In G-protein signaling, an activated receptor catalyzes GDP/GTP exchange on the G α subunit of a heterotrimeric G protein. In an initial step, receptor interaction with G α 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 G α , the extension of which is a high-affinity receptor binding element.signal transduction | structural polymorphism T he α-subunit (G α ) of heterotrimeric G proteins (G αβγ ) mediates signal transduction in a variety of cell signaling pathways (1). Multiple conformational states of G α are involved in the signal transduction pathway shown in Fig. 1A. In the inactive state, the G α subunit contains a bound GDP [G α ðGDPÞ] and has a high affinity for G βγ . When activated by an appropriate signal, a membrane-bound G-protein coupled receptor (GPCR) binds the heterotrimer in a quaternary complex, leading to the dissociation of GDP and formation of an "empty complex" [G α ð0Þ βγ ], which subsequently binds GTP. The affinity of G α ðGTPÞ for G βγ is dramatically reduced relative to G α ðGDPÞ, resulting in functional dissociation of active G α ðGTPÞ from the membrane-bound complex. The active G α ðGTPÞ subsequently binds downstream effector proteins to trigger a variety of regulatory events, depending on the particular system. Thus, the GPCR acts to catalyze GDP/GTP exchange via an empty complex. Crystallographic (2-7), biochemical (8), and biophysical (9-11) studies have elucidated details of the conformational states of G α that correspond to the discrete steps indicated in Fig. 1A, but the mechanism by which receptor interaction leads to release of the bound GDP from G α and the structure of the empty complex remain a major target of research in the field.The G α subunit has two structural domains, namely a nucleotide binding domain and a helical domain that partially occludes the bound nucleotide (Fig. 1B). From the initial G α crystal structure in 1993, Noel et al. (2) recognized that nucleotide release would probably require an opening between the two domains in the empty complex, but in the intervening 18 years there has been little compelling experimental support for this idea. Nevertheless, some constraints on the general topology of the complex are known. For example, numerous studies indicate that the C terminus of G α is bound tightly to the receptor in the empty complex (9). In addition, the N-terminal helix of G α is associated with G βγ and with the membrane via N-terminal myristoylation (12,13). Together, these constraints fix the position of the nucleot...

We present a model of interaction of Gi protein with activated rhodopsin (R*) which pin-points energetic contributions to activation and reconciles the β2AR–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, leading to displacement of the helical domain and GDP release. The model features a less dramatic domain opening than the crystal structure. The α5 helix undergoes a 63º rotation, accompanied by a 5.7Å translation, which 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 receptormediated G protein activation.

Background: GPCRs regulate heterotrimeric G protein activation. However, the intermediate steps regulating GDP release are still unknown. Results: Energy analysis pinpoints information flow through G␣-receptor interaction and GDP release. Conclusion: Hydrophobic interactions around ␣5 helix, ␤2-␤3 strands, and ␣1 helix are key for GDP stability. Significance: G protein activation defines regulation of high affinity receptor interactions and plays a role defining different cellular responses.

The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling modulation that could have implications for the control of dopamine signaling in normal and physiopathological conditions.

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 Gαs 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.

Background: ␤ 2 -Adrenegic receptor (␤ 2 -AR) mediates cAMP accumulation and ERK phosphorylation via different transducers. Results: Some ␤ 2 -AR ligands selectively activate cAMP and ERK responses in HEK-293 cells. Selectivity is cell contact-dependent.

Scaffold proteins tether and orient components of a signaling cascade to facilitate signaling. Although much is known about how scaffolds colocalize signaling proteins, it is unclear whether scaffolds promote signal amplification. Here, we used arrestin-3, a scaffold of the ASK1-MKK4/7-JNK3 cascade, as a model to understand signal amplification by a scaffold protein. We found that arrestin-3 exhibited >15-fold higher affinity for inactive JNK3 than for active JNK3, and this change involved a shift in the binding site following JNK3 activation. We used systems biochemistry modeling and Bayesian inference to evaluate how the activation of upstream kinases contributed to JNK3 phosphorylation. Our combined experimental and computational approach suggested that the catalytic phosphorylation rate of JNK3 at Thr-221 by MKK7 is two orders of magnitude faster than the corresponding phosphorylation of Tyr-223 by MKK4 with or without arrestin-3. Finally, we showed that the release of activated JNK3 was critical for signal amplification. Collectively, our data suggest a “conveyor belt” mechanism for signal amplification by scaffold proteins. This mechanism informs on a long-standing mystery for how few upstream kinase molecules activate numerous downstream kinases to amplify signaling.

G protein coupled receptors (GPCRs) can be activated by various extracellular stimuli, including hormones, peptides, odorants, neurotransmitters, nucleotides or light. After activation, activated receptors interact with heterotrimeric G proteins and catalyze GDP release from the Gα subunit, the rate limiting step in G protein activation, to form a high affinity nucleotide-free GPCR-G protein complex. In vivo, subsequent GTP binding reduces affinity of the Gα protein for the activated receptor. In this study, we investigated the biochemical and structural characteristics of the prototypical GPCR, rhodopsin, and its signaling partner, transducin (Gt), in phospholipid bilayers to better understand the effects of membrane composition on high affinity complex formation, stability, and receptor mediated nucleotide release. Our results demonstrate that the high-affinity complex (rhodopsin-Gt(empty)) forms more readily and has dramatically increased stability when rhodopsin is integrated into bicelles of a defined composition. We increased the half life of functional complex to one week in the presence of negatively charged phospholipids. These data suggest that a membrane-like structure is an important contributor to the formation and stability of functional receptor-G protein complexes, and can extend the range of studies that investigate properties of these complexes.