendra2-tagged receptor remains apparently intact; 2) in a mesoscopic scale (~4μm), ~90% of the receptors are mobile on average, and receptor influx to, and out-flux from a membrane area can be symmetrically explained by a diffusion-like process with an effective diffusion coefficient of ~0.1μm2/s; 3) these mobility parameters are not affected by the activity state of the receptor (assessed by using constitutively active receptor mutants); 4) in the macroscopic scale (4–40μm), although a slowly diffusing fraction of receptors (with D<0.01μm2/s) is identifiable in some cases, the movement of the predominant fraction is perfectly explained by the same effective diffusion process observed in the mesoscopic scale, suggesting that the large scale structure of the cell membrane as felt by the receptor is apparently homogeneous in terms of its mesoscopic properties. We also showed that intracellular compartments and plasma membrane are kinetically connected even at steady-state. [Copyright &y& Elsevier]

In G-protein signaling, an activated receptor catalyzes GDP/GTP exchange on the Ga subunit of a heterotrimeric G protein. In an initial step, receptor interaction with Ga acts to allosterically trigger GDP release from a binding site located between the nucleotide binding domain and a helical domain, but the molecular mechanism is unknown. In this study, site-directed spin labeling and double electron-electron resonance spectroscopy are employed to reveal a large-scale separation of the domains that provides a direct pathway for nucleotide escape. Cross-linking studies show that the domain separation is required for receptor enhancement of nucleotide exchange rates. The interdomain opening is coupled to receptor binding via the C-terminal helix of Ga, the extension of which is a high-affinity receptor binding element. [ABSTRACT FROM AUTHOR]

G protein coupled receptors (GPCRa) can be activated by various extracellular stimuli, iriduding hormones, peptides, odorants, neurotransmitters, nucleutides, or light. After activation, receptors intemct with heterotrimeric G proteins and eatalyee GDP release from the Gα subunit, the rate [uniting step in G protein activation, to loon o high aflinity riucleotide-free GPCR-G protein complex In viva, subsequent GTP binding reduces affinity of the Ga protein for the activated receptor. In this study, we investigated the biochemical and smactural characteristics of the prototypical GPCR5 rhodopsin, and its signaling partner, tranuducin (G1), in bicelles to better understand the effects of membrane composition on high affinity complex formation, stability and receptor mediated nudeotide release. Our results demonstrate that the high-aafinity complex formation-G1(empty)) forms more readily md baa dramatically nereseed stability when rhodopsin is integrated into bicelles of a defined composition. We increased the half-life of functional complex to week in the presence of negatively charged phospholipids. These suggest that a membrane-like structure it an important contributor to the formation and stability of Functional crceptoe-G protein complexes and can extend the range o studies that mvestgate properties of these complexes. [ABSTRACT FROM AUTHOR]