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Neurotensin receptor 1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temperature, weight and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric G.sub.i1 protein, at a resolution of 3 Å. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR-G.sub.i/o complexes (in which the nucleotide-binding pocket adopts more flexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45 degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by molecular dynamics simulations and functional studies, provides insights into the complex process of G-protein activation. Cryo-electron microscopy structures of human neurotensin receptor 1 in complex with G.sub.i1 protein and the agonist JMV449 reveal a non-canonical state that may represent an intermediate form in G-protein activation., Author(s): Hideaki E. Kato [sup.1] [sup.2] , Yan Zhang [sup.1] [sup.3] [sup.9] , Hongli Hu [sup.1] [sup.3] , Carl-Mikael Suomivuori [sup.1] [sup.3] [sup.4] [sup.5] , Francois Marie Ngako Kadji [sup.6] [...]

Heterotrimeric GTP-binding proteins (G proteins) transmit extracellular stimuli perceived by G protein-coupled receptors (GPCRs) to intracellular signaling cascades. Hundreds of GPCRs exist in humans and are the targets of a large percentage of the pharmaceutical drugs used today. Because G proteins are regulated by GPCRs, small molecules that directly modulate G proteins have the potential to become therapeutic agents. However, strategies to develop modulators have been hampered by a lack of structural knowledge of targeting sites for specific modulator binding. Here we present the mechanism of action of the cyclic depsipeptide YM-254890, which is a recently discovered [G.sub.q]-selective inhibitor. YM-254890 specifically inhibits the GDP/GTP exchange reaction of [alpha] subunit of [G.sub.q] protein (G[[alpha].sub.q]) by inhibiting the GDP release from G[[alpha].sub.q]. X-ray crystal structure analysis of the G[[alpha].sub.q][beta][gamma]-YM-254890 complex shows that YM-254890 binds the hydrophobic cleft between two interdomain linkers connecting the GTPase and helical domains of the G[[alpha].sub.q]. The binding stabilizes an inactive GDP-bound form through direct interactions with switch I and impairs the linker flexibility. Our studies provide a novel targeting site for the development of small molecules that selectively inhibit each G[alpha] subunit and an insight into the molecular mechanism of G protein activation. G protein | crystal structure | specific inhibitor doi/ 10.1073/pnas.1003553107

G-protein [beta][gamma] (G[beta][gamma]) subunits interact with a wide range of molecular partners including: G[alpha] subunits, effectors, peptides, and small molecule inhibitors. The molecular mechanisms underlying the ability to accommodate this wide range of structurally distinct binding partners are not well understood. To uncover the role of protein flexibility and alterations in protein conformation in molecular recognition by G[beta][gamma], a method for site-specific [sup.15]N-labeling of G[beta]-Trp residue backbone and indole amines in insect cells was developed. Transverse Relaxation Optimized Spectroscopy-Heteronuclear Single-Quantum Coherence Nuclear Magnetic Resonance (TROSY-HSQC NMR) analysis of [sup.15]N-Trp G[beta][gamma] identified well-dispersed signals for the individual Trp residue side chain and amide positions. Surprisingly, a wide range of signal intensities was observed in the spectrum, likely representing a range of backbone and side chain mobilities. The signal for G[beta]W99 indole was very intense, suggesting a high level of mobility on the protein surface and molecular dynamics simulations indicate that G[beta]W99 is highly mobile on the nanosecond timescale in comparison with other G[beta] tryptophans. Binding of peptides and phosducin dramatically altered the mobility of G[beta]W99 and G[beta]W332 in the binding site and the chemical shifts at sites distant from the direct binding surface in distinct ways. In contrast, binding of G[[alpha].sub.i1]-GDP to G[beta][gamma] had relatively little effect on the spectrum and, most surprisingly, did not significantly alter Trp mobility at the subunit interface. This suggests the inactive heterotrimer in solution adopts a conformation with an open subunit interface a large percentage of the time. Overall, these data show that G[beta][gamma] subunits explore a range of conformations that can be exploited during molecular recognition by diverse binding partners. Beta gamma subunits | clam shell | Hot Spots | Molecular Recognition | subunit interactions www.pnas.org/cgi/doi/10.1073/pnas.0909503107

Heterotrimeric G proteins in physiological and pathological processes have been extensively studied so far. However, little is known about mechanisms regulating the cellular content and compartmentalization of G proteins. Here, we show that the association of nucleoside diphosphate kinase B (NDPK B) with the G protein[beta][gamma] dimer (G[beta][gamma]) is required for G protein function in vivo. In zebrafish embryos, morpholino-mediated knockdown of zebrafish NDPK B, but not NDPK A, results in a severe decrease in cardiac contractility. The depletion of NDPK B is associated with a drastic reduction in G[beta]1[gamma]2 dimer expression. Moreover, the protein levels of the adenylyl cyclase (AC)-regulating Gas and G[alpha]i subunits as well as the caveolae scaffold proteins caveolin-1 and -3 are strongly reduced. In addition, the knockdown of the zebrafish G[beta]1 orthologs, G[beta]1 and G[beta]1like, causes a cardiac phenotype very similar to that of NDPK B morphants. The loss of G[beta]1/G/[beta]11ike is associated with a down-regulation in caveolins, AC-regulating G[alpha]-subunits, and most important, NDPK B. A comparison of embryonic fibroblasts from wild-type and NDPK A/B knockout mice demonstrate a similar reduction of G protein, caveolin-1 and basal cAMP content in mammalian cells that can be rescued by re-expression of human NDPK B. Thus, our results suggest a role for the interaction of NDPK B with G[beta][gamma], dimers and caveolins in regulating membranous G protein content and maintaining normal G protein function in vivo. cAMP | cardiac contractility | G proteins I NDPK | zebrafish

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by G[alpha] subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs--receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with G[alpha] when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of G[alpha], RGS domain binding consequently accelerates G[alpha]-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied G[alpha] substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential G[alpha] selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/G[alpha] complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch Ill and the all-helical domain of the G[alpha] substrate, suggests that unique structural determinants specific to particular RGS protein/G[alpha] pairings exist and could be used to achieve selective inhibition by small molecules. GTPase-accelerating proteins | NMR structure | RGS proteins | x-ray crystallography

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric G[sub.s] protein determined by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7. This conformation is accompanied by a 60 kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the 5-helix of G[sub.s]. Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G-protein coupling via an interaction with the G subunit. This structure provides a new framework for understanding G-protein-coupled receptor function., Author(s): Yi-Lynn Liang [1]; Maryam Khoshouei [2]; Mazdak Radjainia [3]; Yan Zhang [4]; Alisa Glukhova [1]; Jeffrey Tarrasch [4]; David M. Thal [1]; Sebastian G. B. Furness [1]; George Christopoulos [...]

The G protein [alpha]-subunit [G.sub.s][alpha] is required for hormone-stimulated cAMP generation. In pancreatic [beta] cells, [G.sub.s][alpha] mediates the signaling of glucagon-like peptide 1 and other incretin hormones, which are implicated as important regulators of [beta] cell survival and insulin release. Studies have suggested that [G.sub.s][alpha]/cAMP mediates these actions by stimulating insulin receptor substrate 2 (IRS2) expression. Mice with [beta] cell-specific [G.sub.s][alpha] deficiency ([beta]GsKO) were generated by mating [G.sub.s][alpha]-floxed mice to rat insulin II promoter-cre recombinase mice. [beta]GsKO mice had poor survival and postnatal growth with low serum insulin-like growth factor 1 levels. [beta]GsKO mice also developed severe hyperglycemia and glucose intolerance with severe hypoinsulinemia and reduced islet insulin content and glucose-stimulated insulin release. [beta]GsKO mice had markedly reduced average islet size and [beta] cell mass, which was partially explained by reduced [beta] cell size. In addition, [beta]GsKO mice had significantly reduced [beta] cell proliferation and increased [beta] cell apoptosis and markedly reduced expression of the cell cycle protein cyclin D2. The effects on [beta] cell mass and proliferation, but not apoptosis, were present from birth. Unexpectedly expression of Irs2 and the downstream gene Pdx1 were unaffected. These results show that [G.sub.s][alpha]/cAMP pathways are critical regulators of [beta] cell function and proliferation that can work through IRS2-independent mechanisms.

G-protein-coupled receptors (GPCRs) are remarkably versatile signaling molecules. Members of this large family of membrane proteins respond to structurally diverse ligands and mediate most transmembrane signal transduction in response to hormones and neurotransmitters, and in response to the senses of sight, smell and taste. Individual GPCRs can signal through several G-protein subtypes and through G-protein-independent pathways, often in a ligand-specific manner. This functional plasticity can be attributed to structural flexibility of GPCRs and the ability of ligands to induce or to stabilize ligand-specific conformations. Here, we review what has been learned about the dynamic nature of the structure and mechanism of GPCR activation, primarily focusing on spectroscopic studies of purified human [[beta].sub.2] adrenergic receptor.

The interdomain interaction is identified, highlighting the advantage of modular molecules such as R7 family of regulators of G protein signaling (RGSs). The findings reveal that multidomain architecture permits coordination and acceleration of signaling events.

The diverse range of proteins that are identified as Galpah12 and/or Galpha13 interactors is summarised. The expanding role of G12 proteins in the biology of cells is also described.

Extensive configurational sampling was performed for the isolated C-terminal fragment of the [alpha]-subunit of transducin within cavities of photoactivated rhodopsin formed by different energetically feasible conformations of the intracellular loops. The results show that the isolated C-terminal fragment of the [alpha]-subunit of transducin fragment does not necessarily bind rhodopsin in the same binding mode as the same segment in intact [alpha]-subunit.

A model for the structure of the transmembrane domain of perpherin-2 was constructed using methods developed for the G-protein coupled receptor rhodopsin. The fusion-active sequence of perpherin-2 exhibits a sequence motif that matches the viral fusion peptide of influenza hemagglutinin.

The trafficking of leukocytes from the blood to sites of inflammation is the cumulative result of receptor-ligand-mediated signaling events associated with the leukocytes themselves as well as with the underlying vascular endothelium. Our data show that G[[alpha].sub.i] signaling pathways in the vascular endothelium regulate a critical step required for leukocyte diapedesis. In vivo studies using knockout mice demonstrated that a signaling event in a non-lymphohematopoietic compartment of the lung prevented the recruitment of proinflammatory leukocytes. Intravital microscopy showed that blockade was at the capillary endothelial surface and ex vivo studies of leukocyte trafficking demonstrated that a G[[alpha].sub.i]-signaling event in endothelial cells was required for transmigration. Collectively, these data suggest that specific G[[alpha].sub.i2]-mediated signaling between endothelial cells and leukocytes is required for the extravasation of leukocytes and for tissue-specific accumulation. G proteins | inflammation | knockout mice | leukocyte trafficking | pulmonary models

We present evidence that heterotrimeric G protein signaling is involved in cell death associated with the unfolded protein response (UPR) in Arabidopsis. Seedlings of homozygous agb1-2 (G[beta]-null mutation) mutant plants are markedly more resistant to growth inhibition by the protein glycosylation inhibitor tunicamycin (Tm) than either wild-type plants or gpa1-4 (G[alpha]-null mutation) mutants. Leaves of older G[beta] mutant plants show much less cell death when infiltrated with Tm than leaves of wild-type plants. The transcriptional response of G[beta] mutant plants to Tm is less pronounced than that of wild-type plants, as is the accumulation of BiP chaperone proteins. A majority of the Arabidopsis G[beta] protein is associated with the endoplasmic reticulum (ER) and cofractionates with membrane-associated ER luminal BiP. Consistent with its ER localization, G[beta] protein is degraded during the UPR, whereas G[alpha] protein is not. Taken together, these observations imply that the G[beta] protein, which forms a stable heterodimer with the G[gamma] subunit, is involved in the signaling events that trigger UPR-associated cell death. The different Tm sensitivities of G[alpha] and G[beta] mutants, the ER localization of G[beta], and the differential stabilities of G[alpha] and G[beta] proteins during the UPR suggest that the G[beta][gamma] complex serves a signaling function in the ER independent of its function in the G[alpha][beta][gamma] heterotrimer. endoplasmic reticulum | tunicamycin

Arrestins (Arrs) are ubiquitous regulators of the most numerous family of signaling proteins, G protein-coupled receptors. Two models of the Arr--receptor interaction have been proposed: the binding of one Arr to an individual receptor or to two receptors in a dimer. To determine the binding stoichiometry in vivo, we used rod photo-receptors where rhodopsin (Rh) and Arr are expressed at comparably high levels and where Arr localization in the light is determined by its binding to activated Rh. Genetic manipulation of the expression of both proteins shows that the maximum amount of Arr that moves to the Rh-containing compartment exceeds 80%, but not 100%, of the molar amount of Rh present. In vitro experiments with purified proteins confirm that Arr 'saturates' Rh at a 1:1 ratio. Thus, a single Rh molecule is necessary and sufficient to bind Arr. Remarkable structural conservation among receptors and Arrs strongly suggests that all Arr subtypes bind individual molecules of their cognate receptors. protein-protein interactions | signal shutoff | vision | receptor | dimerization

A study focuses on the Arf GTPase activating protein 1 (ArfGAP1), which contains a central motif called ArfGAP1 lipid-packing sensor (ALPS) that preferentially adsorbs on highly curved membranes. The results of the analysis show that the involvement of the ALPS motifs helps the ArfGAP1 to respond to a wide range of membrane curvature with lipid membranes of different compositions.

The respiratory tract is innervated by irritant-responsive sensory nerves, which, on stimulation, release tachykinin neuropeptides in the lung. Tachykinins modulate inflammatory responses to injury by binding to tachykinin (neurokinin) receptors present on various pulmonary cell types. In the present study, the activation of the proinflammatory transcription factor NF-[kappa]B in lung epithelial cells was investigated as a mechanism by which tachykinins stimulate inflammatory processes. In A549 human lung epithelial cells transfected with the tachykinin-1 receptor (Tacr1), treatment with the Tacr1 ligand substance P (SP) resulted in NF-[kappa]B activation, as judged by transcription of an NF-[kappa]B-luciferase reporter gene and production of interleukin-8, a chemokine whose expression is upregulated by NF-[kappa]B. SP caused a dose-dependent activation of NF-[kappa]B that was inhibited by the selective Tacr1 antagonist RP67580. Tacr1 is a G protein-coupled receptor capable of activating both the [G.sub.q] and [G.sub.s] families of G proteins. Expression of inhibitory peptides and constitutively active G protein mutants revealed that [G.sub.q] signaling was both necessary for Tacr1-induced NF-[kappa]B activation and sufficient for NF-[kappa]B activation in the absence of any other treatment. Treatment with pharmacological inhibitors to investigate events downstream of [G.sub.q] revealed that Tacr1-induced NF-[kappa]B activation proceeded through an intracellular signaling pathway that was dependent on phospholipase C, calcium, Ras, Raf-1, MEK, Erk, and proteasome function. These results identify intracellular signaling mechanisms that underlie the proinflammatory effects of tachykinins, which previously have been implicated in lung injury and disease. substance P; A549; Erk; G protein-coupled receptor

One of the major mechanisms in the regulation of G protein-coupled receptors (GPCRs) is their endocytic trafficking, which entails the targeting of receptors to discrete endocytic sites at the plasma membrane followed by receptor internalization and intracellular sorting. The mechanisms that regulate receptor endocytic trafficking, emphasizing the role of GPCR kinases (GRKs) and arrestins in this process are described.

One theory states that [beta]-arrestins contribute to cell polarity during chemotaxis by quenching the signal at the trailing edge of the cell. The second theory states that they scaffold signaling molecules involved in cytoskeletal reorganization to promote localized actin assembly events leading to the formation of a leading edge. The involvement of [beta]-arrestins in chemotaxis both in vivo and in vitro as well as evidence that [beta]-arrestins directly bind and regulate proteins involved in actin reorganization are addressed.

New studies of [beta]-arrestins show that they can function to activate signaling cascades independently of G protein activation as they bring elements of specific signaling pathways into close proximity as well as in nuclear functions. The signaling capacities of these versatile adapter molecules are reviewed and the possible implications for cellular processes such as chemotaxis and apoptosis are discussed.

A study explores the regulation exerted by [Ca.sup.2+] on transamidation activity of TGase-II, and determines whether they are essential for TGase-II to confer survival to human breast cancer cells. Results reveal that the [Ca.sup.2+]-mediated regulation of transmidation activity is essential for the ability of TGase-II to confer cell survival.

The G[alpha] subunits of heterotrimeric G proteins (G[alpha][beta][gamma]) mediate signal transduction via activation by receptors and subsequent interaction with downstream effectors. Crystal structures indicate that conformational changes in 'switch' sequences of G[alpha], controlled by the identity of the bound nucleotide (GDP and GTP), modulate binding affinities to the G[beta][gamma] subunits, receptor, and effector proteins. To investigate the solution structure and dynamics of G[alpha]i1 through the G protein cycle, nitroxide side chains (R1) were introduced at sites in switch II and at a site in helix [alpha]4, a putative effector binding region. In the inactive G[alpha]i1(GDP) state, the EPR spectra are compatible with conformational polymorphism in switch II. Upon complex formation with G[beta][gamma], motions of R1 are highly constrained, reflecting direct contact interactions at the G[alpha]i1-G[beta] interface; remarkably, the presence of R1 at the sites investigated does not substantially affect the binding affinity. Complex formation between the heterotrimer and activated rhodopsin leads to a dramatic change in R1 motion at residue 217 in the receptor-binding [alpha]2/[beta]4 loop and smaller allosteric changes at the G[alpha]i1-G[beta][gamma]/interface distant from the receptor binding surface. Upon addition of GTP[gamma]S, the activated G[alpha]i1(GTP) subunit dissociates from the complex, and switch II is transformed to a unique conformation similar to that in crystal structures but with a flexible backbone. A previously unreported activation-dependent change in [alpha]4, distant from the interaction surface, supports a role for this helix in effector binding. G protein-coupled receptor | signal transduction | site-directed spin labeling | switch II | transducin

Hemodynamic shear stress stimulates a number of intracellular events that both regulate vessel structure and influence development of vascular pathologies. The precise molecular mechanisms by which endothelial cells transduce this mechanical stimulus into intracellular biochemical response have not been established. Here, we show that mechanical perturbation of the plasma membrane leads to ligand-independent conformational transitions in a G protein-coupled receptor (GPCR). By using time-resolved fluorescence microscopy and GPCR conformation-sensitive FRET we found that stimulation of endothelial cells with fluid shear stress, hypotonic stress, or membrane fluidizing agent leads to a significant increase in activity of bradykinin [B.sub.2] GPCR in endothelial cells. The GPCR conformational dynamics was detected by monitoring redistribution of GPCRs between inactive and active conformations in a single endothelial cell under fluid shear stress in real time. We show that this response can be blocked by a [B.sub.2]-selective antagonist. Our data demonstrate that changes in cell membrane tension and membrane fluidity affect conformational dynamics of GPCRs. Therefore, we suggest that GPCRs are involved in mediating primary mechanochemical signal transduction in endothelial cells. We anticipate our experiments to be a starting point for more sophisticated studies of the effects of changes in lipid bilayer environment on GPCR conformational dynamics. Furthermore, because GPCRs are a major target of drug development, a detailed characterization of mechanochemical signaling via the GPCR pathway will be relevant for the development of new antiatherosclerosis drugs. conformational transition | ligand-independent activation | mechanochemical signal transduction | plasma membrane | bradykinin

G/[beta][gamma], a ubiquitous second messenger, relays external signals from G protein-coupled receptors to networks of intracellular effectors, including voltage-dependent calcium channels. Unlike high-voltage-activated [Ca.sup.2+] channels, the inhibition of low-voltage-activated [Ca.sup.2+] channels is subtype-dependent and mediated selectively by G[[beta].sub.2]-containing dimers. Yet, the molecular basis for this exquisite selectivity remains unknown. Here, we used pure recombinant G[beta][gamma] subunits to establish that the G[[beta].sub.2][[gamma].sub.2] dimer can selectively reconstitute the inhibition of [[alpha].sub.1H] channels in isolated membrane patches. This inhibition is the result of a reduction in channel open probability that is not accompanied by a change in channel expression or an alteration in active-channel gating. By exchanging residues between the active G[[beta].sub.2] subunit and the inactive G[[beta].sub.1] subunit, we identified a cluster of amino acids that functionally distinguish G[[beta].sub.2] from other G[beta] subunits. These amino acids on the [beta]-torus identify a region that is distinct from those regions that contact the G[alpha] subunit or other effectors. [alpha]-1H channels | channel regulation | G[beta][gamma] dimers

Cell-surface tissue factor (TF) binds the serine protease factor Vlla to activate coagulation or, alternatively, to trigger signaling through the G protein-coupled, protease-activated receptor 2 (PAR2) relevant to inflammation and angiogenesis. Here we demonstrate that TF*Vlla-mediated coagulation and cell signaling involve distinct cellular pools of TF. The surface-accessible, extracellular [Cys.sup.186]-[Cys.sup.209] disulfide bond of TF is critical for coagulation, and protein disulfide isomerase (PDI) disables coagulation by targeting this disulfide. A TF mutant (TF C209A) with an unpaired [Cys.sup.186] retains TF*Vlla signaling activity, and it has reduced affinity for Vlla, a characteristic of signaling TF on cells with constitutive TF expression. We further show that PDI suppresses TF coagulant activity in a nitric oxide-dependent pathway, linking the regulation of TF thrombogenicity to oxidative stress in the vasculature. Furthermore, a unique monoclonal antibody recognizes only the noncoagulant, cryptic conformation of TF. This antibody inhibits formation of the TF-PAR2 complex and TF.Vlla signaling, but it does not prevent coagulation activation. These experiments delineate an upstream regulatory mechanism that controls TF function, and they provide initial evidence that TF*Vlla signaling can be specifically inhibited with minimal effects on coagulation.

Surface plasmon resonance (SPR) spectroscopy is a technique to study protein-protein interactions in real time; however, application of SPR spectroscopy for investigations of membrane receptors is difficult with respect to functional and uniform immobilization of receptors on a biosensor surface. In the current study, we developed a simple, direct, biosensor-based approach to monitor the molecular interactions between G protein transducin ([G.sub.t]) and rhodopsin (Rho), a prototypical G protein-coupled receptor (GPCR). Detergent-solubilized dark-adapted Rho was captured onto a biosensor surface via lectin interaction, enabling site-directed immobilization of the receptor that made its cytoplasmic surface accessible to a coupling G protein. The system resembled the natural system with respect to receptor density, binding of [G.sub.t] following flash or constant light application, fast GTP-dependent dissociation of [G.sub.t] from Rho, regeneration of Rho, and dependence of [G.sub.t] binding on light intensity and on concentration of [G.sub.t]. The apparent KD of the [G.sub.t]/Rho interaction was 13.6 nM. Our results validate the use of SPR spectroscopy as a tool to study G protein activation in GPCR systems and could be extended for application to other interaction partners of GPCRs.

The metabotropic glutamate receptor 1[alpha] (mGluR1[alpha]) is crucial for some forms of synaptic plasticity, by inducing various cell responses via coupling to various types of G proteins. Upon glutamate binding, an actie conformation, closed-open/active, of the extracellular domain is stabilized, which induces dimeric rearrangement in the intracellular domains, resulting in the initiation of downstream signals. We have confirmed that mGluR1[alpha] functionally interacts with both Gq and Gs pathways; a combination of fluorescent indicators showed that glutamate increased intracellular [Ca.sup.2+] and cAMP concentration ([[[Ca.sup.2+]].sub.i] and [[cAMP].sub.i]). By contrast, [Gd.sup.3+], a different type of ligand whose recognition site on mGluR1[alpha] is distinct from the glutamate site, increased only [[[Ca.sup.2+]].sub.i] and the concentration-activation curve was bell-shaped. FRET analysis revealed that a low concentration of [Gd.sup.3+] induced dimeric rearrangement of the intracellular domains of mGluR1[alpha] as does glutamate, whereas a high concentration of [Gd.sup.3+] reversed the FRET efficiency, which was consistent with a bell-shaped relationship between concentration and Gq activation. These results suggest that [Gd.sup.3+] induces an active and a sort of 'inactivated' conformation in mGluR1[alpha]. The [Gd.sup.3+]-induced active state is considered to correspond to the closed-closed/active conformation, revealed by previous x-ray crystallographic studies. In conclusion, the glutamate-induced closed-open/active state coupled both to Gs and Gq proteins whereas the [Gd.sup.3+]-induced closed-closed/active conformation state preferred Gq to Gs, suggesting that mGluR1[alpha] serves not only as a simple on/off switch but also as a multiple signaling path regulator. FRET | G protein-coupling receptor

The heterotrimeric G protein [G.sub.o] is abundantly expressed in the mammalian nervous system and modulates neural activities in response to various ligands. However, [G.sub.o]'s functions in living animals are less well understood. Here, we demonstrate that GOA-1 [G.sub.o][alpha] has a fundamental role in olfactory adaptation in Caenorhabditis elegans. Impairment of GOA-1 [G.sub.o][alpha] function and excessive activation of EGL-30 [G.sub.o][alpha] cause a defect in adaptation to AWC-sensed odorants. These pathways antagonistically modulate olfactory adaptation in AWC chemosensory neurons. Wild-type animals treated with phorbol esters and double-mutant animals of diacylglycerol (DAG) kinases, dgk-3; dgk-1, also have a defect in adaptation, suggesting that elevated DAG signals disrupt normal adaptation. Constitutively active GOA-1 can suppress the adaptation defect of dgk-3; dgk-1 double mutants, whereas it fails to suppress the adaptation defect of animals with constitutively active EGL-30, implying that GOA-1 acts upstream of EGL-30 in olfactory adaptation. Our results suggest that down-regulation of EGL-30-DAG signaling by GOA-1 underlies olfactory adaptation and plasticity of chemotaxis. chemotaxis | G protein

Previous studies of the specificity of receptor interactions with G protein subunits in living cells have relied on measurements of second messengers or other downstream responses. We have examined the selectivity of interactions between [alpha]2-adrenergic receptors ([alpha]2R) and various combinations of Gi[alpha] and G[beta] subunit isoforms by measuring changes in FRET between Gi[alpha]-yellow fluorescent protein and cyan fluorescent protein-G[beta] chimeras in HeLa cells. All combinations of Gi[alpha]1, -2, or -3 with G[beta]1, -2, or -4 were activated to some degree by endogenous [alpha]2Rs as judged by agonist-dependent decreases in FRET. The degree of G protein activation is determined by the combination of Gi[alpha] and G[beta] subunits rather than by the identity of an individual subunit. RT-PCR analysis and small interfering RNA knockdown of [alpha]2R subtypes, followed by quantification of radiolabeled antagonist binding, demonstrated that HeLa cells express [alpha]2a- and [alpha]2b-adrenergic receptor isoforms in a 2:1 ratio. Increasing receptor number by overexpression of the [alpha]2aR subtype minimized the differences among coupling preferences for Gi[alpha] and G[beta] isoforms. The molecular properties of each Gi[alpha], G[beta], and [alpha]2-adrenergic receptor subtype influence signaling efficiency for the [alpha]2-adrenergic receptor-mediated signaling pathway. fluorescence resonance energy transfer | GTP-binding protein [alpha] subunits | GTP-binding protein [beta] subunits | small interfering RNA

Techniques for fast noninvasive control of neuronal excitability will be of major importance for analyzing and understanding neuronal networks and animal behavior. To develop these tools we demonstrated that two light-activated signaling proteins, vertebrate rat rhodopsin 4 (RO4) and the green algae channelrhodospin 2 (ChR2), could be used to control neuronal excitability and modulate synaptic transmission. Vertebrate rhodopsin couples to the Gi/o, pertussis toxin-sensitive pathway to allow modulation of G protein-gated inward rectifying potassium channels and voltage-gated [Ca.sup.2+] channels. Light-mediated activation of RO4 in cultured hippocampal neurons reduces neuronal firing within ms by hyperpolarization of the somato-dendritic membrane and when activated at presynaptic sites modulates synaptic transmission and paired-pulse facilitation. In contrast, somato-dendritic activation of ChR2 depolarizes neurons sufficiently to induce immediate action potentials, which precisely follow the ChR2 activation up to light stimulation frequencies of 20 Hz. To demonstrate that these constructs are useful for regulating network behavior in intact organisms, embryonic chick spinal cords were electroporated with either construct, allowing the frequency of episodes of spontaneous bursting activity, known to be important for motor circuit formation, to be precisely controlled. Thus light-activated vertebrate RO4 and green algae ChR2 allow the antagonistic control of neuronal function within ms to s in a precise, reversible, and noninvasive manner in cultured neurons and intact vertebrate spinal cords.

A new crystal structure of the pituitary glycoprotein human follicle-stimulating hormone (hFSH) in complex with a fragment of the human FSH receptor ([hFSHR.sub.1-250]) ectodomain has been determined. The hFSH receptor is a Class A rhodopsin-like G-protein-coupled receptor (GPCR). The structure serves as a template for the modeling of the related luteinizing hormone (LH) and thyroid-stimulating hormone (TSH) receptors and explains why these hormones do not cross-react with the FSH receptor. Unexpectedly, the FSH receptor ectodomain in complex with FSH is a dimer but, unlike other cysteine-knot cytokines, FSH does not participate in a receptor-receptor tether. A host of biochemical data can now be placed in the context of the new structure, and will provide new insights that could lead to the development of small-molecule contraceptives and more-active or bifunctional versions of hormones for FSH receptor and other related receptors.

The consequences of a deletion of gtrl on in vivo Pho84 expression, trafficking and activity, and extracellular phosphatase activity were analyzed in strains synthesizing either Pho84-green fluorescent protein or Pho84-myc chimeras. The studies revealed a delayed response in Pho84-mediated phosphate uptake and extracellular phosphate activity under phosphate -limiting conditions.

Knowing the structures of G-protein-coupled receptors, says Lindau keynote speaker Brian Kobilka, should help with drug development. But how is this progressing? Brian Kobilka, with his medical background, said he [...]

The application of the RNA G-quadruplexes for the cap-independent translaton initiation taking place in the human VEGF IRES function is demonstrated.

The properties of intracellular loop 2 (IL2) in G-protein-coupled receptors (GPCRs) are analyzed by using a Monte Carlo-based ab initio method. The P3.57A mutation in ILA has decreased [beta]-arrestin binding and internalization, thus predicting the effects of the mutation and has shown that it has decreased the propensity of IL2 to form helix, where the helical IL2 is identified as a component of the GPCR active form.

A new homogeneous competition assay developed by employing the fluorescence anisotropy change of mant-GTP upon binding to nucleotide-free FtsZ is used to determine the apparent contributions of the guanine, ribose, and the [alpha]-, [beta]-, and [gamma]-phosphates to the free energy change of nucleotide binding. The results offer insight into the FtsZ-nucleotide interactions, which could be useful in the rational design of new inhibitors, especially GTP phosphate mimetics.

The stabilization of G-quadruplex DNA by a scissors-shaped binaphthyl derivative (NPA) by intertwisting the whole G-quadruplex with two long chains, through the cooperation of the two functional groups, binaphthylene and the ethoxy chain is reported. NPA is found to exhibit a good inhibitory effect of telomerase activity as well as excellent cytotoxic activity against HepG-2 human liver cancer cells.

Several approaches are described for characterizing the conformational stability of the human adenosine [A.sub.2]a receptor (h[A.sub.2]aR). These methods have provided a general strategy for analyzing the effect of surfactant and ligand effects on the stability of G protein-coupled receptors (GPCRs).

Thermodynamic and kinetic studies are used for exploring the four c-Myc loops isomers in a [K.sup.+] solution, where the four loop isomers have formed parallel-stranded G-quadruplexes with short loop lengths. The capping structures formed by the extended flanking segments have contributed to the stabilization in [T.sub.m] for the c-Myc promoter G-quadruplex and the thermodynamic stability and kinetic properties of the c-Myc G-quadruplex loops isomers have helped in understanding their biological roles and drug targeting. .

A pair of furan-based cyclic homooligopeptides is prepared to specifically target G-quadruplexes and UV melting has shown that these ligands have effectively induced G-quadruplex structures in the G-rich 22-mer c-MYC DNA sequence and have stabilized the structure. The studies have shown that the present molecules are effective in specifically binding and stabilizing quadruplexes and have provided a suitable scaffold to develop into a quadruplex-targeting therapeutic agent.

NMR experiments are performed to understand the simple oligonucleotide sequence d(TG4T) in solution which forms a tetramolecular G-quadruplex having a parallel orientation of the four strands and four G-quartets with all of the residues in the anti orientation. The results revealed the major forms to be comprised of four G-quartets, while the minor form to exhibit an additional T-quartet at the 5' end.

The NMR solution structures of two G-quadruplex conformations in potassium solution: a (3 + 1) and a parallel-stranded G-quadruplexes were explored to understand their coexistance in guanine-rich stretches of human telomerase, hTERT promoter. The findings from a combination of NMR and CD techniques, complemented with sequence modifications and variations of experimental condition provide better insight into the coexistence of the two G-quadruplex conformations in equilibrium and the way different structural elements adapt to favor a particular form.

Biophysical studies have shown that the 22 base insert has led to the formation of a family of stable G-quadruplex structures that are unaffected by the single nucleotide (G/A) polymorphism. Mutations that destabilize the quadruplex have led to a 15-fold enhancement of translational efficiency, which has shown that a biological role of the insert in exon C of the bovine estrogen receptor [alpha] gene (bov-ESR1) is to regulate translation of the exon.

The electron paramagnetic resonance spectrum of the spin-label 2,2,6,6,-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC)-the C-terminal tail of the transducin [alpha] subunit (Gt[alpha](340-350)) has disclosed greater immobilization of the nitroxide upon binding to rhodopsin upon photoactivation (R*) when compared to that of the N-terminally modified Proxyl-Gt[alpha](340-350) analogue. TOAC and Proxyl spin-labels in the GPCR-G-protein system have provided unique biophysical probes that are used for analyzing the structure and conformational changes at the rhodopsin-G-protein interface.

Umbrella sampling reconstructed free energies of [delta]-opioid receptor (OR) homodimers involving the fourth transmembrane helix is used for predicting their association constant. The results have shown a short lifetime for [delta]OR homodimers in the membrane.