Is haemoglobin Gα Philadelphia linked to α-thalassaemia?

Summary The question, Is Hb G Philadelphia linked to -thalassaemia? was first posed because the abnormal haemoglobin is found in heterozygotes at a concentration greater than 25%, the proportion predicted from a 4 -chain gene model. Globin chain biosynthesis was studied in a West Indian family in which one parent had ⁺ thalassaemia and the other was heterozygous for the G Philadelphia chain gene. The former had a globin chain production ratio / well above 1, while the latter had a ratio significantly less than 1. One child of the marriage had inherited the ⁺ thallassaemia from one parent and the G Philadelphia chain gene from the other and showed the typical picture of /-thalassaemia (/ ratio slightly above normal). It is explained in the discussion that the evidence favours a close linkage of 2 -chain genes.     

PAMAM G4 dendrimers affect the aggregation of α-synuclein

α-Synuclein (ASN) aggregation plays a key role in neurodegenerative disorders including Parkinson's disease, and inhibition of fibril formation is a potential therapeutic strategy for these conditions. The aim of the present study was to investigate polyamidoamine (PAMAM) dendrimers (generations 4 and 3.5) as inhibitors of fibril formation in vitro by examining their interaction with ASN intrinsic tyrosine fluorescence. Furthermore, the effect of dendrimers on ASN aggregation was studied using circular dichroism (CD) spectroscopy and CD studies were complemented by a fluorescence assays using the dye thioflavin T (ThT). The PAMAM G4 dendrimer caused an increase in tyrosine residue fluorescence, and inhibited fibrillation of ASN; inhibited fibrillation was not observed with PAMAM G3.5 dendrimers.     

Screening of integrin-binding peptides from the laminin α4 and α5 chain G domain peptide library

Laminins, a multifunctional protein family of extracellular matrix, interact with various types of integrin. Here, integrin-mediated cell adhesive peptides have been systematically screened in the laminin α4 and α5 chain G domain peptide library consisting of 211 peptides by both the peptide-coated plastic plates and peptide-conjugated Sepharose bead assays using human dermal fibroblasts. Thirteen peptides promoted cell spreading and the activity was specifically inhibited by EDTA. Cell attachment to 11 peptides was inhibited by anti-integrin β1 antibody. Additionally, cell attachment to the A5G81 (AGQWHRVSVRWG) and A5G84 (TWSQKALHHRVP) peptides was specifically inhibited by anti-integrin α3 and α6 antibodies. These results suggest that the A5G81 and A5G84 peptides promote integrin α3β1- and α6β1-mediated cell attachment. Further, most of the integrin-mediated cell adhesive peptides are located in the loop regions in the G domains, suggesting that structure is important for the integrin specific recognition. Integrin binding peptides are useful for understanding laminin functions and have a potential to use for biomaterials and drug development.     

Direct interactions with G α i and G βγ mediate nongenomic signaling by estrogen receptor α

Estrogen induces G protein-dependent nongenomic signaling in a variety of cell types via the activation of a plasma membrane-associated subpopulation of estrogen receptor alpha (ER alpha). Using pull-down experiments with purified recombinant proteins, we now demonstrate that ER alpha binds directly to G alpha i and G betagamma. Mutagenesis and the addition of blocking peptide reveals that this occurs via amino acids 251-260 and 271-595 of ER alpha, respectively. Studies of ER alpha complexed with heterotrimeric G proteins further show that estradiol causes the release of both G alpha i and G betagamma without stimulating GTP binding to G alpha i. Moreover, in COS-7 cells, the disruption of ER alpha-G alpha i interaction by deletion mutagenesis of ER alpha or expression of blocking peptide, as well as G betagamma sequestration with beta-adrenergic receptor kinase C terminus, prevents nongenomic responses to estradiol including src and erk activation. In endothelial cells, the disruption of ER alpha-G alpha i interaction prevents estradiol-induced nitric oxide synthase activation and the resulting attenuation of monocyte adhesion that contributes to estrogen-related cardiovascular protection. Thus, through direct interactions, ER alpha mediates a novel mechanism of G protein activation that provides greater diversity of function of both the steroid hormone receptor and G proteins.     

Domain-Opening and Dynamic Coupling in the α-Subunit of Heterotrimeric G Proteins

Heterotrimeric G proteins are conformational switches that turn on intracellular signaling cascades in response to the activation of G-protein-coupled receptors. Receptor activation by extracellular stimuli promotes a cycle of GTP binding and hydrolysis on the G protein α-subunit (Gα). Important conformational transitions occurring during this cycle have been characterized from extensive crystallographic studies of Gα. However, the link between the observed conformations and the mechanisms involved in G-protein activation and effector interaction remain unclear. Here we describe a comprehensive principal component analysis of available Gα crystallographic structures supplemented with extensive unbiased conventional and accelerated molecular dynamics simulations that together characterize the response of Gα to GTP binding and hydrolysis. Our studies reveal details of activating conformational changes as well as the intrinsic flexibility of the α-helical domain that includes a large-scale 60° domain opening under nucleotide-free conditions. This result is consistent with the recently reported open crystal structure of Gs, the stimulatory G protein for adenylyl cyclase, in complex with the α2 adrenergic receptor. Sets of unique interactions potentially important for the conformational transition are also identified. Moreover simulations reveal nucleotide-dependent dynamical couplings of distal regions and residues potentially important for the allosteric link between functional sites.Heterotrimeric G proteins undergo cycles of GTP-dependent conformational rearrangements and alterations of their oligomeric αβγ form to convey receptor signals to downstream effectors that control diverse cellular processes ranging from movement to division and differentiation. Interaction with activated receptor promotes the exchange of GDP for GTP on the G protein α subunit (Gα) and its separation from its βγ subunit partners (Gβγ). Both isolated Gα and Gβγ then interact with downstream effectors. GTP hydrolysis deactivates Gα, which reassociates with Gβγ, becoming ready to restart the cycle. Each of these stages has been subjected to extensive crystallographic studies with high-resolution structures of Gα in complex with GDP, GTP analog, Gβγ, and, most recently, the G-protein-coupled receptors now available. These studies have provided extensive mechanistic insight. However, a number of important questions remain, including:

• How do the distinct conformations evident in the accumulated structures interconvert?
• How do disease-associated mutations affect the fidelity of these transitions?
• And, critically, how do distal functional sites responsible for nucleotide and protein partner binding allosterically coordinate their activities?

Here we describe a comprehensive analysis of the accumulated Gα crystallographic structures supplemented with extensive conventional (cMD) and accelerated molecular dynamics (aMD) simulations (1) that together map the structural and dynamical features of Gα in different nucleotide states. These enhanced sampling simulations reveal the spontaneous interconversion between GDP and GTP conformations and also characterize large-scale opening motions of the α-helical domain (HD) that were not accessible to previous simulation studies (2–5). Furthermore, the current simulations results reveal a distinctive pattern of collective motions that provide evidence for a nucleotide-dependent network of dynamic communication between the active site and the receptor and effector binding sites.Principal component analysis of 53 Gα experimental structures homologous to transducin (Gαt) reveals that the major variation in accumulated structures is the concerted association/disassociation of three nucleotide-binding site loops termed the switch regions (SI, SII, and SIII). An additional small-scale (<10°) rotation of the HD relative to the main catalytic Ras-like domain (RasD) is also apparent (see Fig. S1 in the Supporting Material). The distinct conformation of SI–SIII regions gives rise to nucleotide-associated segregation of GDP- and GTP-analog-bound experimental structures along the PC1-PC2 plane. Interestingly, both GDP- and GTP-bound structures display a skewed distribution along the PC1-PC2 plane that arises from HD rotation. In comparison, the distribution of the GTP-bound structures becomes more restricted and the skew decreases when the mapping is based on a principle component analysis that excludes the HD region (see Fig. S1).Recently, the HD region of Gαs (the α-subunit of the stimulatory G protein for adenyl cyclase) was shown to adopt a dramatically more open conformation in a crystal structure complex with the β2 adrenergic receptor (β2AR) (6). This clam-shell-like 127° opening in the absence of nucleotide and presence of receptor is consistent with electron microscopy (7) and double electron-electron resonance analysis (8). These results, together with recent hydrogen-deuterium exchange mass spectrometry data (9), indicate that there may be additional functional motions and inherent flexibility in the ensemble of native states beyond those apparent in the accumulated crystal structures of Gαt (9). To address this question, we performed multiple 100-ns aMD simulations of nucleotide-free Gαt. These simulations reveal a spontaneous large-scale opening and closing motion of larger magnitude (>60°) than those evident in the distribution of crystallographic structures (Fig. 1 A and see Fig. S2). In addition, the trajectory reveals two dominant modes of HD opening: an out-of-plane shifting (PC1 in Fig. S3) and an in-plane rotation (PC2 in Fig. S3). It is also notable that nucleotide-free aMD simulations sample both active (GTP-like) and inactive (GDP-like) structures (see Fig. S2) in an analogous manner to the spontaneous GDP to GTP interconversion sampled for Ras and Rho small G proteins with similar methods (10–12).Open in a separate windowFigure 1Nucleotide-associated differences in flexibility and dynamic coupling. (A) Mapping aMD simulation trajectories (blue points) onto the principal components obtained from analysis of Gα crystallographic GDP-bound (green) and GTP-analog bound (red) experimental structures. (Orange) Open β2AR-Gαs complex structure. (B) Results of dynamic coupling analysis mapped onto the average structure for each nucleotide state. (Spheres) Nodes for the nucleotide; the protein cartoon is colored by community structure. (C) Community network graph. (Circles) Communities, colored as in panel B. Radius of the circle indicates the number of residues in the community. Thickness of linking lines is determined by the maximum betweenness of the respective intercommunity edges (see the Supporting Material). (Red, blue, and green edges) Major topological difference between states.The low sequence identity between Gαt and Gαs (44.5%), as well as the absence of the receptor and Gβγ in the simulations, may explain the difference between the predicted ∼60° Gαt-HD rotation and that displayed in the β2AR-Gαs crystallographic structure (see Fig. S3). It is notable that, although the amplitude is much smaller, aMD simulations with bound nucleotide display similar dominant HD motions to those observed in the nucleotide-free simulations (see Fig. S4). This suggests that the interdomain flexibility of RasD and HD is likely an intrinsic feature of Gαt regardless of nucleotide state.The transition between distinct conformations (structural clusters; see Fig. S5) was observed to correspond to significant dynamical changes in side-chain contacts (see Fig. S6). Specifically, we found sequential contacts breaking during the HD in-plane rotation motion starting from the region between HD helix αD and RasD helix αG toward that between HD helix αE and RasD SIII and the P-loop. In comparison, for the out-of-plane shift, we found simultaneous breaking and formation of contacts in the region containing the loop between helices αB and αC, the N-terminus of αA, αE, and αF of HD; α1, SI, and the loop between strand β6 and helix α5 of RasD. Interactions highlighted in these regions as potentially important for the conformational transitions include D137::K276, S140::K273, S140::D227, Q143::R238, N145::E39, and D146::K266, the effect of which can be further evaluated by mutagenesis experiments and simulations.Dynamic network analysis methods developed by Sethi et al. (13) were used to examine whether the motions of one residue were correlated to the motions of another (distant) residue. In this approach, a weighted graph is constructed where each residue represents a node and the weight of the connection between nodes represents their respective correlation value. A clustering of edges is then used to define local communities of highly correlated residues that represent substructures that are highly intraconnected, but loosely interconnected. Applying this approach to multiple 40-ns cMD simulations initiated from GTP-, GDP-, and aMD-derived APO conformations revealed a consistent community composition as well as a distinct pattern of intercommunity connection between nucleotide states (Fig. 1, B and C).The dynamics of the RasD region can be decomposed into two main communities that stem from the nucleotide base and phosphate regions in GDP and GTP states: The first community is composed of residues from the P-loop, helix α1, strands β1–β3, and the phosphates of the nucleotide (orange in Fig. 1, B and C). The second community comprises residues from helix αG, strands β4–β6, and the nucleotide base region (tan in Fig. 1, B and C). This dynamic partitioning of the central β-sheet and central role of the nucleotide is consistent with the bilobal structure and dynamics previously reported for Ras (14). In the presence of GTP, the first community includes or is dynamically coupled to SI, SII, and SIII regions (see the orange node and the red edge in Fig. 1 C). Removal of the γ-phosphate of GTP disrupts this region, leading to decoupling of the switch regions from the nucleotide. Also evident for GDP states is an apparent tighter coupling of RasD and HD regions (blue edges in Fig. 1 C). We note that these findings are robust to the choice of initial simulation conditions and are observed in both cMD and aMD simulations (see Fig. S7 and Fig. S8). Nucleotide-free Gαt simulations display an altered dynamical network with respect to those of nucleotide bound states. In particular, RasD and HD regions lose connecting edges consistent with the large-scale opening of these domains (e.g., SIII-HD green edges in Fig. 1 C).A number of residues highlighted here as potentially important for mediating the coupling between prominent communities (see Table S1 in the Supporting Material) have been shown by previous mutagenesis studies to affect GDP release. For example, the double mutation A322S/R174M was found to significantly enhance the rate of GDP release (15). The current results indicate that these positions are involved in coupling the nucleotide and RasD. Also, mutations R144A and L232Q caused a faster basal GDP release rate in Gαi1 (16). The current analysis indicates that the equivalent positions in Gαt (S140 and M228) couple the RasD and HD, and suggests that their mutation could promote domain-domain motions. We also note the apparent coupling of α5 with the nucleotide base and Ploop-β1 with the phosphate regions of GDP. These direct connections of the receptor connecting N- and C-terminus to GDP are suggestive of potential routes for receptor-mediated GDP release. We expect further study of these sites and of receptor-bound dynamics to be informative in this regard.In conclusion, simulations suggest a flexible HD in Gαt similar to that found for Gαs. In particular, in the absence of nucleotide we observed the spontaneous large-scale opening and closing of HD relative to RasD, which was unseen in previous computational studies. Moreover, we found that the functional states of Gαt are associated with the distinct dynamical couplings of functional regions including SI–SIII, P-loop, α5, and the HD region. Finally, our results indicate that nucleotide may not directly induce large-scale conformational changes but, instead, act as a modulator of intrinsically accessible conformations and as a central participant in their associated dynamical couplings.     

Short chain α-pyrones capable of potentiating penicillin G against Pseudomonas aeruginosa

The dramatic increase in bacterial resistance over the past three decades has greatly reduced the effectiveness of nearly all clinical antibiotics, bringing infectious disease to the forefront as a dire threat to global health. To combat these infections, adjuvant therapies have emerged as a way to reactivate known antibiotics against resistant pathogens. Herein, we report the evaluation of simplified α-pyrone adjuvants capable of potentiating penicillin G against Pseudomonas aeruginosa, a Gram-negative pathogen whose multidrug-resistant strains have been labeled by the Centers for Disease Control and Prevention as a serious threat to public health.     

C-Terminal exchange between Gα o and Gα i3 proteins differently modulates α2A-adrenoceptor activation

Chimeric G proteins, obtained by exchanging their C-terminal portion for that of a G protein from an unrelated class, drive the receptor selectivity to that corresponding to the introduced G protein domain. The _2A-adrenoceptor (_2AAR), which yielded an efficacious and weak [³⁵S]GTPS binding response by respectively G _o and G _i3 protein, was investigated in CHO-K1 cells co-expressing chimeric G proteins for which the six last C-terminal amino acids between G _o and G _i3 proteins, and reciprocally, were permuted. Activation of the chimeric G _{o / i3} protein was highly efficient whereas the G _{i3 / o} protein yielded a weak stimulation. These [³⁵S]GTPS binding responses were not different from their parental wild-type G _o and G _i3 proteins. Similar results were obtained with an _2AAR carrying a facilitating Thr³⁷³Lys mutation in a putative G protein interaction domain. These data indicate that the six terminal G _o protein amino acids do not constitute a major _2AAR interaction domain for G protein activation.     

Cell adhesive peptide screening of the mouse laminin α1 chain G domain

Cell adhesive peptides have been widely applied for therapeutic drugs, drug delivery systems, and biomaterials. Previously, we identified various cell adhesive sequences in the G domains of four laminin α chains (α2-α5) by the systematic soluble peptide screening. We also identified five cell-binding sequences in the laminin α1 chain G domain using synthetic peptide-polystyrene beads. Here, we re-screened cell adhesive peptides in the laminin α1 chain G domain by the systematic soluble peptides screening. The 110 soluble peptides were evaluated for their cell adhesive activities using human fibrosarcoma HT1080 cells and human dermal fibroblasts. Fourteen peptides were newly identified as a cell adhesive. Additionally, four peptides (AG22: SSFHFDGSGYAM, AG42: TFDLLRNSYGVRK, AG76: HQNQMDYATLQLQ, AG86: LGGLPSHYRARNI) promoted integrin-mediated cell adhesion. Further, neurite outgrowth activity with rat pheochromocytoma PC12 cells was evaluated and two peptides (AG20: SIGLWNYIEREGK, AG26: SPNGLLFYLASNG) were newly identified for neurite outgrowth activity. These results suggested that the systematic soluble peptides screening approach is an accurate and powerful strategy for finding biologically active sequences. The active sequences newly identified here could be involved in the biological functions of this domain. The active peptides are useful for evaluating molecular mechanisms of laminin-receptor interactions and for developing cell adhesive biomaterials.     

Differentiation of Human T Cells Alters Their Repertoire of G Protein α-Subunits

Because T cell differentiation leads to an expanded repertoire of chemokine receptors, a subgroup of G protein-coupled receptors, we hypothesized that the repertoire of G proteins might be altered in parallel. We analyzed the abundance of mRNA and/or protein of six G protein α-subunits in human CD4⁺ and CD8⁺ T cell subsets from blood. Although most G protein α-subunits were similarly expressed in all subsets, the abundance of Gα_o, a protein not previously described in hematopoietic cells, was much higher in memory versus naive cells. Consistent with these data, activation of naive CD4⁺ T cells in vitro significantly increased the abundance of Gα_o in cells stimulated under nonpolarizing or T_H17 (but not T_H1 or T_H2)-polarizing conditions. In functional studies, the use of a chimeric G protein α-subunit, Gα_qo5, demonstrated that chemokine receptors could couple to Gα_o-containing G proteins. We also found that Gα_i1, another α-subunit not described previously in leukocytes, was expressed in naive T cells but virtually absent from memory subsets. Corresponding to their patterns of expression, siRNA-mediated knockdown of Gα_o in memory (but not naive) and Gα_i1 in naive (but not memory) CD4⁺ T cells inhibited chemokine-dependent migration. Moreover, although even in Gα_o- and Gα_i1-expressing cells mRNAs of these α-subunits were much less abundant than Gα_i2 or Gα_i3, knockdown of any of these subunits impaired chemokine receptor-mediated migration similarly. Together, our data reveal a change in the repertoire of Gα_i/o subunits during T cell differentiation and suggest functional equivalence among Gα_i/o subunits irrespective of their relative abundance.     

Integrin αIIbβ3: A novel effector of Gα13

Under physiological conditions, circulating platelets are discoid in shape.¹ On these platelets, the fibrinogen receptor (integrin α_IIbβ₃) is in a low-affinity state, unable to bind soluble fibrinogen (Fg). Activation by agonists such as ADP and thrombin leads to a change in the conformation of the integrin α_IIbβ₃ through a process known as inside-out signaling. This enables the integrin to bind soluble Fg, which initiates a cascade of events referred to as outside-in signaling.² Outside-in signaling control processes, such as platelet spreading and clot retraction, by regulating small G-proteins such as RhoA, Rac and cdc42.Key words: platelets, integrin α_IIbβ₃, Galpha13, RhoA, clot retraction, thrombin, fibrinogenThe majority of the physiological platelet agonists (except collagen) induce inside-out signaling by binding to specific G-protein-coupled receptors (GPCRs). A G-protein plays a crucial role in translating the signal from GPCR to downstream effector molecules, ultimately leading to affinity modulation of integrin α_IIbβ₃. Platelets express nine Gα subunits; namely G_q, G_i1, G_i2, G_i3, G_z, G₁₂, G₁₃, G_s and G₁₆. Previous studies have shown that a small G-protein, RhoA, is activated by the G_12/13 family and plays a crucial role in calcium-independent platelet shape change.³ However, RhoA is also activated by α_IIbβ₃ and inhibits platelet spreading to trigger clot retraction.⁴ Recently, in a series of elegant experiments, Gong et al. have described the dynamic regulation of RhoA through a signaling crosstalk between Gα₁₃ and α_IIbβ₃.⁵By generating mice in which the platelets were depleted of Gα₁₃ using siRNA technology, Gong et al. investigated the role of Gα₁₃-mediated signaling on platelet spreading on immobilized Fg.⁵ The confocal images very clearly showed that, in the absence of Gα₁₃, platelets spread poorly on Fg, which was rescued by pretreatment with the Rho-kinase inhibitor Y27632, confirming previous findings that RhoA activated downstream of integrin α_IIbβ₃ inhibits platelet spreading. Interestingly, Gα₁₃-depleted platelets failed to activate c-Src but accelerated RhoA activation. From these observations, the authors infer that Gα₁₃ is important for integrin-mediated c-Src activation and RhoA inhibition, leading to increased cell spreading.⁵Since Gα₁₃ regulates integrin-mediated cell spreading and c-Src activation, Gong et al. examined the interaction of Gα₁₃ with α_IIbβ₃ using co-immunoprecipitation and GST pull-down assays.⁵ They found that the GTP-bound form of Gα₁₃ shows enhanced interaction with the integrin β₃ subunit. This interaction is required for the activation of c-Src and the inhibition of RhoA. However, they found that the inhibition of RhoA is transient. RhoA activation is suppressed for the first 15 min of platelet spreading, after which RhoA is activated. This initial suppression is rescued by blocking Gα₁₃ and β₃ cytoplasmic domain (β3-CD) interaction. Furthermore, they observed that RhoA activation parallels clot retraction.⁵ These findings indicate that Gα₁₃ is a key regulator of platelet spreading and clot retraction phenomena.According to Gong et al., thrombin-induced inside-out signaling through GPCR leads to GTP loading of Gα₁₃ (Fig. 1A). This GTP-bound Gα₁₃ interacts with integrin β3-CD of ligand-bound integrin, thus facilitating c-Src activation, which leads to platelet spreading. Blockade of the interaction between Gα₁₃ and β3-CD or cleavage of β3-CD by calpain results in clot retraction (Fig. 1B).Open in a separate windowFigure 1Schematic representation of the dynamic regulation of RhoA by Gα₁₃ during platelet activation. (A) Activation of platelets by thrombin receptors coupled to Gα₁₃ leads to the activation of RhoA, leading to platelet shape change. (B) The change in the conformation of integrin to a high-affinity form results in fibrinogen binding to α_IIbβ₃. Active Gα13 binds to the cytoplasmic domain of β₃ leading to the activation of c-Src, resulting in platelet spreading. The rise in intracellular calcium activates calpain, which cleaves the β₃ cytoplasmic domain, releasing c-Src, which, resulting in the activation of RhoA, leads to cell retraction. *Denotes GTP-bound active form of G-proteins.Perhaps the most significant and novel finding of the study is the identification of integrin α_IIbβ₃ as an effector of Gα₁₃. The study also convincingly shows that Gα₁₃ bound to integrin regulates RhoA via c-Src. Furthermore, achieving 80% knockdown of Gα₁₃ in an in vivo setting using siRNA represents a technological advancement. Since Gα₁₃ binds to integrin β3-CD in a 1:1 stoichiometry, it appears that only a small population of integrin is regulated by Gα₁₃, as there are far less Gα₁₃ molecules in a single platelet than the number of α_IIbβ₃ molecules. This will require further investigation. Gong et al. also finds that an appreciable amount of Gα₁₃ is associated with β₃ in resting platelets, which requires some explanation.⁵ It is also not clear if Gα₁₃ remains bound to β3-CD or dissociates from the integrin during clot retraction.Overall, this is a paradigm-shifting study that establishes the importance of the dynamic regulation of RhoA by Gα₁₃ in order to achieve efficient platelet spreading and clot retraction.     

Identification and Characterization of a G Protein-binding Cluster in α7 Nicotinic Acetylcholine Receptors

α7 nicotinic acetylcholine receptors (nAChRs) play an important role in synaptic transmission and inflammation. In response to ligands, this receptor channel opens to conduct cations into the cell but desensitizes rapidly. In recent studies we show that α7 nAChRs bind signaling proteins such as heterotrimeric GTP-binding proteins (G proteins). Here, we demonstrate that direct coupling of α7 nAChRs to G proteins enables a downstream calcium signaling response that can persist beyond the expected time course of channel activation. This process depends on a G protein-binding cluster (GPBC) in the M3-M4 loop of the receptor. A mutation of the GPBC in the α7 nAChR (α7_345–348A) abolishes interaction with Gα_q as well as Gβγ while having no effect on receptor synthesis, cell-surface trafficking, or α-bungarotoxin binding. Expression of α7_345–348A, however, did significantly attenuate the α7 nAChR-induced Gα_q calcium signaling response as evidenced by a decrease in PLC-β activation and IP₃R-mediated calcium store release in the presence of the α7 selective agonist choline. Taken together, the data provides new evidence for the existence of a GPBC in nAChRs serving to promote intracellular signaling.     

Backbone resonance assignments for G protein αi3 subunit in the GTP-bound state

Guanine-nucleotide binding proteins (G proteins) act as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of GDP-bound form of the G protein α subunit (Gα(GDP)) and G protein βγ subunit (Gβγ). Ligand binding to GPCRs promotes the GDP-GTP exchange on Gα, leading to the dissociation of the GTP-bound form of Gα (Gα(GTP)) and Gβγ. Then, Gα(GTP) and Gβγ bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Gα hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gβγ. G proteins are classified with four major families based on the amino acid sequences of Gα: i/o, s, q/11, and 12/13. Each family transduces the signaling from different GPCRs to the specific effectors. Here, we established the backbone resonance assignments of human Gα_i3, a member of the i/o family, with a molecular weight of 41 K in complex with a GTP analogue, GTPγS.     

Backbone resonance assignments for G protein αi3 subunit in the GDP-bound state

Guanine-nucleotide binding proteins (G proteins) serve as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of the G protein α subunit with GDP (Gα·GDP) and the G protein βγ subunit (Gβγ). Ligand binding to GPCRs promotes the GDP–GTP exchange on Gα, leading to the dissociation of the GTP-bound form of Gα (Gα·GTP) and Gβγ. Then, Gα·GTP and Gβγ bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Gα hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gβγ. The G proteins are classified with four major families based on the amino acid sequences of Gα: i/o, s, q/11, and 12/13. Here, we established the backbone resonance assignments of human Gα_i3, a member of the i/o family with a molecular weight of 41 K, in complex with GDP. The chemical shifts were compared with those of Gα_i3 in complex with a GTP-analogue, GTPγS, which we recently reported, indicating that the residues with significant chemical shift differences are mostly consistent with the regions with the structural differences between the GDP- and GTPγS-bound states, as indicated in the crystal structures. The assignments of Gα_i3·GDP would be useful for the analyses of the dynamics of Gα_i3 and its interactions with various target molecules.     

Synthesis of new α-heterocyclic α-aminoesters

alpha-Heterocyclic alpha-aminoesters were obtained in good yields by reaction of a glycine cation equivalent and different heterocyclic nucleophiles; diastereoselectivity using a carbohydrate (galactopyranose) as N-protecting group was modest.     

Familial hypertrophic cardiomyopathy related E180G mutation increases flexibility of human cardiac α-tropomyosin

α-Tropomyosin (αTm) is central to Ca²⁺-regulation of cardiac muscle contraction. The familial hypertrophic cardiomyopathy mutation αTm E180G enhances Ca²⁺-sensitivity in functional assays. To investigate the molecular basis, we imaged single molecules of human cardiac αTm E180G by direct probe atomic force microscopy. Analyses of tangent angles along molecular contours yielded persistence length corresponding to ∼35% increase in flexibility compared to wild-type. Increased flexibility of the mutant was confirmed by fitting end-to-end length distributions to the worm-like chain model. This marked increase in flexibility can significantly impact systolic and possibly diastolic phases of cardiac contraction, ultimately leading to hypertrophy.     

The α-arrestin ARRDC3 mediates ALIX ubiquitination and G protein–coupled receptor lysosomal sorting

The sorting of G protein–coupled receptors (GPCRs) to lysosomes is critical for proper signaling and cellular responses. We previously showed that the adaptor protein ALIX regulates lysosomal degradation of protease-activated receptor-1 (PAR1), a GPCR for thrombin, independent of ubiquitin-binding ESCRTs and receptor ubiquitination. However, the mechanisms that regulate ALIX function during PAR1 lysosomal sorting are not known. Here we show that the mammalian α-arrestin arrestin domain–containing protein-3 (ARRDC3) regulates ALIX function in GPCR sorting via ubiquitination. ARRDC3 colocalizes with ALIX and is required for PAR1 sorting at late endosomes and degradation. Depletion of ARRDC3 by small interfering RNA disrupts ALIX interaction with activated PAR1 and the CHMP4B ESCRT-III subunit, suggesting that ARRDC3 regulates ALIX activity. We found that ARRDC3 is required for ALIX ubiquitination induced by activation of PAR1. A screen of nine mammalian NEDD4-family E3 ubiquitin ligases revealed a critical role for WWP2. WWP2 interacts with ARRDC3 and not ALIX. Depletion of WWP2 inhibited ALIX ubiquitination and blocked ALIX interaction with activated PAR1 and CHMP4B. These findings demonstrate a new role for the α-arrestin ARRDC3 and the E3 ubiquitin ligase WWP2 in regulation of ALIX ubiquitination and lysosomal sorting of GPCRs.     

Immunohistochemical expression of G protein α-subunit isoforms in rat and monkey Merkel cell-neurite complexes

The true function of Merkel cells (MCs) is still enigmatic, though the localization of various kinds of neurotransmitter-like substances in MCs has been revealed by immunohistochemistry. Most of the neurotransmitters act on target cells via seven-transmembrane receptors coupled to heterotrimeric G proteins. The heterotrimeric G proteins include various subfamilies that contribute to different signal transduction pathways. Therefore investigation of specific types of G proteins in MCs and related axon terminals (MC-axon terminals) should contribute to the elucidation of the function of MCs. In this study, we investigated the expression patterns of alpha-subunit isoforms of G proteins in MC-neurite complexes of the rat and monkey by enzymatic and fluorescence immunohistochemistry. MC-axon terminals of the rat and monkey showed positive immunoreactions of Galphao and Galphai1. Those of the monkey also showed a weak immunoreaction of Galphas. On the other hand, MCs of both animals showed positive immunoreactions of Galphao, Galphai1, Galphaq, and Galphaz. In addition, MCs of the monkey showed weak immunoreactions of Galphas. Galphao- and Galphai1-like immunoreactions in the MC-axon terminals suggest that MCs suppressively regulate receptive functions of type I mechanosensory nerve terminals. On the other hand, the localization of Galpha-subunits in MCs suggests that these cells are regulated with hormones, neurotransmitter-like substances, or growth factors.