Glutamine-induced signaling pathways via amino acid receptors in enteroendocrine L cell lines.

Glucagon-like peptide-1 (GLP-1), secreted by gastrointestinal enteroendocrine L cells, induces insulin secretion and is important for glucose homeostasis. GLP-1 secretion is induced by various luminal nutrients, including amino acids. Intracellular Ca2+ and cAMP dynamics play an important role in GLP-1 secretion regulation; however, several aspects of the underlying mechanism of amino acid-induced GLP-1 secretion are not well characterized. We investigated the mechanisms underlying the L-glutamine-induced increase in Ca2+ and cAMP intracellular concentrations ([Ca2+]i and [cAMP]i, respectively) in murine enteroendocrine L cell line GLUTag cells. Application of L-glutamine to cells under low extracellular [Na+] conditions, which inhibited the function of the sodium-coupled L-glutamine transporter, did not induce an increase in [Ca2+]i. Application of G protein-coupled receptor family C group 6 member A and calcium-sensing receptor antagonist showed little effect on [Ca2+]i and [cAMP]i; however, taste receptor type 1 member 3 (TAS1R3) antagonist suppressed the increase in [cAMP]i. To elucidate the function of TAS1R3, which forms a heterodimeric umami receptor with taste receptor type 1 member 1 (TAS1R1), we generated TAS1R1 and TAS1R3 mutant GLUTag cells using the CRISPR/Cas9 system. TAS1R1 mutant GLUTag cells exhibited L-glutamine-induced increase in [cAMP]i, whereas some TAS1R3 mutant GLUTag cells did not exhibit L-glutamine-induced increase in [cAMP]i and GLP-1 secretion. These findings suggest that TAS1R3 is important for L-glutamine-induced increase in [cAMP]i and GLP-1 secretion. Thus, TAS1R3 may be coupled with Gs and related to cAMP regulation.

Neuronal processing of amino acids in Drosophila: from taste sensing to behavioural regulation.

Finding and feeding on appropriate food are crucial for all animals. Carbohydrates and amino acids are both essential nutrients, albeit with distinct roles: the former are the main energy source whereas the latter are the building blocks of proteins and are used as neurotransmitters. Despite their crucial role, neither the sensing nor the neuronal processing of amino acids is well understood. Studies in Drosophila melanogaster have only recently gained momentum in shedding new light on the molecular and neuronal mechanisms of peripheral and internal amino acid sensing, as well as the organization of amino acid feeding behaviour. Furthermore, amino acids have been shown to act as rewards in associative learning. Focusing on recent studies in Drosophila, we summarize what is known so far about the perception of, and the behavioural responses to, amino acids in insects, and try to identify key questions for future research.

Neuronal D-serine regulates dendritic architecture in the somatosensory cortex.

D-Serine, which is synthesized by the enzyme serine racemase (SR), is a co-agonist at the N-methyl-D-aspartate receptor (NMDAR). In an animal model of NMDAR hypofunction, the constitutive SR knockout (SR-/-) mouse, pyramidal neurons in primary somatosensory cortex (S1) have reductions in the complexity, total length, and spine density of apical and basal dendrites. We wondered whether the dendritic pathology required deprivation of D-serine throughout development or reflected the loss of D-serine only in adulthood. To address this question, we used mice homozygous for floxed SR in which we bred CaMKIICre2834, which is expressed in forebrain glutamatergic neurons starting at 3-4 weeks post-partum (nSR-/-). Our prior studies demonstrated that the majority of cortical SR is expressed in glutamatergic neurons. We found that similar to SR-/- mice, pyramidal neurons in S1 of nSR-/- also had significantly reduced dendritic arborization and spine density, albeit to a lesser degree. S1 neurons of nSR-/- mice had reduced total basal dendritic length that was accompanied by less complex arborization. These characteristics were unaltered in the apical dendritic compartment. In contrast, spine density on S1 neurons was significantly reduced on apical, but not basal dendrites of nSR-/- mice. These results demonstrate that in adulthood neuronally derived D-serine, which is required for optimal activation of post-synaptic NMDAR activity, regulates pyramidal neuron dendritic arborization and spine density. Moreover, they highlight the glycine modulatory site (GMS) of the NMDAR as a potential target for therapeutic intervention in diseases characterized by synaptic deficits, like schizophrenia.

Rules of engagement: factors that regulate activity-dependent synaptic plasticity during neural network development.

Overproduction and pruning during development is a phenomenon that can be observed in the number of organisms in a population, the number of cells in many tissue types, and even the number of synapses on individual neurons. The sculpting of synaptic connections in the brain of a developing organism is guided by its personal experience, which on a neural level translates to specific patterns of activity. Activity-dependent plasticity at glutamatergic synapses is an integral part of neuronal network formation and maturation in developing vertebrate and invertebrate brains. As development of the rodent forebrain transitions away from an over-proliferative state, synaptic plasticity undergoes modification. Late developmental changes in synaptic plasticity signal the establishment of a more stable network and relate to pronounced perceptual and cognitive abilities. In large part, activation of glutamate-sensitive N-methyl-d-aspartate (NMDA) receptors regulates synaptic stabilization during development and is a necessary step in memory formation processes that occur in the forebrain. A developmental change in the subunits that compose NMDA receptors coincides with developmental modifications in synaptic plasticity and cognition, and thus much research in this area focuses on NMDA receptor composition. We propose that there are additional, equally important developmental processes that influence synaptic plasticity, including mechanisms that are upstream (factors that influence NMDA receptors) and downstream (intracellular processes regulated by NMDA receptors) from NMDA receptor activation. The goal of this review is to summarize what is known and what is not well understood about developmental changes in functional plasticity at glutamatergic synapses, and in the end, attempt to relate these changes to maturation of neural networks.

Broad-spectrum amino acid-sensing class C G-protein coupled receptors: molecular mechanisms, physiological significance and options for drug development.

In this article, we consider the molecular mechanisms that underlie broad-spectrum amino acid sensing by a discrete subgroup of class C G-protein-coupled receptors that includes the calcium-sensing receptor, GPRC6A and heterodimers composed of two closely related receptor subunits, T1R(1) and T1R(3). We consider their physiological significance highlighting their diverse spectrum of cellular responses and the phenotypes of global and conditional knock-out mice. In addition, we consider strategies for the development of new drugs that target these receptors.

When and why amino acids?

This article reviews especially the early history of glutamate and GABA as neurotransmitters in vertebrates. The proposal that some amino acids could mediate synaptic transmission in the CNS initially met with much resistance. Both GABA and its parent glutamate are abundant in the brain; but, unlike glutamate, GABA had no obvious metabolic function. By the late 1950s, the switch of interest from electrical to chemical transmission invigorated the search for central transmitters. Its identification with Factor I, a brain extract that inhibited crustacean muscle, focused interest on GABA as a possible inhibitory transmitter. In the first microiontophoretic tests, though GABA strongly inhibited spinal neurons, these effects were considered 'non-specific'. Strong excitation by glutamate (and other acidic amino acids) led to the same conclusion. However, their great potency and rapid actions on cortical neurons convinced other authors that these endogenous amino acids are probably synaptic transmitters. This was partly confirmed by showing that both IPSPs and GABA greatly increased Cl() conductance, their effects having similar reversal potentials. Many anticonvulsants proving to be GABA antagonists, by the 1970s GABA became widely accepted as a mediator of IPSPs. Progress was much slower for glutamate. Being generated on distant dendrites, EPSPs could not be easily compared with glutamate-induced excitation, and the search for specific antagonists was long hampered by the lack of blockers and the variety of glutamate receptors. These difficulties were gradually overcome by the application of powerful techniques, such as single channel recording, cloning receptors, as well as new pharmacological tools.

Engineered socket study of signaling through a four-helix bundle: evidence for a yin-yang mechanism in the kinase control module of the aspartate receptor.

The chemoreceptors of Escherichia coli and Salmonella typhimurium form stable oligomers that associate with the coupling protein CheW and the histidine kinase CheA to form an ultrasensitive, ultrastable signaling lattice. Attractant binding to the periplasmic domain of a given receptor dimer triggers a transmembrane conformational change transmitted through the receptor to its cytoplasmic kinase control module, a long four-helix bundle that binds and regulates CheA kinase. The kinase control module comprises three functional regions: the adaptation region possessing the receptor adaptation sites, a coupling region that transmits signals between other regions, and the protein interaction region possessing contact sites for receptor oligomerization and for CheA-CheW binding. On the basis of the spatial clustering of known signal locking Cys substitutions and engineered disulfide bonds, this study develops the yin-yang hypothesis for signal transmission through the kinase control module. This hypothesis proposes that signals are transmitted through the four-helix bundle via changes in helix-helix packing and that the helix packing changes in the adaptation and protein interaction regions are tightly and antisymmetrically coupled. Specifically, strong helix packing in the adaptation region stabilizes the receptor on state, while strong helix packing in the protein interaction region stabilizes the off state. To test the yin-yang hypothesis, conserved sockets likely to strengthen specific helix-helix contacts via knob-in-hole packing interactions were identified in the adaptation, coupling, and protein interaction regions. For 32 sockets, the knob side chain was truncated to Ala to weaken the knob-in-hole packing and thereby destabilize the local helix-helix interaction provided by that socket. We term this approach a "knob truncation scan". Of the 32 knob truncations, 28 yielded stable receptors. Functional analysis of the signaling state of these receptors revealed seven lock-off knob truncations, all located in the adaptation region, that trap the receptor in its "off" signaling state (low kinase activity, high methylation activity). Also revealed were five lock-on knob truncations, all located in the protein interaction region, that trap the "on" state (high kinase activity, low methylation activity). These findings provide strong evidence that a yin-yang coupling mechanism generates concerted, antisymmetric helix-helix packing changes within the adaptation and protein interaction regions during receptor on-off switching. Conserved sockets that stabilize local helix-helix interactions play a central role in this mechanism: in the on state, sockets are formed in the adaptation region and disrupted in the protein interaction region, while the opposite is true in the off state.

Involvement of hypothalamic paraventricular nucleus non-N-methyl-D-aspartate receptors in the pressor response to noradrenaline microinjected into the bed nucleus of the stria terminalis of unanesthetized rats.

Microinjection of noradrenaline into the bed nucleus of the stria terminalis (BST) has been reported to cause a pressor response in unanesthetized rats, which was shown to be mediated by acute vasopressin release into the systemic circulation. In the present study we verified the involvement of magnocellular neurons of the hypothalamic paraventricular (PVN) or supraoptic (SON) nuclei and the local neurotransmitter involved in the pressor response to noradrenaline microinjection into the BST. The PVN pretreatment with the non-selective neurotransmission blocker CoCl2 (1 nmol/100 nL) inhibited the noradrenaline-evoked pressor response. However, responses were not affected by SON treatment with CoCl2. Further experiments were carried out to test if glutamatergic neurotransmission in the PVN mediates the pressor response evoked by noradrenaline microinjection into the BST. Pretreatment of the PVN with the selective N-methyl-d-aspartate (NMDA) receptor antagonist LY235959 (2 nmol/100 nL) did not affect the noradrenaline-evoked pressor response. However, PVN pretreatment with the selective non-NMDA receptor antagonist NBQX (2 nmol/100 nL) significantly reduced the pressor response to noradrenaline microinjection into the BST. In conclusion, our results suggest that pressor responses to noradrenaline microinjection into the BST are mediated by PVN magnocellular neurons without involvement of SON neurons. They also suggest that a glutamatergic neurotransmission through non-NMDA glutamate receptors in the PVN mediates the response.

The region preceding the C-terminal NWETF pentapeptide modulates baseline activity and aspartate inhibition of Escherichia coli Tar.

The Tar chemoreceptor-CheA-CheW ternary complex of Escherichia coli is a transmembrane allosteric enzyme in which binding of ligands to the periplasmic domain modulates the activity of CheA kinase. Kinase activity is also affected by reversible methylation of four glutamyl residues in the cytoplasmic domain of the receptor. E. coli Tar contains 553 residues. Residues 549-553 comprise the NWETF pentapeptide that binds the CheR methyltransferase and CheB methylesterase. The crystal structure of the similar Tsr chemoreceptor predicts that residues 263-289 and 490-515 of Tar form the most membrane-proximal portion of the extended CD1-CD2 four-helix bundle of the cytoplasmic domain. The last methylation site, Glu-491, is in the C19 heptad, and the N22-19 and C22-19 heptads are present in all classes of bacterial transmembrane chemoreceptors. Residues 516-548 probably serve as a flexible tether for the NWETF pentapeptide. Here, we present a mutational analysis of residues 505-548. The more of this region that is deleted, the less sensitive Tar is to inhibition by aspartate. Tar deleted from residue 505 through the NWETF sequence stimulates CheA in vitro but is not inhibited by aspartate. Thus, interaction of the last two heptads (C21 and C22) of CD2 with the first two heptads (N22 and N21) of CD1 must be important for transmitting an inhibitory signal from the HAMP domain to the four-helix bundle. The R514A, K523A, R529A, R540A, and R542A substitutions, singly or together, increase the level of activation of CheA in vitro, whereas the R505A substitution decreases the level of CheA stimulation by 40% and lowers the aspartate K(i) 7-fold. The R505E substitution completely abolishes stimulation of CheA in vitro. Glu-505 may interact electrostatically with Asp-273 to destabilize the "on" signaling state by loosening the four-helix bundle.

Molecular dynamics of postsynaptic receptors and scaffold proteins.

The activity of neurotransmitter receptors determines the strength of synaptic transmission. Therefore, the clustering of receptors at synapses is an important mechanism underlying synaptic plasticity. The dynamic exchange of receptors between synaptic and extrasynaptic membranes is dependent on their interaction with synaptic scaffold proteins. Here, we review the recent advances and emerging concepts related to the dynamics of synaptic proteins at inhibitory and excitatory synapses. These include the imaging techniques that enable the study of protein dynamics in cells, the differences and similarities of receptor dynamics at excitatory and inhibitory synapses, the relationship between the exchange of receptor and scaffold proteins, as well as the role of receptor fluxes in the modulation of synaptic strength.

Dietary protein and bone health: roles of amino acid-sensing receptors in the control of calcium metabolism and bone homeostasis.

In this article, we review the evidence that dietary protein has a positive influence on bone health, reduces hip fracture risk, and promotes postfracture recovery, and we consider the molecular, cellular, and endocrine bases of the interactions that link protein and calcium metabolism, including effects via IGF-1 and PTH. In addition, we consider the roles of amino acid-sensing mechanisms in coupling dietary protein intake to metabolic change as well as the central role of calcium-sensing receptors (CaRs) in the control of calcium metabolism. Finally, we consider how recently identified broad-spectrum amino acid-sensing receptors from class 3 of the G-protein coupled receptor superfamily including, remarkably, the CaR itself may contribute to the impact of dietary protein on bone.

N-methyl-D-aspartate receptors and amnesia in mice with depression-like state.

We studied the effect of activation (N-methyl-D-aspartic acid and D-cycloserine) and blockade (dizocilpine and 7-chlorokynurenic acid) of N-methyl-D-aspartate receptors on the development of amnesia in intact and depressive mice under conditions of conditioned passive avoidance response. Agonists and antagonists of N-methyl-D-aspartate receptors produce a strong antiamnesic effect in mice with behavioral despair. In intact animals, only N-methyl-D-aspartic acid and D-cycloserine improved passive avoidance performance.

Role of platelet-derived growth factor and vascular endothelial growth factor in obliterative airway disease.

RATIONALE: Platelet-derived growth factor (PDGF) is an important smooth muscle cell mitogen, and vascular endothelial growth factor (VEGF) is a known angiogenic and proinflammatory growth factor. We hypothesized that specific therapy aimed at these growth factors might inhibit the development of experimental obliterative airway disease (OAD). METHODS: In fully mismatched rat tracheal allografts, we used imatinib and PTK/ZK, either alone or in combination, to block PDGF and VEGF receptor protein tyrosine kinase (RTK) action, respectively. Prophylaxis was initiated at the time of transplantation. Early treatment was commenced on Day 7 during the inflammatory phase and late treatment on Day 14 during the fibroproliferative phase of OAD. No immunosuppression was administered. MEASUREMENTS AND MAIN RESULTS: Prophylaxis with either PTK/ZK or imatinib alone significantly reduced OAD, and combined prophylaxis completely prevented its development. Early treatment with PTK/ZK and imatinib also effectively reduced the development of OAD. Late treatment failed to show significant efficacy. Blocking VEGF RTK action with PTK/ZK reduced the activation of allograft blood vessels and the number of lymph vessels in the allograft airway wall, and significantly diminished allograft inflammation, whereas PDGF blockade with imatinib inhibited the growth of smooth muscle cells in the proliferating lesion. CONCLUSIONS: Combined prophylactic PDGF and VEGF RTK blockade completely prevents the development of OAD. Also, when early treatment with PTK/ZK and imatinib is commenced during the inflammatory phase of OAD development, it significantly attenuates the development of tracheal occlusion, suggesting that these drugs could potentially be used to treat bronchiolitis obliterans syndrome in its early phase.

Competitive intra- and extracellular nutrient sensing by the transporter homologue Ssy1p.

Recent studies of Saccharomyces cerevisiae revealed sensors that detect extracellular amino acids (Ssy1p) or glucose (Snf3p and Rgt2p) and are evolutionarily related to the transporters of these nutrients. An intriguing question is whether the evolutionary transformation of transporters into nontransporting sensors reflects a homeostatic capability of transporter-like sensors that could not be easily attained by other types of sensors. We previously found SSY1 mutants with an increased basal level of signaling and increased apparent affinity to sensed extracellular amino acids. On this basis, we propose and test a general model for transporter- like sensors in which occupation of a single, central ligand binding site increases the activation energy needed for the conformational shift between an outward-facing, signaling conformation and an inward-facing, nonsignaling conformation. As predicted, intracellular leucine accumulation competitively inhibits sensing of extracellular amino acids. Thus, a single sensor allows the cell to respond to changes in nutrient availability through detection of the relative concentrations of intra- and extracellular ligand.

Medullary lateral tegmental field neurons influence the timing and pattern of phrenic nerve activity in cats.

In an effort to characterize the role of the medullary lateral tegmental field (LTF) in regulating respiration, we tested the effects of selective blockade of excitatory (EAA) and inhibitory amino acid (IAA) receptors in this region on phrenic nerve activity (PNA) of vagus-intact and vagotomized cats anesthetized with dial-urethane. We found distinct patterns of changes in central respiratory rate, duration of inspiratory and expiratory phases of PNA (Ti and Te, respectively), and I-burst amplitude after selective blockade of EAA and IAA receptors in the LTF. First, blockade of N-methyl-D-aspartate (NMDA) receptors significantly (P < 0.05) decreased central respiratory rate primarily by increasing Ti but did not alter I-burst amplitude. Second, blockade of non-NMDA receptors significantly reduced I-burst amplitude without affecting central respiratory rate. Third, blockade of GABAA receptors significantly decreased central respiratory rate by increasing Te and significantly reduced I-burst amplitude. Fourth, blockade of glycine receptors significantly decreased central respiratory rate by causing proportional increases in Ti and Te and significantly reduced I-burst amplitude. These changes in PNA were markedly different from those produced by blockade of EAA or IAA receptors in the pre-Bötzinger complex. We propose that a proper balance of excitatory and inhibitory inputs to several functionally distinct pools of LTF neurons is essential for maintaining the normal pattern of PNA in anesthetized cats.

Conserved glycine residues in the cytoplasmic domain of the aspartate receptor play essential roles in kinase coupling and on-off switching.

The aspartate receptor of the bacterial chemotaxis pathway serves as a scaffold for the formation of a multiprotein signaling complex containing the receptor and the cytoplasmic pathway components. Within this complex, the receptor regulates the autophosphorylation activity of histidine kinase CheA, thereby controlling the signals sent to the flagellar motor and the receptor adaptation system. The receptor cytoplasmic domain, which controls the on-off switching of CheA, possesses 14 glycine residues that are highly conserved in related receptors. In principle, these conserved glycines could be required for static turns, bends, or close packing in the cytoplasmic domain, or they could be required for conformational dynamics during receptor on-off switching. To determine which glycines are essential and to probe their functional roles, we have substituted each conserved glycine with both alanine and cysteine, and then measured the effects on receptor function in vivo and in vitro. The results reveal a subset of six glycines which are required for receptor function during cellular chemotaxis. Two of these essential glycines (G388 and G391) are located at a hairpin turn at the distal end of the folded cytoplasmic domain, where they are required for the tertiary fold of the signaling subdomain and for CheA kinase activation. Three other essential glycines (G338, G339, and G437) are located at the border between the adaptation and signaling subdomains, where they play key roles in CheA kinase activation and on-off switching. These three glycines form a ring around the four-helix bundle that comprises the receptor cytoplasmic domain, yielding a novel architectural feature termed a bundle hinge. The final essential glycine (G455) is located in the adaptation subdomain where it is required for on-off switching. Overall, the findings confirm that six of the 14 conserved cytoplasmic glycines are essential for receptor function because they enable helix turns and bends required for native receptor structure, and in some cases for switching between the on and off signaling states. An initial working model proposes that the novel bundle hinge enables the four-helix bundle to bend, perhaps during the assembly of the receptor trimer of dimers or during on-off switching. More generally, the findings predict that certain human disease states, including specific cancers, could be triggered by lock-on mutations at essential glycine positions that control the on-off switching of receptors and signaling proteins.

Adaptation mechanism of the aspartate receptor: electrostatics of the adaptation subdomain play a key role in modulating kinase activity.

The aspartate receptor of the Escherichia coli and Salmonella typhimurium chemotaxis pathway generates a transmembrane signal that regulates the activity of the cytoplasmic kinase CheA. Previous studies have identified a region of the cytoplasmic domain that is critical to receptor adaptation and kinase regulation. This region, termed the adaptation subdomain, contains a high density of acidic residues, including specific glutamate residues that serve as receptor adaptation sites. However, the mechanism of signal propagation through this region remains poorly understood. This study uses site-directed mutagenesis to neutralize each acidic residue within the subdomain to probe the hypothesis that electrostatics in this region play a significant role in the mechanism of kinase activation and modulation. Each point mutant was tested for its ability to regulate chemotaxis in vivo and kinase activity in vitro. Four point mutants (D273N, E281Q, D288N, and E477Q) were found to superactivate the kinase relative to the wild-type receptor, and all four of these kinase-activating substitutions are located along the same intersubunit interface as the adaptation sites. These activating substitutions retained the wild-type ability of the attractant-occupied receptor to inhibit kinase activity. When combined in a quadruple mutant (D273N/E281Q/D288N/E477Q), the four charge-neutralizing substitutions locked the receptor in a kinase-superactivating state that could not be fully inactivated by the attractant. Similar lock-on character was observed for a charge reversal substitution, D273R. Together, these results implicate the electrostatic interactions at the intersubunit interface as a major player in signal transduction and kinase regulation. The negative charge in this region destabilizes the local structure in a way that enhances conformational dynamics, as detected by disulfide trapping, and this effect is reversed by charge neutralization of the adaptation sites. Finally, two substitutions (E308Q and E463Q) preserved normal kinase activation in vitro but blocked cellular chemotaxis in vivo, suggesting that these sites lie within the docking site of an adaptation enzyme, CheR or CheB. Overall, this study highlights the importance of electrostatics in signal transduction and regulation of kinase activity by the cytoplasmic domain of the aspartate receptor.

Receptor clustering and signal processing in E. coli chemotaxis.

Chemotaxis in Escherichia coli is one of the most thoroughly studied model systems for signal transduction. Receptor-kinase complexes, organized in clusters at the cell poles, sense chemoeffector stimuli and transmit signals to flagellar motors by phosphorylation of a diffusible response regulator protein. Despite the apparent simplicity of the signal transduction pathway, the high sensitivity, wide dynamic range and integration of multiple stimuli of this pathway remain unexplained. Recent advances in computer modeling and in quantitative experimental analysis suggest that cooperative protein interactions in receptor clusters play a crucial role in the signal processing during bacterial chemotaxis.