G-protein βγ subunits are positive regulators of Kv7.4 and native vascular Kv7 channel activity

Kv7.4 channels are a crucial determinant of arterial diameter both at rest and in response to endogenous vasodilators. However, nothing is known about the factors that ensure effective activity of these channels. We report that G-protein βγ subunits increase the amplitude and activation rate of whole-cell voltage-dependent K⁺ currents sensitive to the Kv7 blocker linopirdine in HEK cells heterologously expressing Kv7.4, and in rat renal artery myocytes. In excised patch recordings, Gβγ subunits (2–250 ng /mL) enhanced the open probability of Kv7.4 channels without changing unitary conductance. Kv7 channel activity was also augmented by stimulation of G-protein–coupled receptors. Gallein, an inhibitor of Gβγ subunits, prevented these stimulatory effects. Moreover, gallein and two other structurally different Gβγ subunit inhibitors (GRK2i and a β-subunit antibody) abolished Kv7 channel currents in the absence of either Gβγ subunit enrichment or G-protein–coupled receptor stimulation. Proximity ligation assay revealed that Kv7.4 and Gβγ subunits colocalized in HEK cells and renal artery smooth muscle cells. Gallein disrupted this colocalization, contracted whole renal arteries to a similar degree as the Kv7 inhibitor linopirdine, and impaired isoproterenol-induced relaxations. Furthermore, mSIRK, which disassociates Gβγ subunits from α subunits without stimulating nucleotide exchange, relaxed precontracted arteries in a linopirdine-sensitive manner. These results reveal that Gβγ subunits are fundamental for Kv7.4 activation and crucial for vascular Kv7 channel activity, which has major consequences for the regulation of arterial tone.Increased arterial constriction and lack of responsiveness to endogenous vasodilators is a hallmark of vascular disease leading to poor health prognosis. Defining the factors that determine vascular smooth muscle (VSM) activity and modulation by vasorelaxant molecules is therefore imperative for a better understanding of vascular disease. Potassium channels are key regulators of VSM tone because they promote membrane hyperpolarization that limits the activity of voltage-dependent calcium channels known to precipitate vasoconstriction (1). The Kv7 family of voltage-dependent potassium channels and the Kv7.4 isoform, in particular, has a fundamental role in this process. There are five Kv7 isoforms (Kv7.1–Kv7.5) of which Kv7.1, Kv7.4, and Kv7.5 are consistently expressed within VSM, where the predominant molecular architecture is a Kv7.4/Kv7.5 heterotetramer (2, 3). Activation of Kv7 channels produces relaxation of numerous arteries (4–8), whereas blockade of Kv7 channels results in contraction of vessels at rest (7, 9–11) or an inhibition of endogenously derived vasorelaxations (2, 11–13). In addition, molecular reduction of Kv7.4 reduces responses to various Gs-coupled vasodilators in a number of arteries (2, 11). Crucially, Kv7.4 abundance is reduced in various arteries from hypertensive animals (6, 11, 12) where relaxant responses to endogenous vasodilators are also impaired (11, 12). Despite the key role of Kv7.4 channels in the regulation of VSM, and their involvement in mediating Gs-coupled vasodilator responses, the factors that regulate channel activity are poorly understood, and the signals linking Kv7.4 to Gs-receptor activation remain to be elucidated.G-protein–coupled receptor (GPCR) activation promotes the exchange of GDP for GTP resulting in disassociation of the heterotrimeric Gαβγ complex from the receptor into Gα-GTP and Gβγ (14). It is now established that the Gβγ complex as well as the Gα–GTP activates various intracellular signaling pathways (see refs. 15, 16 for reviews). Gβγ subunits also modulate various ion channels directly, a phenomenon of which there are only a handful of examples, with the positive regulation of an inwardly rectifying K⁺ channel in the heart the best characterized (17, 18). In this study, we explored whether Gβγ subunits modulated Kv7.4 channels and therefore function as signaling intermediates following receptor stimulation. Our results show that not only are Gβγ subunits able to enhance Kv7 channels, but also that they are a crucial requirement for the basal activity of the Kv7.4 channel.     

Molecular architecture of the αβ T cell receptor–CD3 complex

αβ T-cell receptor (TCR) activation plays a crucial role for T-cell function. However, the TCR itself does not possess signaling domains. Instead, the TCR is noncovalently coupled to a conserved multisubunit signaling apparatus, the CD3 complex, that comprises the CD3εγ, CD3εδ, and CD3ζζ dimers. How antigen ligation by the TCR triggers CD3 activation and what structural role the CD3 extracellular domains (ECDs) play in the assembled TCR–CD3 complex remain unclear. Here, we use two complementary structural approaches to gain insight into the overall organization of the TCR–CD3 complex. Small-angle X-ray scattering of the soluble TCR–CD3εδ complex reveals the CD3εδ ECDs to sit underneath the TCR α-chain. The observed arrangement is consistent with EM images of the entire TCR–CD3 integral membrane complex, in which the CD3εδ and CD3εγ subunits were situated underneath the TCR α-chain and TCR β-chain, respectively. Interestingly, the TCR–CD3 transmembrane complex bound to peptide–MHC is a dimer in which two TCRs project outward from a central core composed of the CD3 ECDs and the TCR and CD3 transmembrane domains. This arrangement suggests a potential ligand-dependent dimerization mechanism for TCR signaling. Collectively, our data advance our understanding of the molecular organization of the TCR–CD3 complex, and provides a conceptual framework for the TCR activation mechanism.T cells are key mediators of the adaptive immune response. Each αβ T cell contains a unique αβ T-cell receptor (TCR), which binds antigens (Ags) displayed by major histocompatibility complexes (MHCs) and MHC-like molecules (1). The TCR serves as a remarkably sensitive driver of cellular function: although TCR ligands typically bind quite weakly (1–200 μM), even a handful of TCR ligands are sufficient to fully activate a T cell (2, 3). The TCR does not possess intracellular signaling domains, uncoupling Ag recognition from T-cell signaling. The TCR is instead noncovalently associated with a multisubunit signaling apparatus, consisting of the CD3εγ and CD3εδ heterodimers and the CD3ζζ homodimer, which collectively form the TCR–CD3 complex (4, 5). The CD3γ/δ/ε subunits each consist of a single extracellular Ig domain and a single immunoreceptor tyrosine-based activation motif (ITAM), whereas CD3ζ has a short extracellular domain (ECD) and three ITAMs (6–11). The TCR–CD3 complex exists in 1:1:1:1 stoichiometry for the αβTCR:CD3εγ:CD3εδ:CD3ζζ dimers (12). Phosphorylation of the intracellular CD3 ITAMs and recruitment of the adaptor Nck lead to T-cell activation, proliferation, and survival (13, 14). Understanding the underlying principles of TCR–CD3 architecture and T-cell signaling is of therapeutic interest. For example, TCR–CD3 is the target of therapeutic antibodies such as the immunosuppressant OKT3 (15), and there is increasing interest in manipulating T cells in an Ag-dependent manner by using naturally occurring and engineered TCRs (16).Assembly of the TCR–CD3 complex is primarily driven by each protein’s transmembrane (TM) region, enforced through the interaction of evolutionarily conserved, charged, residues in each TM region (4, 5, 12). What, if any, role interactions between TCR and CD3 ECDs play in the assembly and function of the complex remains controversial (5): there are several plausible proposed models of activation, which are not necessarily mutually exclusive (5, 17–19). Although structures of TCR–peptide–MHC (pMHC) complexes (2), TCR–MHC-I–like complexes (1), and the CD3 dimers (6–10) have been separately determined, how the αβ TCR associates with the CD3 complex is largely unknown. Here, we use two independent structural approaches to gain an understanding of the TCR–CD3 complex organization and structure.     

αIIbβ3 variants defined by next-generation sequencing: Predicting variants likely to cause Glanzmann thrombasthenia

Next-generation sequencing is transforming our understanding of human genetic variation but assessing the functional impact of novel variants presents challenges. We analyzed missense variants in the integrin αIIbβ3 receptor subunit genes ITGA2B and ITGB3 identified by whole-exome or -genome sequencing in the ThromboGenomics project, comprising ∼32,000 alleles from 16,108 individuals. We analyzed the results in comparison with 111 missense variants in these genes previously reported as being associated with Glanzmann thrombasthenia (GT), 20 associated with alloimmune thrombocytopenia, and 5 associated with aniso/macrothrombocytopenia. We identified 114 novel missense variants in ITGA2B (affecting ∼11% of the amino acids) and 68 novel missense variants in ITGB3 (affecting ∼9% of the amino acids). Of the variants, 96% had minor allele frequencies (MAF) < 0.1%, indicating their rarity. Based on sequence conservation, MAF, and location on a complete model of αIIbβ3, we selected three novel variants that affect amino acids previously associated with GT for expression in HEK293 cells. αIIb P176H and β3 C547G severely reduced αIIbβ3 expression, whereas αIIb P943A partially reduced αIIbβ3 expression and had no effect on fibrinogen binding. We used receiver operating characteristic curves of combined annotation-dependent depletion, Polyphen 2-HDIV, and sorting intolerant from tolerant to estimate the percentage of novel variants likely to be deleterious. At optimal cut-off values, which had 69–98% sensitivity in detecting GT mutations, between 27% and 71% of the novel αIIb or β3 missense variants were predicted to be deleterious. Our data have implications for understanding the evolutionary pressure on αIIbβ3 and highlight the challenges in predicting the clinical significance of novel missense variants.Next-generation sequencing is transforming our understanding of human genetic variation (1) and providing profound insights into the impact of both inherited and de novo variants on human health (2, 3). At the same time, the data from these studies present serious challenges in providing information to individuals who are found to have variant forms of different proteins. To highlight these challenges, in this report we describe our experience in analyzing missense variants of the platelet αIIbβ3 integrin receptor from The Human Genome Mutation Database (HGMD), the 1000 Genomes project (1000G), the United Kingdom 10K Whole Exome Sequencing project (U.K.10KWES), the United Kingdom 10K Whole Genome Sequencing project (U.K.10KWGS), and The National Heart, Lung and Blood Institute Exome Sequencing Project (ESP); the latter four sources encompass ∼32,000 alleles derived from 16,108 individuals.The αIIbβ3 receptor has a number of virtues as a model system. First, it is required for hemostasis because platelet aggregation requires cross-linking of the activated form of αIIbβ3 by macromolecular ligands (4). Thus, defects in its biogenesis, activation, or ligand binding lead to the rare bleeding diathesis Glanzmann thrombasthenia (GT), an autosomal recessive disorder (5, 6). Patients with GT come to medical attention because of their hemorrhagic symptoms, and thus have been carefully analyzed clinically and with tests of platelet function for nearly 50 y (5, 7). The biochemical and molecular abnormalities in GT have been studied for nearly 40 y (4, 6, 8–10). In the past 10 y, high-resolution crystallography, electron microscopy, and computational studies of the αIIbβ3 receptor have provided atomic-level information on the correlation between receptor structure and function (11–21). In addition, ethnic groups with relatively high prevalence of GT have been defined that share the same genetic abnormality based on founder mutations, and the dates that some of the mutations entered the population have been estimated (22–28). An on-line registry of GT abnormalities, including patient phenotypes was developed in 1997 (29) and currently contains 51 αIIb and 43 β3 missense variants linked to the disorder (sinaicentral.mssm.edu/intranet/research/glanzmann). The frequency of GT in the general population has not been established but it has a world-wide distribution, and based on data from hematologic practices, it is rare except in areas with a high rate of consanguineous mating (30).Second, alloimmune disorders, including neonatal thrombocytopenia and posttransfusion purpura, due to amino acid substitutions in either αIIb or β3, have been characterized at the molecular biological level and correlated with mechanisms of immunologic recognition (31).Third, inherited macrothrombocytopenia and anisothrombocytopenia have been associated with heterozygous missense variants or deletions in αIIb or β3. All of these appear to induce constitutive activation of the receptor and impair proplatelet formation (32–38).Fourth, αIIbβ3 contributes to pathological platelet thrombus formation in human ischemic cardiovascular disease and αIIbβ3 is a validated target for antithrombotic therapy (39–41).Fifth, αIIbβ3 is a member of the large integrin family of receptors, which includes 24 receptors derived from 18 α- and 8 β-subunits (41, 42). These receptors are involved in important biologic processes, including development, cell migration, homing, cell survival, and adaptive immunity (41–43). More is known about the structure–function relationships of αIIbβ3 than the other members of the group, and so it serves as the paradigmatic integrin receptor (44, 45).Sixth, 3D molecular models have been built based on crystallographic and NMR data to analyze the effects of novel amino acid substitutions on receptor structure and function and the generation of alloantigens (15, 46–53). The data from these models and assessments of the severity of the amino acid change in the variants have the potential to aid in predicting whether a novel variant is likely to affect receptor function and immunogenicity (54–59).     

Oncogenic activity of the regulatory subunit p85β of phosphatidylinositol 3-kinase (PI3K)

Expression of the regulatory subunit p85β of PI3K induces oncogenic transformation of primary avian fibroblasts. The transformed cells proliferate at an increased rate compared with nontransformed controls and show elevated levels of PI3K signaling. The oncogenic activity of p85β requires an active PI3K-TOR signaling cascade and is mediated by the p110α and p110β isoforms of the PI3K catalytic subunit. The data suggest that p85β is a less effective inhibitor of the PI3K catalytic subunit than p85α and that this reduced level of p110 inhibition accounts for the oncogenic activity of p85β.Class IA PI3Ks (phosphatidylinositol 3-kinase) are dimeric enzymes consisting of a catalytic subunit and a regulatory subunit. The two major regulatory subunits are p85α and p85β (1). They stabilize and inhibit the catalytic subunit p110 by domain-specific interactions (2–6). The p85α and p85β proteins share core functions, but also display unique activities (7–10). Both p85α and p85β are found mutated in several cancers. These mutants show oncogenic activity; most of the p85α mutations disrupt inhibitory interactions between p85 and p110 or destabilize PTEN (phosphatase and tensin homolog) and result in increased PI3K signaling (5, 11–14). The molecular mechanisms by which p85β mutations activate PI3K signaling have not been fully explored. Elevated expression of wild-type p85β is found in several cancers, and in an experimental setting drives tumor progression (15). A recent study has revealed a role of p85β in the formation of invadopodia with possible effects on metastatic cellular behavior (16). Here we show that expression of p85β induces cellular transformation of primary fibroblasts, increased cell proliferation and elevated PI3K signaling. The oncogenic activity of p85β depends on active PI3K and TOR (target of rapamycin) signaling and is mediated by two PI3K catalytic isoforms, p110α and p110β. Our data are compatible with the conclusion that p85β exerts a reduced inhibitory activity on p110 compared with p85α.     

Neurosteroids promote phosphorylation and membrane insertion of extrasynaptic GABAA receptors

Neurosteroids are synthesized within the brain and act as endogenous anxiolytic, anticonvulsant, hypnotic, and sedative agents, actions that are principally mediated via their ability to potentiate phasic and tonic inhibitory neurotransmission mediated by γ-aminobutyric acid type A receptors (GABA_ARs). Although neurosteroids are accepted allosteric modulators of GABA_ARs, here we reveal they exert sustained effects on GABAergic inhibition by selectively enhancing the trafficking of GABA_ARs that mediate tonic inhibition. We demonstrate that neurosteroids potentiate the protein kinase C-dependent phosphorylation of S443 within α4 subunits, a component of GABA_AR subtypes that mediate tonic inhibition in many brain regions. This process enhances insertion of α4 subunit-containing GABA_AR subtypes into the membrane, resulting in a selective and sustained elevation in the efficacy of tonic inhibition. Therefore, the ability of neurosteroids to modulate the phosphorylation and membrane insertion of α4 subunit-containing GABA_ARs may underlie the profound effects these endogenous signaling molecules have on neuronal excitability and behavior.Neurosteroids are synthesized de novo in the brain from cholesterol, or steroid hormone precursors. Raising neurosteroid levels in the CNS causes anxiolysis, sedation/hypnosis, anticonvulsant action, and anesthesia and reduces depressive-like behaviors (1–3). Accordingly, dysregulation of neurosteroid signaling is associated with premenstrual dysphoric disorder, panic disorder, depression, schizophrenia, and bipolar disorder. Neurosteroids exert the majority of their actions via potentiating the activity of γ-aminobutyric acid receptors (GABA_ARs), which mediate the majority of fast synaptic inhibition in the adult brain. Accordingly, at low nanomolar concentrations they potentiate GABA-dependent currents, whereas at micromolar concentrations they directly activate GABA_ARs (4–8).GABA_ARs are Cl⁻-preferring pentameric ligand-gated ion channels that assemble from eight families of subunits: α(1–6), β(1–3), γ(1–3), δ, ε, ө, π, and ρ(1–3) (9, 10). Receptor subtypes composed of α1–3βγ subunits largely mediate synaptic or phasic inhibition, whereas those constructed from α4–6β1–3, with or without γ/δ subunits, are principal determinants of tonic inhibition (11–13). Neurosteroids have been shown to bind GABA_ARs at an allosteric site distinct from that of GABA, benzodiazepines, or barbiturates (9, 14). Hosie et al. identified residues located within the transmembrane domain of GABA_AR α and β subunits that are critical for the direct activation (α1–6; Threonine 236, β1–3; Tyrosine 284) and allosteric potentiation (α1–6 Asparagine 407, and α1–6 Glutamine 246) of neurosteroids (15–17). Accordingly, mutation of glutamine 241 (Q241) within the α1–6 subunits prevents allosteric potentiation of GABA_AR composed of αβγ and αβδ subunits by neurosteroids (15, 16).In addition to modulating channel gating, neurosteroids exert potent effects on the expression levels of GABA_ARs (1, 18–20). Moreover, in the hippocampus, prolonged exposure to physiological concentrations of neurosteroids has been shown to enhance the tonic conductance mediated by extrasynaptic GABA_ARs containing the α4/δ subunits, while having little effect on the phasic conductance mediated by synaptic GABA_ARs (6, 21). However, the molecular mechanisms by which neurosteroids regulate GABA_AR expression levels remain unknown.Here, we reveal that neurosteroids act to increase the PKC-dependent phosphorylation of serine 443 (S443) within the intracellular domain of the α4 subunit. This process leads to increased insertion of α4 subunit-containing GABA_ARs into the plasma membrane and a selective enhancement of tonic inhibition. Thus, our experiments reveal a previously unidentified molecular mechanism by which neurosteroids exert sustained effects on GABAergic inhibition by selectively increasing α4-containing GABA_ARs in the membrane and therefore potentiate tonic inhibition.     

Apoptotic pore formation is associated with in-plane insertion of Bak or Bax central helices into the mitochondrial outer membrane

The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores.Commitment of cells to apoptosis is determined primarily by interactions within the B-cell lymphoma-2 (Bcl-2) protein family on the mitochondrial outer membrane (MOM) (1–4). The proapoptotic members Bcl-2 antagonist/killer (Bak) and Bcl-2–associated X protein (Bax) mediate the pivotal step of MOM permeabilization, which releases proteins, such as cytochrome c, that promote the proteolytic demolition by caspases. Two other Bcl-2 subfamilies tightly control Bak and Bax activation. Their activation is promoted by the Bcl-2 homology domain 3 (BH3)-only proteins, such as BH3-interacting domain death agonist (Bid), the truncated form of which (tBid) can directly bind both. Conversely, prosurvival family members can bind and inhibit activated Bak and Bax, as well as the BH3-only proteins.Like their prosurvival relatives, Bak and Bax in healthy cells are globular monomers, comprising similar helical bundles with a hydrophobic α-helix (α5) surrounded by amphipathic helices (5, 6). Their C-terminal helix (α9) is a hydrophobic transmembrane (TM) domain that anchors them in the MOM. In healthy cells Bak is already anchored there, presumably solely by α9, whereas Bax is primarily cytosolic (5), accumulating at the MOM after an apoptotic signal and inserting its α9. Other major conformational changes in both Bak and Bax, reviewed in ref 4, include exposure of their BH3 (α2) and its reburial within the surface groove of another activated Bak or Bax molecule (7–10). These novel “symmetric” homo-dimers can multimerize via association of α6 helices (8, 11, 12).Although oligomeric Bak and Bax are highly implicated in MOM permeabilization, how they interact with the membrane to form pores remains a mystery. The first structure of a Bcl-2 family member, the prosurvival protein Bcl-x_L (13), and later those of Bax (5) and Bak (6), provided a tantalizing clue: similarities with the pore-forming domains of bacterial toxins, such as diphtheria toxin or colicin A. To form pores, these toxins are thought to insert their two hydrophobic core helices as a hairpin across the membrane (14), suggesting that the central helices of Bak and Bax (α5 and α6) might penetrate the MOM similarly (reviewed in refs 3, 15, and 16). Consistent with this hairpin insertion model, α5 and α6 peptides can permeabilize membranes (17–19). More pertinently, Bax α5 and α6 were reported to insert into and span the MOM as a hairpin before oligomerization (20).This longstanding model, however, does not fit well with recent evidence on the structure of Bak and Bax oligomers, as recently reviewed (2, 4). Analysis of Bak oligomers in liposomes by electron paramagnetic resonance (EPR) suggests that α6 inserts only shallowly in the lipid bilayer (21). Additionaly, the first 3D structures of activated forms of Bax (10) suggest that, early in its activation, α5 and α6 separate. Moreover, a Bax core domain containing only helices α2 to α5 generated a BH3:groove symmetric dimer in which two α4 and two α5 helices form an aromatic face that might sit on the bilayer (10). These findings fit better with an “in-plane model” in which only α9 is a TM domain and other helices (including α5 and α6) insert only shallowly into the bilayer.The nature of the apoptotic pores remains uncertain. Some findings favor a proteinaceous pore (22), but studies with model membranes suggest that Bax oligomers can perturb the bilayer and produce lipidic pores (i.e., pores not bounded entirely by protein) (23–26).These important unresolved questions about the pivotal event in apoptosis prompted us to explore the membrane topology of Bak, before, during, and after an apoptotic signal, and to reinvestigate that of Bax. In accord with recent Bax structures (10) and recent EPR studies on Bak (21, 27), the results show that neither oligomeric Bak nor Bax inserts an α5–α6 hairpin across the MOM. We propose instead that the α5 and α6 helices lie in the bilayer plane and disrupt membrane integrity by imposing tension and curvature to the membrane that provoke its permeabilization.     

From the Cover: PNAS Plus: Nicastrin functions to sterically hinder γ-secretase–substrate interactions driven by substrate transmembrane domain

γ-Secretase is an intramembrane-cleaving protease that processes many type-I integral membrane proteins within the lipid bilayer, an event preceded by shedding of most of the substrate’s ectodomain by α- or β-secretases. The mechanism by which γ-secretase selectively recognizes and recruits ectodomain-shed substrates for catalysis remains unclear. In contrast to previous reports that substrate is actively recruited for catalysis when its remaining short ectodomain interacts with the nicastrin component of γ-secretase, we find that substrate ectodomain is entirely dispensable for cleavage. Instead, γ-secretase–substrate binding is driven by an apparent tight-binding interaction derived from substrate transmembrane domain, a mechanism in stark contrast to rhomboid—another family of intramembrane-cleaving proteases. Disruption of the nicastrin fold allows for more efficient cleavage of substrates retaining longer ectodomains, indicating that nicastrin actively excludes larger substrates through steric hindrance, thus serving as a molecular gatekeeper for substrate binding and catalysis.Regulated intramembrane proteolysis (RIP) involves the cleavage of a wide variety of integral membrane proteins within their transmembrane domains (TMDs) by a highly diverse family of intramembrane-cleaving proteases (I-CLiPs) (1). I-CLiPs are found in all forms of life and govern many important biological functions, including but not limited to organism development (2), lipid homeostasis (3), the unfolded protein response (4), and bacterial quorum sensing (5). As the name implies, RIP must be tightly regulated to ensure that the resultant signaling events occur only when prompted by the cell and to prevent cleavage of the many nonsubstrate “bystander” proteins present within cellular membranes. Despite this, very little is known about the molecular mechanisms by which I-CLiPs achieve their exquisite specificity. Although traditional soluble proteases maintain substrate specificity by recognizing distinct amino acid sequences flanking the scissile bond, substrates for intramembrane proteases have little to no sequence similarity.Recent work on rhomboid proteases has demonstrated that this family of I-CLiPs achieves substrate specificity via a mechanism that is dependent on the transmembrane dynamics of the substrate rather than its sequence of amino acids (6, 7). Here, rhomboid possesses a very weak binding affinity for substrate and, in a rate-driven reaction, only cleaves those substrates that have unstable TMD helices that have had time to unfold into the catalytic active site, where they are cleaved before they can dissociate from the enzyme–substrate complex. Although it may be tempting to speculate that this is a conserved mechanism for all I-CLiPs, rhomboid is the only family of I-CLiPs that does not require prior activation of substrate through an initial cleavage by another protease (8). Specifically, site-2 protease substrates must be first cleaved by site-1 protease (9), signal peptide peptidase substrates are first cleaved by signal peptidase (10), and ectodomain shedding by α- or β-secretase is required before γ-secretase cleavage of its substrates (11, 12). These facts suggest that the diverse families of I-CLiPs likely have evolved fundamentally different mechanisms by which they recognize and cleave their substrates.Presenilin/γ-secretase is the founding member of the aspartyl family of I-CLiPs. The importance of γ-secretase function in biology and medicine is highlighted by its cleavage of the notch family of receptors, which is required for cell fate determination in all metazoans (2, 13–16), and of the amyloid precursor protein (APP), which is centrally implicated in Alzheimer’s disease (AD) (14, 17). In addition to APP and notch, γ-secretase has over 90 other reported substrates, many of which are involved in important signaling events (12, 18). Despite this, little is known about the mechanism by which γ-secretase binds and cleaves its substrates. Currently, the only known prerequisite for a substrate to be bound and hydrolyzed by γ-secretase is that it be a type-I integral membrane protein that first has most of its ectodomain removed by a sheddase, either α- or β-secretases (11, 12, 19). How γ-secretase selectively recognizes ectodomain-shed substrates and recruits them for catalysis while at the same time preventing cleavage of nonsubstrates remains unsettled.γ-Secretase is a multimeric complex composed of four integral membrane proteins both necessary and sufficient for full activity: presenilin, nicastrin, Aph-1, and Pen-2 (20–24). Presenilin is the proteolytic component, housing catalytic aspartates on TMDs 6 and 7 of its nine TMDs (17, 25, 26). After initial complex formation, the mature proteolytically active complex is formed when presenilin undergoes auto-proteolysis, resulting in N- and C-terminal fragments (NTF and CTF, respectively) (17, 27, 28), a process thought to be stimulated by the three-TMD component Pen-2 (29). The seven-TMD protein Aph-1 is believed to play a scaffolding role in complex formation (30, 31). Nicastrin is a type-I integral membrane protein with a large, heavily glycosylated ectodomain (32–34) that contains multiple stabilizing disulfide bridges (24, 34).The ectodomain of nicastrin is structurally homologous to a bacterial amino peptidase (34). Although nicastrin lacks the specific amino acids required for peptidase activity, it has been proposed to bind the N terminus of ectodomain-shed substrate, thereby directing substrate TMD to the γ-secretase active site for cleavage (35, 36). This mechanism has been suggested to depend on a key binding interaction between the free amine at the N terminus of the shortened substrate ectodomain and E333 of the vestigial amino peptidase domain of nicastrin (35, 36). However, the importance of nicastrin in substrate recognition has been questioned (37, 38), and although an initial high-resolution structure of γ-secretase suggested a role for nicastrin in substrate recognition (24), the most recent structures of the γ-secretase complex and the nicastrin ectodomain reveal that E333 is actually buried within the interior of nicastrin and resides on the opposite side of the complex relative to the active site (39, 40). Although this makes it unlikely that nicastrin is involved in direct substrate binding barring a large, energy-intensive conformational change, the basic mechanism of substrate recognition by γ-secretase remains controversial and requires resolution.Here, we demonstrate that nicastrin functions to sterically exclude substrates based on ectodomain size rather than actively recruit them for catalysis. This blocking mechanism allows γ-secretase to distinguish substrate from nonsubstrate and explains why substrate ectodomain shedding by α- or β-secretases is a prerequisite for γ-secretase catalysis. In contrast to rhomboid, γ-secretase apparently binds substrate TMD tightly, making the nicastrin steric hindrance mechanism necessary to prevent cleavage of nonectodomain-shed substrates and nonsubstrates alike.     

PP2A methylation controls sensitivity and resistance to β-amyloid–induced cognitive and electrophysiological impairments

Elevated levels of the β-amyloid peptide (Aβ) are thought to contribute to cognitive and behavioral impairments observed in Alzheimer’s disease (AD). Protein phosphatase 2A (PP2A) participates in multiple molecular pathways implicated in AD, and its expression and activity are reduced in postmortem brains of AD patients. PP2A is regulated by protein methylation, and impaired PP2A methylation is thought to contribute to increased AD risk in hyperhomocysteinemic individuals. To examine further the link between PP2A and AD, we generated transgenic mice that overexpress the PP2A methylesterase, protein phosphatase methylesterase-1 (PME-1), or the PP2A methyltransferase, leucine carboxyl methyltransferase-1 (LCMT-1), and examined the sensitivity of these animals to behavioral and electrophysiological impairments caused by exogenous Aβ exposure. We found that PME-1 overexpression enhanced these impairments, whereas LCMT-1 overexpression protected against Aβ-induced impairments. Neither transgene affected Aβ production or the electrophysiological response to low concentrations of Aβ, suggesting that these manipulations selectively affect the pathological response to elevated Aβ levels. Together these data identify a molecular mechanism linking PP2A to the development of AD-related cognitive impairments that might be therapeutically exploited to target selectively the pathological effects caused by elevated Aβ levels in AD patients.Multiple observations suggest a role for the serine/threonine protein phosphatase 2A (PP2A) in the molecular pathways that underlie Alzheimer’s disease (AD). Analyses conducted on postmortem AD brains have found reduced PP2A expression and activity, and studies conducted in animal models have found that inhibiting PP2A produces AD-like tau pathology and cognitive impairment (1–3). One of the ways in which PP2A may affect AD is through its role as the principal tau phosphatase (4–7). PP2A also interacts with a number of kinases implicated in AD including glycogen synthase kinase 3β (GSK3β), cyclin-dependent kinase 5 (CDK5), and ERK and JNK as well as amyloid precursor protein and the NMDA and metabotropic glutamate receptors (reviewed in ref. 2).PP2A is a heterotrimeric protein composed of a catalytic, scaffolding, and regulatory subunit. Each subunit is encoded by multiple genes and splice isoforms, and the subunit composition of a particular PP2A molecule determines its subcellular distribution and substrate specificity (reviewed in ref. 2). One of the ways in which PP2A activity is regulated is through C-terminal methylation of the catalytic subunit (reviewed in refs. 8 and 9). Impaired methyl-donor metabolism is a risk factor for AD (10, 11), and PP2A dysregulation caused by impaired methylation is thought to be one of the molecular mechanisms contributing to this increased risk (12–14). Methylation promotes the formation of PP2A holoenzymes that contain Bα regulatory subunits (7, 13, 15–19), and these forms of PP2A exhibit the greatest tau phosphatase activity (6, 7).PP2A methylation is catalyzed in vivo by the methyl transferase, leucine carboxyl methyltransferase 1 (LCMT-1) (20–22), and its demethylation is catalyzed by the methylesterase, protein phosphatase methylesterase 1 (PME-1) (23–25). To explore the role of PP2A in AD further, we generated lines of transgenic mice that overexpress these enzymes and tested their effect on the sensitivity of animals to electrophysiogical and behavioral impairments caused by β-amyloid (Aβ). We found that LCMT-1 overexpression protected animals from Aβ-induced impairments, whereas overexpression of PME-1 worsened Aβ neurotoxicity. Neither transgene affected endogenous Aβ levels, suggesting that they acted by altering the response to Aβ rather than Aβ production. We also found that PME-1 and LCMT-1 overexpression were without effect on the electrophysiological response to picomolar Aβ application, suggesting that they selectively affected the response to pathological Aβ concentrations. Together these data indicate that this pathway has potential as a therapeutic avenue for AD that acts not by targeting Aβ production but by selectively altering the response to pathological levels of Aβ.     

Intramuscular injection of α-synuclein induces CNS α-synuclein pathology and a rapid-onset motor phenotype in transgenic mice

It has been hypothesized that α-synuclein (αS) misfolding may begin in peripheral nerves and spread to the central nervous system (CNS), leading to Parkinson disease and related disorders. Although recent data suggest that αS pathology can spread within the mouse brain, there is no direct evidence for spread of disease from a peripheral site. In the present study, we show that hind limb intramuscular (IM) injection of αS can induce pathology in the CNS in the human Ala53Thr (M83) and wild-type (M20) αS transgenic (Tg) mouse models. Within 2–3 mo after IM injection in αS homozygous M83 Tg mice and 3–4 mo for hemizygous M83 Tg mice, these animals developed a rapid, synchronized, and predictable induction of widespread CNS αS inclusion pathology, accompanied by astrogliosis, microgliosis, and debilitating motor impairments. In M20 Tg mice, starting at 4 mo after IM injection, we observed αS inclusion pathology in the spinal cord, but motor function remained intact. Transection of the sciatic nerve in the M83 Tg mice significantly delayed the appearance of CNS pathology and motor symptoms, demonstrating the involvement of retrograde transport in inducing αS CNS inclusion pathology. Outside of scrapie-mediated prion disease, to our knowledge, this findiing is the first evidence that an entire neurodegenerative proteinopathy associated with a robust, lethal motor phenotype can be initiated by peripheral inoculation with a pathogenic protein. Furthermore, this facile, synchronized rapid-onset model of α-synucleinopathy will be highly valuable in testing disease-modifying therapies and dissecting the mechanism(s) that drive αS-induced neurodegeneration.Synucleinopathies are a group of diseases defined by the presence of amyloidogenic α-synuclein (αS) inclusions that can occur in neurons and glia of the central nervous system (CNS) (1–4). In Parkinson disease (PD), a causative role for αS has been established via the discovery of mutations in the αS gene SNCA resulting in autosomal-dominant PD (4–11). Although αS inclusions (e.g., Lewy bodies) are the hallmark pathology of PD, how they contribute to disease pathogenesis remains controversial (1, 3, 4, 12).Postmortem studies have suggested that αS pathology may spread following neuroanatomical tracts (13–15) and between cells (16–18). αS pathology has also been found in the peripheral nervous system (PNS): for example, in the enteric and pelvic plexus (19, 20). And it has been suggested that αS pathology might originate in the nerves of the PNS and spread to the CNS (14). Experimentally, it has been reported that intracerebral injections of preformed amyloidogenic αS fibrils in nontransgenic (nTg) and αS transgenic (Tg) mice induce the formation of intracellular αS inclusions that appear to progress from the site of injection (21–26). Collectively, these studies support the notion that αS inclusion pathology may propagate via a prion-like conformational self-templating mechanism (27, 28). A caveat of the direct intracerebral injection of αS is that this CNS invasive surgical procedure directly alters brain homeostasis that could influence or facilitate the formation of brain pathologies, especially because incidents such as traumatic brain injury can promote the formation of αS pathology (29). Here, we report that the intramuscular (IM) injection of fibrillar (fib) αS in M83 Tg mice expressing human Ala53Thr (A53T) αS can result in the rapid and synchronized development of hind limb motor weakness and robust widespread CNS αS pathology. Additionally, similar injection into M20 Tg mice expressing human wild-type αS, which do not intrinsically develop pathology, leads to the induction of CNS αS pathology as early as 4 mo postinjection.     

Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels

Being activated by depolarizing voltages and increases in cytoplasmic Ca²⁺, voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.High-conductance voltage- and calcium-activated potassium (BK) channels are homotetrameric proteins of α-subunits encoded by the slo1 gene (1). These channels are expressed in virtually all mammalian tissues, where they detect and integrate membrane voltage and calcium concentration changes dampening the responsiveness of cells when confronted with excitatory stimuli. They are abundant in the CNS and nonneuronal tissues, such as smooth muscle or hair cells. This wide distribution is associated with an outstandingly large functional diversity, in which BK channel activity appears optimally adapted to the particular physiological demands of each cell type (2). On the other hand, small alterations in BK channel function may contribute to the pathophysiology of hypertension, asthma, cancer, epilepsy, diabetes, and other conditions in humans (3–8). Alternative splicing, posttranslational modifications, and regulation by auxiliary proteins have been proposed to contribute to this functional diversity (1, 2, 9–16).The BK channel α-subunit is formed by a single polypeptide of about 1,200 amino acids that contains all of the key structural elements for ion permeation, gating, and modulation by ions and other proteins. Tetramers of α-subunits form functional BK channels. Each subunit has seven hydrophobic transmembrane segments (S0–S6), where the voltage-sensor domain (VSD) and pore domain (PD) reside (2). The N terminus faces the extracellular side of the membrane, whereas the C terminus is intracellular. The latter contains four hydrophobic α-helices (S7–S10) and the main Ca²⁺ binding sites (2). VSDs formed by segments S1–S4 harbor a series of charged residues across the membrane that contributes to voltage sensing (2). Upon membrane depolarization, each VSD undergoes a rearrangement (17) that prompts the opening of a highly K⁺-selective pore formed by the four PDs that come together at the symmetry center of the tetramer.Although BK channel expression is ubiquitous, in most physiological scenarios their functioning is provided by their coassembly with auxiliary proteins, such as β-subunits. This coassembly brings channel activity into the proper cell/tissue context (11, 13). Four different β-subunits have been cloned (β1–β4) (18–24), all of which have been observed to modify BK channel function. Albeit to a different extent, all β-subunits modify the Ca²⁺ sensitivity, voltage dependence, and gating properties of BK channels, hence modifying plasma membrane excitability balance. Regarding auxiliary β-subunits, β1- and β2-subunits increase apparent Ca²⁺ sensitivity and decelerate macroscopic current kinetics (14, 20, 21, 25–30); β2 and β3 induce fast inactivation as well as an instantaneous outward rectification (20, 21, 24, 31, 32); and β4 slows down activation and deactivation kinetics (12, 23) and modifies Ca²⁺ sensitivity (12, 33, 34).It should be kept in mind that β-subunits are potential targets for different molecules that modulate channel function, such as alcohol (35), estrogens (15), hormones (36), and fatty acids (37, 38). Additionally, scorpion toxin affinity in BK channels would tend to increase when β1 is coexpressed with the α-subunit (22).To identify the molecular elements that give β1 the ability to modulate the voltage sensor of BK channels, we constructed chimeric proteins of β1/β2- and β1/β3-subunits by swapping their N and C termini, the transmembrane (TM) segments, and the extracellular loops and recorded their gating currents. Two lysine residues that are unique to the N terminus of β1 were identified to be sufficient for BK voltage-sensor modulation.     

The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1

Heterotrimeric G proteins are activated by exchange of GDP for GTP at the G protein alpha subunit (Gα), most notably by G protein-coupled transmembrane receptors. Ric-8A is a soluble cytoplasmic protein essential for embryonic development that acts as both a guanine nucleotide exchange factor (GEF) and a chaperone for Gα subunits of the i, q, and 12/13 classes. Previous studies demonstrated that Ric-8A stabilizes a dynamically disordered state of nucleotide-free Gα as the catalytic intermediate for nucleotide exchange, but no information was obtained on the structures involved or the magnitude of the structural fluctuations. In the present study, site-directed spin labeling (SDSL) together with double electron-electron resonance (DEER) spectroscopy is used to provide global distance constraints that identify discrete members of a conformational ensemble in the Gαi1:Ric-8A complex and the magnitude of structural differences between them. In the complex, the helical and Ras-like nucleotide-binding domains of Gαi1 pivot apart to occupy multiple resolved states with displacements as large as 25 Å. The domain displacement appears to be distinct from that observed in Gαs upon binding of Gs to the β₂ adrenergic receptor. Moreover, the Ras-like domain exhibits structural plasticity within and around the nucleotide-binding cavity, and the switch I and switch II regions, which are known to adopt different conformations in the GDP- and GTP-bound states of Gα, undergo structural rearrangements. Collectively, the data show that Ric-8A induces a conformationally heterogeneous state of Gαi and provide insight into the mechanism of action of a nonreceptor Gα GEF.Heterotrimeric G proteins are activated by exchange of GDP for GTP at the alpha subunit (Gα), a reaction with a high-activation energy barrier (1). Guanine dinucleotides and trinucleotides bind tightly to Gα with affinities in the low nanomolar range (2, 3), and contribute substantially to the overall stability of Gα tertiary structure. Indeed, nucleotide-free Gα exhibits properties characteristic of a molten globule (4). In cells, agonist-stimulated 7-transmembrane helical G protein-coupled receptors (GPCRs) catalyze nucleotide exchange from G protein heterotrimers, in which Gα•GDP is bound to a heterodimer of Gβ and Gγ subunits (5). The cytosolic proteins Ric-8A and Ric-8B, which are structurally unrelated to GPCRs, have been shown to have guanine nucleotide exchange (GEF) activity toward Gα•GDP subunits in the absence of Gβγ (6, 7), thus functionally activating the subunit. In Caenorhabditis elegans, Drosophila, and mouse, Ric-8 homologs have been shown to be essential for asymmetric cell division, where they are assumed to function as GEFs (8–12). Ric-8 proteins also promote efficient folding and membrane localization of certain Gα subunits (13, 14), and inhibit their ubiquitination and degradation (15, 16). With respect to these activities, Ric-8A acts specifically on Gα subunits of the i, q, and 12/13 classes, whereas Ric-8B is active toward Gαs (17).Gα subunits are composed of two structural domains (3). The Ras-like domain is homologous to guanine nucleotide-binding domains of the Ras superfamily. Within the Ras-like domain are three so-called switch segments that integrate catalytic (guanine nucleotide-binding and GTP hydrolysis) with regulatory function (effector regulation). The conformations of these peptide segments differ between the GTP and GDP-bound states of Gα (3). In crystals of Gαi1–nucleotide complexes, switch I and switch II are well-ordered in the GTP-bound state, but partially (switch I) or fully (switch II) disordered when GDP is bound (18, 19). Inserted into switch I of the Ras domain is a helical domain that is unique to the family of heterotrimeric G proteins. The helical domain flanks the guanine nucleotide-binding site and, while it makes few direct contacts with the nucleotide, shields it from solvent and may affect the rate of its dissociation (20, 21).We have shown that in the complex of nucleotide-free Gαi1 and Ric-8A, an intermediate in the nucleotide exchange reaction (6, 22), Gαi1 is conformationally heterogeneous and dynamic, but the structures involved and the magnitude of the structural fluctuations were not determined (4). The nucleotide-free Gαi1:Ric-8A complex is stable and can be readily isolated. In the present study, we used site-directed spin labeling (SDSL) and both continuous wave (CW) and double electron-electron resonance (DEER) (23, 24) spectroscopy to map sequence-specific structural and dynamical changes in Gαi1 upon complex formation with Ric-8A. The data reveal that binding of Ric-8A to Gαi1 induces structural heterogeneity due to new conformations in which the helical domain has pivoted away from the Ras-like domain, exposing the nucleotide-binding site to solvent, thus providing an escape (and entry) pathway for the nucleotide. A similar change is induced in Gαi1 upon formation of the nucleotide-free complex with the activated GPCR rhodopsin (20), but is distinctly different from that in the crystal structure of Gαs in the complex with β₂ adrenergic receptor (β2R) (25). In addition to the global changes in tertiary structure, binding of Ric-8A also triggers deformation within the Ras-like domain, particularly of structural elements that surround the nucleotide-binding pocket. Together, these changes reveal salient features of a mechanism underlying the GEF activity of Ric-8A.     

α2A adrenergic receptor promotes amyloidogenesis through disrupting APP-SorLA interaction

Accumulation of amyloid β (Aβ) peptides in the brain is the key pathogenic factor driving Alzheimer’s disease (AD). Endocytic sorting of amyloid precursor protein (APP) mediated by the vacuolar protein sorting (Vps10) family of receptors plays a decisive role in controlling the outcome of APP proteolytic processing and Aβ generation. Here we report for the first time to our knowledge that this process is regulated by a G protein-coupled receptor, the α_2A adrenergic receptor (α_2AAR). Genetic deficiency of the α_2AAR significantly reduces, whereas stimulation of this receptor enhances, Aβ generation and AD-related pathology. Activation of α_2AAR signaling disrupts APP interaction with a Vps10 family receptor, sorting-related receptor with A repeat (SorLA), in cells and in the mouse brain. As a consequence, activation of α_2AAR reduces Golgi localization of APP and concurrently promotes APP distribution in endosomes and cleavage by β secretase. The α_2AAR is a key component of the brain noradrenergic system. Profound noradrenergic dysfunction occurs consistently in patients at the early stages of AD. α_2AAR-promoted Aβ generation provides a novel mechanism underlying the connection between noradrenergic dysfunction and AD. Our study also suggests α_2AAR as a previously unappreciated therapeutic target for AD. Significantly, pharmacological blockade of the α_2AAR by a clinically used antagonist reduces AD-related pathology and ameliorates cognitive deficits in an AD transgenic model, suggesting that repurposing clinical α₂AR antagonists would be an effective therapeutic strategy for AD.Excess amyloid β (Aβ) peptides in the brain are a neuropathological hallmark of Alzheimer’s disease (AD) and are generally accepted as the key pathogenic factor of the disease (1). Aβ is generated by two sequential cleavages of amyloid precursor protein (APP) by β and γ secretase, whereas cleavage by α secretase within the Aβ domain precludes Aβ generation (2, 3). APP and the secretases undergo endocytic sorting into various organelles, such as the trans-Golgi network, the plasma membrane, and endosomes (2–6). The initial step of APP processing by α versus β secretase preferentially occurs in distinct compartments of the cell. Although α secretase-mediated cleavage of APP occurs on the plasma membrane, β secretase primarily interacts with and cleaves APP in endosomes (2–6). Therefore, endocytic sorting of APP into different membranous compartments, causing it to coreside or avoid a particular secretase, plays a decisive role in APP proteolytic processing. Consistent with this notion, abnormalities of the endocytic pathway have been found to precede Aβ deposition in late-onset AD (7).Retrograde sorting of APP from endosomes to trans-Golgi network mediated by the vacuolar protein sorting-10 (Vps10) family proteins and the retromer complex represents a critical mechanism to prevent amyloidogenic processing of APP (8–10) and has recently emerged as a potential target for therapeutic intervention (11). In particular, the sorting-related receptor with A repeat (SorLA) directs retrograde transport of APP to trans-Golgi network by binding to both APP and the retromer complex (12, 13) and retains APP in the Golgi (14), thus preventing its proteolytic processing. A connection between SorLA and AD was first revealed in patients with late-onset AD, in whom the levels of SorLA at the steady state are markedly reduced (15). Further human genetic studies identified variations of SORL1 (the gene encoding SorLA) resulting in a lower level of expression that are associated with late-onset sporadic AD (12, 16, 17). Moreover, nonsense and missense mutations of SORL1 cause autosomal dominant early-onset AD (18), supporting an etiological role of SorLA in AD. The function of SorLA in inhibiting Aβ production is confirmed by mouse genetic studies showing that loss of SorLA significantly increases Aβ levels in the brain (14) and enhances AD-related early pathology (19). Despite the importance of SorLA-dependent APP sorting in controlling Aβ metabolism and AD pathogenesis, how this process may be targeted by extracellular stimuli, such as neurotransmitters and hormones, to modulate amyloidogenesis remains largely unstudied.The α_2A adrenergic receptor (AR) belongs to the G protein-coupled receptor (GPCR) superfamily and is a crucial component of the brain noradrenergic (NA) system, controlling both NA input to the cerebral cortex and the resulting response in this brain region (20). Profound dysfunction of the NA system consistently occurs at the early stage of AD (21), raising the possibility of involvement of the α_2AAR in AD pathogenesis. Here we report for the first time to our knowledge that α_2AAR signaling regulates SorLA-dependent APP sorting and promotes amyloidogenic processing of APP by beta-site amyloid precursor protein cleaving enzyme (BACE) 1. The initial cleavage of APP by BACE1 is the rate-limiting factor of Aβ generation (22, 23). Furthermore, blockade of α_2AAR by a clinical antagonist reduces AD-related pathology and rescues cognitive deficits in an AD transgenic model, suggesting that repurposing clinical α₂AR antagonists would be a novel effective strategy for AD treatment.     

Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling

Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein–protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein–protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.Scaffolding proteins act as flexible organizing centers to consolidate and propagate the cellular information flow from activated cell-surface receptors to intracellular effector cascades. Activated G protein-coupled receptors (GPCRs) engage pleiotropic scaffolds to recruit cytoplasmic downstream effector molecules (1–4). Thus, compartmentalized GTPases, kinases, and phosphatases act as GPCR-linked molecular switches to spatially and temporally control signal propagation. In the classic view of GPCR signaling, extracellular ligands bind to the receptor, which catalyzes the intracellular GDP/GTP exchange and activation of receptor-associated trimeric G protein (α:β:γ) combinations. GPCR signaling and trafficking involve G protein-dependent and -independent intracellular interactions with scaffolds, such as β-arrestin and A-kinase anchoring proteins (AKAPs) (1, 2, 5, 6). Receptor-interacting proteins and kinase activities contribute to the fine-tuning of GPCR localization and activities (4, 7–9). Different AKAPs coordinate and compartmentalize diffusible second-messenger responses through anchoring of cAMP-dependent type I or type II protein kinase A (PKA) holoenzymes, composed of a regulatory subunit (R) dimer and two catalytic (PKAc) subunits, to discrete subcellular localizations (1, 10, 11). The four R subunits (RIα/β or RIIα/β) have different expression patterns and are functionally nonredundant. The growing family of AKAPs are functionally diverse; however, all AKAPs contain an amphipathic helix, which accounts for nanomolar binding affinities to PKA R subunit dimers (12, 13). Moreover, additional components of the cAMP signaling machinery, such as GPCRs, adenylyl cyclases, and phosphodiesterases, physically interact with AKAPs (1, 5, 11, 14). Mutations activating cAMP/PKA signaling have been shown to contribute to carcinogenesis or degenerative diseases, and inactivating mutations have been linked to hormone resistance (15–20). In-depth analyses of transient protein–protein interactions (PPIs) of PKA, along with the phosphorylation dynamics, have the potential to reveal conditional signal flow and thus may help to explain pathological implications of cAMP/PKA signaling. Such a strategy will lead to a better understanding of cAMP-signaling, which boosts proliferation in many cell types but inhibits cell growth in others (21, 22). Using a proteomics approach, we discovered that an AKAP motif is embedded within the C-terminal tail of Gpr161, an orphan GPCR that is associated with the primary cilia (23). We confirmed the direct functional interaction of Gpr161 with PKA and showed that it has absolute specificity for RI subunits. We show that Gpr161 is not only a RI-specific AKAP but also a PKA substrate. Finally, in experiments with zebrafish embryos we demonstrate that Gpr161 receptors recruit RI to primary cilia.     

α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation

Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration.α-Synuclein is an abundant presynaptic protein that physiologically acts to promote soluble NSF attachment protein receptor (SNARE) complex assembly in vitro and in vivo (1–3). Point mutations in α-synuclein (A30P, E46K, H50Q, G51D, and A53T) as well as α-synuclein gene duplications and triplications produce early-onset Parkinson''s disease (PD) (4–10). Moreover, α-synuclein is a major component of intracellular protein aggregates called Lewy bodies, which are pathological hallmarks of neurodegenerative disorders such as PD, Lewy body dementia, and multiple system atrophy (11–14). Strikingly, neurotoxic α-synuclein aggregates propagate between neurons during neurodegeneration, suggesting that such α-synuclein aggregates are not only intrinsically neurotoxic but also nucleate additional fibrillization (15–18).α-Synuclein is highly concentrated in presynaptic terminals where α-synuclein exists in an equilibrium between a soluble and a membrane-bound state, and is associated with synaptic vesicles (19–22). The labile association of α-synuclein with membranes (23, 24) suggests that binding of α-synuclein to synaptic vesicles, and its dissociation from these vesicles, may regulate its physiological function. Membrane-bound α-synuclein assumes an α-helical conformation (25–32), whereas cytosolic α-synuclein is natively unfolded and monomeric (refs. 25, 26, 31, and 32; however, see refs. 33 and 34 and Discussion for a divergent view). Membrane binding by α-synuclein is likely physiologically important because in in vitro experiments, α-synuclein remodels membranes (35, 36), influences lipid packing (37, 38), and induces vesicle clustering (39). Moreover, membranes were found to be important for the neuropathological effects of α-synuclein (40–44).However, the relation of membrane binding to the in vivo function of α-synuclein remains unexplored, and it is unknown whether α-synuclein binds to membranes as a monomer or oligomer. Thus, in the present study we have investigated the nature of the membrane-bound state of α-synuclein and its relation to its physiological function in SNARE complex assembly. We found that soluble monomeric α-synuclein assembles into higher-order multimers upon membrane binding and that membrane binding of α-synuclein is required for its physiological activity in promoting SNARE complex assembly at the synapse.