Segregation of Odor Identity and Intensity during Odor Discrimination in Drosophila Mushroom Body

Molecular and cellular studies have begun to unravel a neurobiological basis of olfactory processing, which appears conserved among vertebrate and invertebrate species. Studies have shown clearly that experience-dependent coding of odor identity occurs in “associative” olfactory centers (the piriform cortex in mammals and the mushroom body [MB] in insects). What remains unclear, however, is whether associative centers also mediate innate (spontaneous) odor discrimination and how ongoing experience modifies odor discrimination. Here we show in naïve flies that G_αq-mediated signaling in MB modulates spontaneous discrimination of odor identity but not odor intensity (concentration). In contrast, experience-dependent modification (conditioning) of both odor identity and intensity occurs in MB exclusively via G_αs-mediated signaling. Our data suggest that spontaneous responses to odor identity and odor intensity discrimination are segregated at the MB level, and neural activity from MB further modulates olfactory processing by experience-independent G_αq-dependent encoding of odor identity and by experience-induced G_αs-dependent encoding of odor intensity and identity.     

β-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1

FFAR1/GPR40 is a seven-transmembrane domain receptor (7TMR) expressed in pancreatic β cells and activated by FFAs. Pharmacological activation of GPR40 is a strategy under consideration to increase insulin secretion in type 2 diabetes. GPR40 is known to signal predominantly via the heterotrimeric G proteins G_q/11. However, 7TMRs can also activate functionally distinct G protein-independent signaling via β-arrestins. Further, G protein- and β-arrestin-based signaling can be differentially modulated by different ligands, thus eliciting ligand-specific responses (“biased agonism”). Whether GPR40 engages β-arrestin-dependent mechanisms and is subject to biased agonism is unknown. Using bioluminescence resonance energy transfer-based biosensors for real-time monitoring of cell signaling in living cells, we detected a ligand-induced GPR40-β-arrestin interaction, with the synthetic GPR40 agonist TAK-875 being more effective than palmitate or oleate in recruiting β-arrestins 1 and 2. Conversely, TAK-875 acted as a partial agonist of G_q/11-dependent GPR40 signaling relative to both FFAs. Pharmacological blockade of G_q activity decreased FFA-induced insulin secretion. In contrast, knockdown or genetic ablation of β-arrestin 2 in an insulin-secreting cell line and mouse pancreatic islets, respectively, uniquely attenuated the insulinotropic activity of TAK-875, thus providing functional validation of the biosensor data. Collectively, these data reveal that in addition to coupling to G_q/11, GPR40 is functionally linked to a β-arrestin 2-mediated insulinotropic signaling axis. These observations expose previously unrecognized complexity for GPR40 signal transduction and may guide the development of biased agonists showing improved clinical profile in type 2 diabetes.     

A Gs-RhoGEF interaction: An old G protein finds a new job

The heterotrimeric G proteins are known to have a variety of downstream effectors, but G_s was long thought to be specifically coupled to adenylyl cyclases. A new study indicates that activated G_s can also directly interact with a guanine nucleotide exchange factor for Rho family small GTPases, PDZ-RhoGEF. This novel interaction mediates activation of the small G protein Cdc42 by G_s-coupled GPCRs, inducing cytoskeletal rearrangements and formation of filopodia-like structures. Furthermore, overexpression of a minimal PDZ-RhoGEF fragment can down-regulate cAMP signaling, suggesting that this effector competes with canonical signaling. This first demonstration that the Gα_s subfamily regulates activity of Rho GTPases extends our understanding of Gα_s activity and establishes RhoGEF coupling as a universal Gα function.

The canonical G protein pathway consists of a cell surface receptor, a heterotrimeric G protein, and an effector protein that controls signaling within the cells. This fundamental paradigm, familiar to every biologist, is rooted in discoveries by the laboratories of Sutherland, Rodbell, and Gilman, which in the 1970s and 1980s dissected biochemical mechanisms of adenylyl cyclase activation by hormones. Their breakthrough came after experiments showing that the G protein G_s is essential to transfer agonist stimulation from the receptor to adenylyl cyclase (1). This G protein consists of the ∼42-kDa α subunit, which binds and hydrolyzes GTP, and the permanently associated dimer of 35-kDa β and ∼10-kDa γ subunits (Gβγ). Their findings helped establish a canonical model in which the agonist-bound receptor causes the G protein to release GDP, and the heterotrimer dissociates into Gα-GTP and free Gβγ; in this state, the G protein can activate its effector (i.e. Gα_s will activate adenylyl cyclase until GTP is hydrolyzed). Although the rod photoreceptor G protein, transducin, was discovered by that time (2), the ubiquitously expressed G_s can be considered the founding member of the G protein family.The subsequent cloning and identification of the other three families (G_i, G_q, and G₁₂) completed the rough map of G protein–mediated transduction. These initial studies suggested that the α subunits were responsible for activation of one type of effector (e.g. Gα_s for adenylyl cyclase and cAMP; Gα_q for phospholipase C, phosphoinositides, and Ca²⁺; and Gα_i for ion channels and inhibition of adenylyl cyclase), whereas the free Gβγ complexes interact with a remarkably large number of binding partners, including some effector enzymes and ion channels (3). Later, Gα₁₂ and Gα₁₃ were found to regulate a distinct type of effectors, the RhoGEFs (4, 5). These multidomain proteins contain pleckstrin homology (PH) domains, which facilitate their membrane localization, and Dbl homology (DH) domains, which catalyze GDP-for-GTP exchange (guanine nucleotide exchange factor; GEF) in the Rho family of small (∼20-kDa) G proteins. At the time, the G₁₂-RhoGEF pathway seemed odd as it contained two G proteins: the receptor-activated “large” G₁₂ class protein and the “small” Rho G protein, which is activated by RhoGEF. However, it was then discovered that Gα_q could activate a RhoGEF called Trio (6), and that Gβγ complexes activate other RhoGEFs, indicating that this pathway, if unusual, is at least popular. Gα_s, however, mostly appeared to be faithful to its originally determined role—to stimulate adenylyl cyclase(s)—possibly contributing to the enduring perception that regulation of a second messenger–generating enzyme is the “real” function of a heterotrimeric G protein.In the current issue of JBC, Castillo-Kauil et al. (7) force a reexamination of the existing canon, presenting data that show Gα_s can also interact with a specific RhoGEF, in this case PDZ-RhoGEF (PRG). The authors made this discovery as part of an examination of the regulation of cell shape by the Rho family. They began by expressing a series of short constructs of three RhoGEF proteins, p115RhoGEF, PRG, and LARG, all of which activated RhoA as expected, promoting cell contraction. However, they noticed that the DH/PH domain of PRG also activated Cdc42 and induced filopodia-like cell protrusions. To investigate which G protein is responsible for activation of this Cdc42-mediated pathway, they overexpressed constitutively active mutants of different Gα subunits. These mutants are stabilized in the active GTP-bound state due to substitution of the glutamine residue crucial for GTP hydrolysis. Surprisingly, the PRG-Cdc42 pathway was stimulated by Gα_sQ227L, the one Gα subtype not known for interaction with RhoGEFs. Furthermore, they showed that binding of PRG to Cdc42 was promoted only by G_s-coupled receptors, and not by G_q- or G_i-coupled GPCRs. The authors then investigated the PRG site responsible for the interaction with Gα_s, narrowing it down to the isolated PRG DH and PH domains and their linker region. A construct encompassing these domains was able to inhibit (i) GPCR-mediated activation of Cdc42, (ii) the Gα_sQ227L-promoted interaction of PRG with Cdc42, and (iii) some protein phosphorylation events downstream of the canonical cAMP pathway. Taken together, their work identifies PRG as a novel effector for G_s; the Gα_s-PRG interaction mediates activation of Rho family protein Cdc42, leading to cytoskeletal remodeling.The unexpected results of Castillo-Kauil et al. open up new opportunities to explore this mechanism at different levels of biology. The experiments described in the paper were performed in vitro using cultured cells, imaging, and pulldown of protein complexes containing the overexpressed Gα_s Q227L mutant. Considering the multitude of G_s-coupled receptors and RhoGEFs in the body (8, 9), it will be important to understand the physiological context where the new G_s-mediated pathway plays a significant role. This will require experimentation in vivo and possibly reevaluation of the phenotypes associated with known pathogenic mutations in Gα_s (GNAS) and other relevant genes. At the molecular level, it would be important to delineate the biochemical mechanisms of Gα_s interaction with PRG. For example, at what stage of the GTP/GDP cycle does Gα_s bind to PRG: in the GTP-bound state, which also activates adenylate cyclase, or in the transition state (i.e. just before the terminal phosphate of GTP is removed)? Indeed, there is precedent for proteins that bind preferentially with the transition state—specifically RGS proteins, which accelerate the GTPase reaction. Another possibility is that, by analogy with p115RhoGEF, which stimulates GTPase activity of Gα₁₂ and Gα₁₃, PRG (and other RhoGEFs with similar DH-PH sequences) can influence interaction of Gα_s with nucleotides, Gβγ, and other partners.Since defining the receptor, G protein, and effector as the three essential members of the G protein pathway, researchers have discovered many additional proteins that regulate the amplitude and duration of the stimulus and/or participate in cross-talk with other signaling circuits. These “new” proteins include arrestins, receptor kinases, nonreceptor exchange factors, GTPase-activating proteins, special chaperones, etc. Thus, in a way, discovering a novel binding partner for a signaling molecule is not as surprising as it would have been 20 years ago. However, the new partner identified by Castillo-Kauil et al. makes the result of extra significance; until now, we knew that three of four G protein subfamilies could regulate Rho GTPases by activating RhoGEFs: G₁₂ and G_q via their α subunits and G_i via the Gβγ subunits (10). The demonstration that the G_s subfamily is no exception shows that activation of RhoGEFs by heterotrimeric G proteins may be a truly universal mechanism (Fig. 1). The significance of this insight is that the multitude of biological processes regulated by Rho-GTPase networks can potentially respond to the entire repertoire of GPCR-mediated stimuli.Open in a separate windowFigure 1.Activation of the Rho family by heterotrimeric G proteins. The Rho family of small GTPases is activated by RhoGEF proteins, some of which can be stimulated by heterotrimeric G proteins. Of four families of heterotrimeric G proteins, three (G₁₂, G_q, and G_i, shown in shades of gray) were known to activate certain RhoGEFs. The new results (highlighted in orange) (7) show that G_s, the G protein known to stimulate production of cAMP, can also stimulate a particular RhoGEF; this suggests that the Rho GTPases can potentially be stimulated by the multitude of signals from the entire class of GPCRs, including those coupled to G_s. IP₃, inositol 1,4,5-trisphosphate.

Funding and additional information—This work was supported in part by National Institutes of Health Grant R56DK119262 (to V. Z. S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Abbreviations—The abbreviations used are:

PH

pleckstrin homology

DH

Dbl homology

GEF

guanine nucleotide exchange factor

PRG

PDZ-RhoGEF

GPCR

G protein–coupled receptor.

     

Tyrosine 308 Is Necessary for Ligand-directed Gs Protein-biased Signaling of β2-Adrenoceptor

Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, β₂-adrenoceptor (β₂-AR) couples dually to G_s and G_i proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of β₂-AR causes a switch in receptor coupling from G_s to G_i. More recent studies have demonstrated that phosphorylation of β₂-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the G_i coupling. We have previously shown that although most β₂-AR agonists cause both G_s and G_i activation, (R,R′)-fenoterol preferentially activates β₂-AR-G_s signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on β₂-AR essential for G_s-biased signaling. Following stimulation with a β₂-AR-G_s-biased agonist (R,R′)-4′-aminofenoterol, the G_i disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type β₂-AR. Interestingly, Y308F substitution on β₂-AR enabled (R,R′)-4′-aminofenoterol to activate G_i and to produce these responses in a pertussis toxin-sensitive manner without altering β₂-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of β₂-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of β₂-AR stabilize receptor conformations favoring the receptor-G_s protein coupling and subsequently result in G_s-biased agonism.     

Differential Stimulation of Insulin Secretion by GLP-1 and Kisspeptin-10

β-cells in the pancreatic islet respond to elevated plasma glucose by secreting insulin to maintain glucose homeostasis. In addition to glucose stimulation, insulin secretion is modulated by numerous G-protein coupled receptors (GPCRs). The GPCR ligands Kisspeptin-10 (KP) and glucagon-like peptide-1 (GLP-1) potentiate insulin secretion through G_q and G_s-coupled receptors, respectively. Despite many studies, the signaling mechanisms by which KP and GLP-1 potentiate insulin release are not thoroughly understood. We investigated the downstream signaling pathways of these ligands and their affects on cellular redox potential, intracellular calcium activity ([Ca²⁺]_i), and insulin secretion from β-cells within intact murine islets. In contrast to previous studies performed on single β-cells, neither KP nor GLP-1 affect [Ca²⁺]_i upon stimulation with glucose. KP significantly increases the cellular redox potential, while no effect is observed with GLP-1, suggesting that KP and GLP-1 potentiate insulin secretion through different mechanisms. Co-treatment with KP and the G_βγ-subunit inhibitor gallein inhibits insulin secretion similar to that observed with gallein alone, while co-treatment with gallein and GLP-1 does not differ from GLP-1 alone. In contrast, co-treatment with the G_βγ activator mSIRK and either KP or GLP-1 stimulates insulin release similar to mSIRK alone. Neither gallein nor mSIRK alter [Ca²⁺]_i activity in the presence of KP or GLP-1. These data suggest that KP likely alters insulin secretion through a G_βγ-dependent process that stimulates glucose metabolism without altering Ca²⁺ activity, while GLP-1 does so, at least partly, through a G_α-dependent pathway that is independent of both metabolism and Ca²⁺.     

Modification of Heterotrimeric G-Proteins in Swiss 3T3 Cells Stimulated with Pasteurella multocida Toxin

Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s). The Pasteurella multocida toxin (PMT) mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, G_q, G₁₂ and G_i. PMT has been shown by others to lead to the deamidation of recombinant Gα_i at Gln-205 to inhibit its intrinsic GTPase activity. We have investigated modification of native Gα subunits mediated by PMT in Swiss 3T3 cells using 2-D gel electrophoresis and antibody detection. An acidic change in the isoelectric point was observed for the Gα subunit of the G_q and G_i families following PMT treatment of Swiss 3T3 cells, which is consistent with the deamidation of these Gα subunits. Surprisingly, PMT also induced a similar modification of Gα₁₁, a member of the G_q family of G-proteins that is not activated by PMT. Furthermore, an alkaline change in the isoelectric point of Gα₁₃ was observed following PMT treatment of cells, suggesting differential modification of this Gα subunit by PMT. G_s was not affected by PMT treatment. Prolonged treatment with PMT led to a reduction in membrane-associated Gα_i, but not Gα_q. We also show that PMT inhibits the GTPase activity of G_q.     

Differential Signaling of the Endogenous Agonists at the β2-Adrenergic Receptor

The concept of “functional selectivity” or “biased signaling” suggests that a ligand can have distinct efficacies with regard to different signaling pathways. We have investigated the question of whether biased signaling may be related to distinct agonist-induced conformational changes in receptors using the β₂-adrenergic receptor (β₂AR) and its two endogenous ligands epinephrine and norepinephrine as a model system. Agonist-induced conformational changes were determined in a fluorescently tagged β₂AR FRET sensor. In this β₂AR sensor, norepinephrine caused signals that amounted to only ≈50% of those induced by epinephrine and the standard “full” agonist isoproterenol. Furthermore, norepinephrine-induced changes in the β₂AR FRET sensor were slower than those induced by epinephrine (rate constants, 47 versus 128 ms). A similar partial β₂AR activation signal was revealed for the synthetic agonists fenoterol and terbutaline. However, norepinephrine was almost as efficient as epinephrine (and isoproterenol) in causing activation of G_s and adenylyl cyclase. In contrast, fenoterol was quite efficient in triggering β-arrestin2 recruitment to the cell surface and its interaction with β₂AR, as well as internalization of the receptors, whereas norepinephrine caused partial and slow changes in these assays. We conclude that partial agonism of norepinephrine at the β₂AR is related to the induction of a different active conformation and that this conformation is efficient in signaling to G_s and less efficient in signaling to β-arrestin2. These observations extend the concept of biased signaling to the endogenous agonists of the β₂AR and link it to distinct conformational changes in the receptor.     

Modulation of distinct isoforms of L-type calcium channels by Gq-coupled receptors in Xenopus oocytes: Antagonistic effects of Gβγ and protein kinase C

L-type voltage dependent Ca²⁺ channels (L-VDCCs; Ca_v1.2) are crucial in cardiovascular physiology. In heart and smooth muscle, hormones and transmitters operating via G_q enhance L-VDCC currents via essential protein kinase C (PKC) involvement. Heterologous reconstitution studies in Xenopus oocytes suggested that PKC and G_q-coupled receptors increased L-VDCC currents only in cardiac long N-terminus (NT) isoforms of α_1C, whereas known smooth muscle short-NT isoforms were inhibited by PKC and G_q activators. We report a novel regulation of the long-NT α_1C isoform by Gβγ. Gβγ inhibited whereas a Gβγ scavenger protein augmented the G_q- but not phorbol ester-mediated enhancement of channel activity, suggesting that Gβγ acts upstream from PKC. In vitro binding experiments reveal binding of both Gβγ and PKC to α_1C-NT. However, PKC modulation was not altered by mutations of multiple potential phosphorylation sites in the NT, and was attenuated by a mutation of C-terminally located serine S1928. The insertion of exon 9a in intracellular loop 1 rendered the short-NT α_1C sensitive to PKC stimulation and to Gβγ scavenging. Our results suggest a complex antagonistic interplay between G_q-activated PKC and Gβγ in regulation of L-VDCC, in which multiple cytosolic segments of α_1C are involved.     

Development and Characterization of Pepducins as Gs-biased Allosteric Agonists

The β₂-adrenergic receptor (β₂AR) is a prototypical G protein-coupled receptor that mediates many hormonal responses, including cardiovascular and pulmonary function. β-Agonists used to combat hypercontractility in airway smooth muscle stimulate β₂AR-dependent cAMP production that ultimately promotes airway relaxation. Chronic stimulation of the β₂AR by long acting β-agonists used in the treatment of asthma can promote attenuated responsiveness to agonists and an increased frequency of fatal asthmatic attacks. β₂AR desensitization to β-agonists is primarily mediated by G protein-coupled receptor kinases and β-arrestins that attenuate receptor-G_s coupling and promote β₂AR internalization and degradation. A biased agonist that can selectively stimulate G_s signaling without promoting receptor interaction with G protein-coupled receptor kinases and β-arrestins should serve as an advantageous asthma therapeutic. To identify such molecules, we screened ∼50 lipidated peptides derived from the intracellular loops of the β₂AR, known as pepducins. This screen revealed two classes of G_s-biased pepducins, receptor-independent and receptor-dependent, as well as several β-arrestin-biased pepducins. The receptor-independent G_s-biased pepducins operate by directly stimulating G protein activation. In contrast, receptor-dependent G_s-biased pepducins appear to stabilize a G_s-biased conformation of the β₂AR that couples to G_s but does not undergo G protein-coupled receptor kinase-mediated phosphorylation or β-arrestin-mediated internalization. Functional studies in primary human airway smooth muscle cells demonstrate that G_s-biased pepducins are not subject to conventional desensitization and thus may be good candidates for the development of next generation asthma therapeutics. Our study reports the first G_s-biased activator of the β₂AR and provides valuable tools for the study of β₂AR function.     

Divergent Transducer-specific Molecular Efficacies Generate Biased Agonism at a G Protein-coupled Receptor (GPCR)

The concept of “biased agonism” arises from the recognition that the ability of an agonist to induce a receptor-mediated response (i.e. “efficacy”) can differ across the multiple signal transduction pathways (e.g. G protein and β-arrestin (βarr)) emanating from a single GPCR. Despite the therapeutic promise of biased agonism, the molecular mechanism(s) whereby biased agonists selectively engage signaling pathways remain elusive. This is due in large part to the challenges associated with quantifying ligand efficacy in cells. To address this, we developed a cell-free approach to directly quantify the transducer-specific molecular efficacies of balanced and biased ligands for the angiotensin II type 1 receptor (AT₁R), a prototypic GPCR. Specifically, we defined efficacy in allosteric terms, equating shifts in ligand affinity (i.e. K_Lo/K_Hi) at AT₁R-G_q and AT₁R-βarr2 fusion proteins with their respective molecular efficacies for activating G_q and βarr2. Consistent with ternary complex model predictions, transducer-specific molecular efficacies were strongly correlated with cellular efficacies for activating G_q and βarr2. Subsequent comparisons across transducers revealed that biased AT₁R agonists possess biased molecular efficacies that were in strong agreement with the signaling bias observed in cellular assays. These findings not only represent the first measurements of the thermodynamic driving forces underlying differences in ligand efficacy between transducers but also support a molecular mechanism whereby divergent transducer-specific molecular efficacies generate biased agonism at a GPCR.     

M3 Muscarinic Receptor Interaction with Phospholipase C β3 Determines Its Signaling Efficiency

Phospholipase Cβ (PLCβ) enzymes are activated by G protein-coupled receptors through receptor-catalyzed guanine nucleotide exchange on Gαβγ heterotrimers containing G_q family G proteins. Here we report evidence for a direct interaction between M3 muscarinic receptor (M3R) and PLCβ3. Both expressed and endogenous M3R interacted with PLCβ in coimmunoprecipitation experiments. Stimulation of M3R with carbachol significantly increased this association. Expression of M3R in CHO cells promoted plasma membrane localization of YFP-PLCβ3. Deletion of the PLCβ3 C terminus or deletion of the PLCβ3 PDZ ligand inhibited coimmunoprecipitation with M3R and M3R-dependent PLCβ3 plasma membrane localization. Purified PLCβ3 bound directly to glutathione S-transferase (GST)-fused M3R intracellular loops 2 and 3 (M3Ri2 and M3Ri3) as well as M3R C terminus (M3R/H8-CT). PLCβ3 binding to M3Ri3 was inhibited when the PDZ ligand was removed. In assays using reconstituted purified components in vitro, M3Ri2, M3Ri3, and M3R/H8-CT potentiated Gα_q-dependent but not Gβγ-dependent PLCβ3 activation. Disruption of key residues in M3Ri3N and of the PDZ ligand in PLCβ3 inhibited M3Ri3-mediated potentiation. We propose that the M3 muscarinic receptor maximizes the efficiency of PLCβ3 signaling beyond its canonical role as a guanine nucleotide exchange factor for Gα.     

Loss of Gsα in the Postnatal Skeleton Leads to Low Bone Mass and a Blunted Response to Anabolic Parathyroid Hormone Therapy

Parathyroid hormone (PTH) is an important regulator of osteoblast function and is the only anabolic therapy currently approved for treatment of osteoporosis. The PTH receptor (PTH1R) is a G protein-coupled receptor that signals via multiple G proteins including G_sα. Mice expressing a constitutively active mutant PTH1R exhibited a dramatic increase in trabecular bone that was dependent upon expression of G_sα in the osteoblast lineage. Postnatal removal of G_sα in the osteoblast lineage (P-G_sα^OsxKO mice) yielded markedly reduced trabecular and cortical bone mass. Treatment with anabolic PTH(1–34) (80 μg/kg/day) for 4 weeks failed to increase trabecular bone volume or cortical thickness in male and female P-G_sα^OsxKO mice. Surprisingly, in both male and female mice, PTH administration significantly increased osteoblast numbers and bone formation rate in both control and P-G_sα^OsxKO mice. In mice that express a mutated PTH1R that activates adenylyl cyclase and protein kinase A (PKA) via G_sα but not phospholipase C via G_q/11 (D/D mice), PTH significantly enhanced bone formation, indicating that phospholipase C activation is not required for increased bone turnover in response to PTH. Therefore, although the anabolic effect of intermittent PTH treatment on trabecular bone volume is blunted by deletion of G_sα in osteoblasts, PTH can stimulate osteoblast differentiation and bone formation. Together these findings suggest that alternative signaling pathways beyond G_sα and G_q/11 act downstream of PTH on osteoblast differentiation.     

Spared Piriform Cortical Single-Unit Odor Processing and Odor Discrimination in the Tg2576 Mouse Model of Alzheimer's Disease

Alzheimer''s disease is a neurodegenerative disorder that is the most common cause of dementia in the elderly today. One of the earliest reported signs of Alzheimer''s disease is olfactory dysfunction, which may manifest in a variety of ways. The present study sought to address this issue by investigating odor coding in the anterior piriform cortex, the primary cortical region involved in higher order olfactory function, and how it relates to performance on olfactory behavioral tasks. An olfactory habituation task was performed on cohorts of transgenic and age-matched wild-type mice at 3, 6 and 12 months of age. These animals were then anesthetized and acute, single-unit electrophysiology was performed in the anterior piriform cortex. In addition, in a separate group of animals, a longitudinal odor discrimination task was conducted from 3–12 months of age. Results showed that while odor habituation was impaired at all ages, Tg2576 performed comparably to age-matched wild-type mice on the olfactory discrimination task. The behavioral data mirrored intact anterior piriform cortex single-unit odor responses and receptive fields in Tg2576, which were comparable to wild-type at all age groups. The present results suggest that odor processing in the olfactory cortex and basic odor discrimination is especially robust in the face of amyloid β precursor protein (AβPP) over-expression and advancing amyloid β (Aβ) pathology. Odor identification deficits known to emerge early in Alzheimer''s disease progression, therefore, may reflect impairments in linking the odor percept to associated labels in cortical regions upstream of the primary olfactory pathway, rather than in the basic odor processing itself.     

Voltage-dependent regulation of CaV2.2 channels by Gq-coupled receptor is facilitated by membrane-localized β subunit

G protein–coupled receptors (GPCRs) signal through molecular messengers, such as Gβγ, Ca²⁺, and phosphatidylinositol 4,5-bisphosphate (PIP₂), to modulate N-type voltage-gated Ca²⁺ (Ca_V2.2) channels, playing a crucial role in regulating synaptic transmission. However, the cellular pathways through which G_qPCRs inhibit Ca_V2.2 channel current are not completely understood. Here, we report that the location of Ca_V β subunits is key to determining the voltage dependence of Ca_V2.2 channel modulation by G_qPCRs. Application of the muscarinic agonist oxotremorine-M to tsA-201 cells expressing M₁ receptors, together with Ca_V N-type α1B, α2δ1, and membrane-localized β2a subunits, shifted the current-voltage relationship for Ca_V2.2 activation 5 mV to the right and slowed current activation. Muscarinic suppression of Ca_V2.2 activity was relieved by strong depolarizing prepulses. Moreover, when the C terminus of β-adrenergic receptor kinase (which binds Gβγ) was coexpressed with N-type channels, inhibition of Ca_V2.2 current after M₁ receptor activation was markedly reduced and delayed, whereas the delay between PIP₂ hydrolysis and inhibition of Ca_V2.2 current was decreased. When the Gβγ-insensitive Ca_V2.2 α1C-1B chimera was expressed, voltage-dependent inhibition of calcium current was virtually abolished, suggesting that M₁ receptors act through Gβγ to inhibit Ca_V2.2 channels bearing membrane-localized Ca_V β2a subunits. Expression of cytosolic β subunits such as β2b and β3, as well as the palmitoylation-negative mutant β2a(C3,4S), reduced the voltage dependence of M₁ muscarinic inhibition of Ca_V2.2 channels, whereas it increased inhibition mediated by PIP₂ depletion. Together, our results indicate that, with membrane-localized Ca_V β subunits, Ca_V2.2 channels are subject to Gβγ-mediated voltage-dependent inhibition, whereas cytosol-localized β subunits confer more effective PIP₂-mediated voltage-independent regulation. Thus, the voltage dependence of G_qPCR regulation of calcium channels can be determined by the location of isotype-specific Ca_V β subunits.     

Diversity and Activity of Methanotrophic Bacteria in Different Upland Soils

Samples from diverse upland soils that oxidize atmospheric methane were characterized with regard to methane oxidation activity and the community composition of methanotrophic bacteria (MB). MB were identified on the basis of the detection and comparative sequence analysis of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. MB commonly detected in soils were closely related to Methylocaldum spp., Methylosinus spp., Methylocystis spp., or the “forest sequence cluster” (USC α), which has previously been detected in upland soils and is related to pmoA sequences of type II MB (Alphaproteobacteria). As well, a novel group of sequences distantly related (<75% derived amino acid identity) to those of known type I MB (Gammaproteobacteria) was often detected. This novel “upland soil cluster γ” (USC γ) was significantly more likely to be detected in soils with pH values of greater than 6.0 than in more acidic soils. To identify active MB, four selected soils were incubated with ¹³CH₄ at low mixing ratios (<50 ppm of volume), and extracted methylated phospholipid fatty acids (PLFAs) were analyzed by gas chromatography-online combustion isotope ratio mass spectrometry. Incorporation of ¹³C into PLFAs characteristic for methanotrophic Gammaproteobacteria was observed in all soils in which USC γ sequences were detected, suggesting that the bacteria possessing these sequences were active methanotrophs. A pattern of labeled PLFAs typical for methanotrophic Alphaproteobacteria was obtained for a sample in which only USC α sequences were detected. The data indicate that different MB are present and active in different soils that oxidize atmospheric methane.     

New distinct compartments in the G2 phase of the cell cycle defined by the levels of γH2AX.

Induction of DNA double strand breaks leads to phosphorylation and focus-formation of H2AX. However, foci of phosphorylated H2AX (γH2AX) appear during DNA replication also in the absence of exogenously applied injury. We measured the amount and the number of foci of γH2AX in different phases of the cell cycle by flow cytometry, sorting and microscopy in 4 malignant B-lymphocyte cell lines. There were no detectable γH2AX and no γH2AX-foci in G₁ cells in exponentially growing cells and cells treated with PARP inhibitor (PARPi) for 24 h to create damage and reduce DNA repair. The amount of γH2AX increased immediately upon S phase entry, and about 10 and 30 γH2AX foci were found in mid-S phase control and PARPi-treated cells, respectively. The γH2AX-labeled damage caused by DNA replication was not fully repaired before entry into G₂. Intriguingly, G₂ cells populated a continuous distribution of γH2AX levels, from cells with a high content of γH2AX and the same number of foci as S phase cells (termed “G₂H” compartment), to cells that there were almost negative and had about 2 foci (termed “G₂L” compartment). EdU-labeling of S phase cells revealed that G₂H was directly populated from S phase, while G₂L was populated from G₂H, but in control cells also directly from S phase. The length of G₂H in particular increased after PARPi treatment, compatible with longer DNA-repair times. Our results show that cells repair replication-induced damage in G₂H, and enter mitosis after a 2–3 h delay in G₂L.     

Olfactory Dysfunction and Neurotransmitter Disturbance in Olfactory Bulb of Transgenic Mice Expressing Human A53T Mutant α-Synuclein

Parkinson disease is a multi-system neurodegenerative disease characterized by both motor and non-motor symptoms. Hyposmia is one of the early non-motor symptoms occurring in more than 90% of Parkinson disease cases, which can precede motor symptoms even several years. Up to now, the relationship between hyposmia and Parkinson disease remains elusive. Lack of proper animal models of hyposmia restricts the investigation. In this study we assessed olfactory function in Prp-A53T-α-synuclein transgenic (αSyn^A53T) mice which had been reported to show age-dependent motor impairments and intracytoplasmic inclusions. We also examined cholinergic and dopaminergic systems in olfactory bulb of αSyn^A53T mice by immunofluorescent staining, enzyme linked immunosorbent assay and western blot. We found that compared to wild type littermates, αSyn^A53T mice at 6 months or older displayed a deficit of odor discrimination and odor detection. No significant changes were found in olfactory memory and odor habituation. Furthermore compared to wildtype littermates, in olfactory bulb of αSyn^A53T mice at 10 months old we detected a marked decrease of cholinergic neurons in mitral cell layer and a decrease of acetylcholinesterase activity, while dopaminergic neurons were found increased in glomerular layer, accompanied with an increase of tyrosine hydroxylase protein. Our studies indicate that αSyn^A53T mice have olfactory dysfunction before motor deficits occur, and the cholinergic and dopaminergic disturbance might be responsible for the Parkinson disease-related olfactory dysfunction.     

A Mouse Model for Osseous Heteroplasia

GNAS/Gnas encodes G_sα that is mainly biallelically expressed but shows imprinted expression in some tissues. In Albright Hereditary Osteodystrophy (AHO) heterozygous loss of function mutations of GNAS can result in ectopic ossification that tends to be superficial and attributable to haploinsufficiency of biallelically expressed G_sα. Oed-Sml is a point missense mutation in exon 6 of the orthologous mouse locus Gnas. We report here both the late onset ossification and occurrence of benign cutaneous fibroepithelial polyps in Oed-Sml. These phenotypes are seen on both maternal and paternal inheritance of the mutant allele and are therefore due to an effect on biallelically expressed G_sα. The ossification is confined to subcutaneous tissues and so resembles the ossification observed with AHO. Our mouse model is the first with both subcutaneous ossification and fibroepithelial polyps related to G_sα deficiency. It is also the first mouse model described with a clinically relevant phenotype associated with a point mutation in G_sα and may be useful in investigations of the mechanisms of heterotopic bone formation. Together with earlier results, our findings indicate that G_sα signalling pathways play a vital role in repressing ectopic bone formation.     

Central Nervous System Imprinting of the G Protein Gsα and Its Role in Metabolic Regulation

In Albright hereditary osteodystrophy, a monogenic obesity disorder linked to heterozygous mutations of G_sα, the G protein that mediates receptor-stimulated cAMP generation, obesity develops only when the mutation is on the maternal allele. Likewise, mice with maternal (but not paternal) germline G_sα mutation develop obesity, insulin resistance, and diabetes. These parent-of-origin effects are due to G_sα imprinting, with preferential expression from the maternal allele in some tissues. As G_sα is ubiquitously expressed, the tissue involved in this metabolic imprinting effect is unknown. Using brain-specific G_sα knockout mice, we show that G_sα imprinting within the central nervous system underlies these effects and that G_sα is imprinted in the paraventricular nucleus of the hypothalamus. Maternal G_sα mutation impaired melanocortin stimulation of energy expenditure but did not affect melanocortin's effect on food intake, suggesting that melanocortins may regulate energy balance in the central nervous system through both G_sα-dependent and -independent pathways.