Nitric oxide-mediated posttranslational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability.

Nitric oxide (NO) regulates neuronal function and thus is critical for tuning neuronal communication. Mechanisms by which NO modulates protein function and interaction include posttranslational modifications (PTMs) such as S-nitrosylation. Importantly, cross signaling between S-nitrosylation and prenylation can have major regulatory potential. However, the exact protein targets and resulting changes in function remain elusive. Here, we interrogated the role of NO-dependent PTMs and farnesylation in synaptic transmission. We found that NO compromises synaptic function at the Drosophila neuromuscular junction (NMJ) in a cGMP-independent manner. NO suppressed release and reduced the size of available vesicle pools, which was reversed by glutathione (GSH) and occluded by genetic up-regulation of GSH-generating and de-nitrosylating glutamate-cysteine-ligase and S-nitroso-glutathione reductase activities. Enhanced nitrergic activity led to S-nitrosylation of the fusion-clamp protein complexin (cpx) and altered its membrane association and interactions with active zone (AZ) and soluble N-ethyl-maleimide-sensitive fusion protein Attachment Protein Receptor (SNARE) proteins. Furthermore, genetic and pharmacological suppression of farnesylation and a nitrosylation mimetic mutant of cpx induced identical physiological and localization phenotypes as caused by NO. Together, our data provide evidence for a novel physiological nitrergic molecular switch involving S-nitrosylation, which reversibly suppresses farnesylation and thereby enhances the net-clamping function of cpx. These data illustrate a new mechanistic signaling pathway by which regulation of farnesylation can fine-tune synaptic release.

Ligand-Specific Signaling Profiles and Resensitization Mechanisms of the Neuromedin U2 Receptor.

The structurally related, but distinct neuropeptides, neuromedin U (NmU) and neuromedin S (NmS) are ligands of two G protein-coupled NmU receptors (NMU1 and NMU2). Hypothalamic NMU2 regulates feeding behavior and energy expenditure and has therapeutic potential as an anti-obesity target, making an understanding of its signaling and regulation of particular interest. NMU2 binds both NmU and NmS with high affinity, resulting in receptor-ligand co-internalization. We have investigated whether receptor trafficking events post-internalization are biased by the ligand bound and can therefore influence signaling function. Using recombinant cell lines expressing human NMU2, we demonstrate that acute Ca2+ signaling responses to NmU or NmS are indistinguishable and that restoration of responsiveness (resensitization) requires receptor internalization and endosomal acidification. The rate of NMU2 resensitization is faster following NmU compared with NmS exposure, but is similar if endothelin-converting enzyme-1 activity is inhibited or knocked down. Although acute activation of extracellular signal-regulated kinase (ERK) is also similar, activation by NMU2 is longer lasting if NmS is the ligand. Furthermore, when cells are briefly challenged before removal of free, but not receptor-bound ligand, activation of ERK and p38 mitogen-activated protein kinase by NmS is more sustained. However, only NmU responses are potentiated and extended by endothelin-converting enzyme-1 inhibition. These data indicate that differential intracellular ligand processing produces different signaling and receptor resensitization profiles and add to the findings of other studies demonstrating that intracellular ligand processing can shape receptor behavior and signal transduction.

Small-Molecule G Protein-Coupled Receptor Kinase Inhibitors Attenuate G Protein-Coupled Receptor Kinase 2-Mediated Desensitization of Vasoconstrictor-Induced Arterial Contractions.

Vasoconstrictor-driven G protein-coupled receptor (GPCR)/phospholipase C (PLC) signaling increases intracellular Ca2+ concentration to mediate arterial contraction. To counteract vasoconstrictor-induced contraction, GPCR/PLC signaling can be desensitized by G protein-coupled receptor kinases (GRKs), with GRK2 playing a predominant role in isolated arterial smooth muscle cells. In this study, we use an array of GRK2 inhibitors to assess their effects on the desensitization of UTP and angiotensin II (AngII)-mediated arterial contractions. The effects of GRK2 inhibitors on the desensitization of UTP- or AngII-stimulated mesenteric third-order arterial contractions, and PLC activity in isolated mesenteric smooth muscle cells (MSMC), were determined using wire myography and Ca2+ imaging, respectively. Applying a stimulation protocol to cause receptor desensitization resulted in reductions in UTP- and AngII-stimulated arterial contractions. Preincubation with the GRK2 inhibitor paroxetine almost completely prevented desensitization of UTP- and attenuated desensitization of AngII-stimulated arterial contractions. In contrast, fluoxetine was ineffective. Preincubation with alternative GRK2 inhibitors (Takeda compound 101 or CCG224063) also attenuated the desensitization of UTP-mediated arterial contractile responses. In isolated MSMC, paroxetine, Takeda compound 101, and CCG224063 also attenuated the desensitization of UTP- and AngII-stimulated increases in Ca2+, whereas fluoxetine did not. In human uterine smooth muscle cells, paroxetine reversed GRK2-mediated histamine H1 receptor desensitization, but not GRK6-mediated oxytocin receptor desensitization. Utilizing various small-molecule GRK2 inhibitors, we confirm that GRK2 plays a central role in regulating vasoconstrictor-mediated arterial tone, highlighting a potentially novel strategy for blood pressure regulation through targeting GRK2 function.

An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory.

Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser(228)) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser(228) These data supported the hypothesis that phosphorylation at Ser(228) was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser(228) on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser(228) phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser(228) not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning.

Defining the roles of arrestin2 and arrestin3 in vasoconstrictor receptor desensitization in hypertension.

Prolonged vasoconstrictor-stimulated phospholipase C activity can induce arterial constriction, hypertension, and smooth muscle hypertrophy/hyperplasia. Arrestin proteins are recruited by agonist-occupied G protein-coupled receptors to terminate signaling and counteract changes in vascular tone. Here we determine whether the development of hypertension affects arrestin expression in resistance arteries and how such changes alter arterial contractile signaling and function. Arrestin2/3 expression was increased in mesenteric arteries of 12-wk-old spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto (WKY) controls, while no differences in arrestin expression were observed between 6-wk-old SHR and WKY animals. In mesenteric artery myography experiments, high extracellular K(+)-stimulated contractions were increased in both 6- and 12-wk-old SHR animals. Concentration-response experiments for uridine 5'-triphosphate (UTP) acting through P2Y receptors displayed a leftward shift in 12-wk, but not 6-wk-old animals. Desensitization of UTP-stimulated vessel contractions was increased in 12-wk-old (but not 6-wk-old) SHR animals. Dual IP3/Ca(2+) imaging in mesenteric arterial cells showed that desensitization of UTP and endothelin-1 (ET1) responses was enhanced in 12-wk-old (but not 6-wk-old) SHR compared with WKY rats. siRNA-mediated depletion of arrestin2 for UTP and arrestin3 for ET1, reversed the desensitization of PLC signaling. In conclusion, arrestin2 and 3 expression is elevated in resistance arteries during the emergence of the early hypertensive phenotype, which underlies an enhanced ability to desensitize vasoconstrictor signaling and vessel contraction. Such regulatory changes may act to compensate for increased vasoconstrictor-induced vessel contraction.

Nitric oxide synthesis and cGMP production is important for neurite growth and synapse remodeling after axotomy.

Nitric oxide (NO) is an important signaling molecule with a variety of functions in the CNS, including a potential role in modulating neuronal growth and synapse formation. In the present study, we used tractable, identified neurons in the CNS of the pond snail Lymnaea stagnalis to study the role of endogenous NO signaling in neuronal growth and synaptic remodeling after nerve injury. Axonal damage of L. stagnalis neurons B1 and B2 induces extensive central growth of neurites that is accompanied by changes in existing electrical connections, the transient formation of novel electrical connections, and the formation of a novel excitatory chemical synapse from B2 to B1 neurons. Partial chronic inhibition of endogenous NO synthesis reduces neurite growth in NO-synthase-expressing B2, but has only minor effects on NOS-negative B1 neurons. Chronic application of an NO donor while inhibiting endogenous NO synthesis rescues neurite extension in B2 neurons and boosts growth of B1 neurons. Blocking soluble guanylate cyclase activity completely suppresses neurite extension and synaptic remodeling after nerve crush, demonstrating the importance of cGMP in these processes. Interestingly, inhibition of cGMP-dependent protein kinase only suppresses chemical synapse formation without effects on neuronal growth and electrical synapse remodeling. We conclude that NO signaling via cGMP is an important modulator of both neurite growth and synaptic remodeling after nerve crush. However, differential effects of cGMP-dependent protein kinase inhibition on neurite growth and synaptic remodeling suggest that these effects are mediated by separate signaling pathways.

Long-term channel block is required to inhibit cellular transformation by human ether-à-go-go-related gene (hERG1) potassium channels.

Both human ether-à-go-go-related gene (hERG1) and the closely related human ether-à-go-go (hEAG1) channel are aberrantly expressed in a large proportion of human cancers. In the present study, we demonstrate that transfection of hERG1 into mouse fibroblasts is sufficient to induce many features characteristic of malignant transformation. An important finding of this work is that this transformation could be reversed by chronic incubation (for 2-3 weeks) with the hERG channel blocker dofetilide (100 nM), whereas more acute applications (for 1-2 days) were ineffective. The hERG1 expression resulted in a profound loss of cell contact inhibition, multiple layers of overgrowing cells, and high saturation densities. Cells also changed from fibroblast-like to a more spindle-shaped morphology, which was associated with a smaller cell size, a dramatic increase in cell polarization, a reduction in the number of actin stress fibers, and less punctate labeling of focal adhesions. Analysis of single-cell migration and scratch-wound closure clearly demonstrated that hERG1-expressing cells migrated more rapidly than vector-transfected control cells. In contrast to previous studies on hEAG1, there were no increases in rates of proliferation, or loss of growth factor dependency; however, hERG1-expressing cells were capable of substrate-independent growth. Allogeneic transplantation of hERG1-expressing cells into nude mice resulted in an increased incidence of tumors. In contrast to hEAG1, the mechanism of cellular transformation is dependent on ion conduction. Trafficking-deficient and conduction-deficient hERG1 mutants also prevented cellular transformation. These results provide evidence that hERG1 expression is sufficient to induce cellular transformation by a mechanism distinct from hEAG1. The most important conclusion of this study is that selective hERG1 channel blockers have therapeutic potential in the treatment of hERG1-expressing cancers.

Novel structural and functional insights into M3 muscarinic receptor dimer/oligomer formation.

Class A G protein-coupled receptors (GPCRs) are able to form homodimers and/or oligomeric arrays. We recently proposed, based on bioluminescence resonance energy transfer studies with the M3 muscarinic receptor (M3R), a prototypic class A GPCR, that the M3R is able to form multiple, structurally distinct dimers that are probably transient in nature (McMillin, S. M., Heusel, M., Liu, T., Costanzi, S., and Wess, J. (2011) J. Biol. Chem. 286, 28584-28598). To provide more direct experimental support for this concept, we employed a disulfide cross-linking strategy to trap various M3R dimeric species present in a native lipid environment (transfected COS-7 cells). Disulfide cross-linking studies were carried out with many mutant M3Rs containing single cysteine (Cys) substitutions within two distinct cytoplasmic M3R regions, the C-terminal portion of the second intracellular loop (i2) and helix H8 (H8). The pattern of cross-links that we obtained, in combination with molecular modeling studies, was consistent with the existence of two structurally distinct M3R dimer interfaces, one involving i2/i2 contacts (TM4-TM5-i2 interface) and the other one characterized by H8-H8 interactions (TM1-TM2-H8 interface). Specific H8-H8 disulfide cross-links led to significant impairments in M3R-mediated G protein activation, suggesting that changes in the structural orientation or mobility of H8 are critical for efficient receptor-G protein coupling. Our findings provide novel structural and functional insights into the mechanisms involved in M3R dimerization (oligomerization). Because the M3R shows a high degree of sequence similarity with many other class A GPCRs, our findings should be of considerable general interest.

[Ca2+]i oscillations in ASM: relationship with persistent airflow obstruction in asthma.

The cause of airway smooth muscle (ASM) hypercontractility in asthma is not fully understood. The relationship of spontaneous intracellular calcium oscillation frequency in ASM to asthma severity was investigated. Oscillations were increased in subjects with impaired lung function abolished by extracellular calcium removal, attenuated by caffeine and unaffected by verapamil or nitrendipine. Whether modulation of increased spontaneous intracellular calcium oscillations in ASM from patients with impaired lung function represents a therapeutic target warrants further investigation.

Structural aspects of M3 muscarinic acetylcholine receptor dimer formation and activation.

To explore the structural mechanisms underlying the assembly and activation of family A GPCR dimers, we used the rat M(3) muscarinic acetylcholine receptor (M3R) as a model system. Studies with Cys-substituted mutant M3Rs expressed in COS-7 cells led to the identification of several mutant M3Rs that exclusively existed as cross-linked dimers under oxidizing conditions. The cross-linked residues were located at the bottom of transmembrane domain 5 (TM5) and within the N-terminal portion of the third intracellular loop (i3 loop). Studies with urea-stripped membranes demonstrated that M3R disulfide cross-linking did not require the presence of heterotrimeric G proteins. Molecular modeling studies indicated that the cross-linking data were in excellent agreement with the existence of a low-energy M3R dimer characterized by a TM5-TM5 interface. [(35)S]GTPγS binding/Gα(q/11) immunoprecipitation assays revealed that an M3R dimer that was cross-linked within the N-terminal portion of the i3 loop (264C) was functionally severely impaired (∼50% reduction in receptor-G-protein coupling, as compared to control M3R). These data support the novel concept that agonist-induced activation of M3R dimers requires a conformational change of the N-terminal segment of the i3 loop. Given the high degree of structural homology among family A GPCRs, these findings should be of broad significance.

Increased nicotinamide adenine dinucleotide phosphate oxidase 4 expression mediates intrinsic airway smooth muscle hypercontractility in asthma.

RATIONALE: Asthma is characterized by disordered airway physiology as a consequence of increased airway smooth muscle contractility. The underlying cause of this hypercontractility is poorly understood. OBJECTIVES: We sought to investigate whether the burden of oxidative stress in airway smooth muscle in asthma is heightened and mediated by an intrinsic abnormality promoting hypercontractility. METHODS: We examined the oxidative stress burden of airway smooth muscle in bronchial biopsies and primary cells from subjects with asthma and healthy controls. We determined the expression of targets implicated in the control of oxidative stress in airway smooth muscle and their role in contractility. MEASUREMENTS AND MAIN RESULTS: We found that the oxidative stress burden in the airway smooth muscle in individuals with asthma is heightened and related to the degree of airflow obstruction and airway hyperresponsiveness. This was independent of the asthmatic environment as in vitro primary airway smooth muscle from individuals with asthma compared with healthy controls demonstrated increased oxidative stress-induced DNA damage together with an increased production of reactive oxygen species. Genome-wide microarray of primary airway smooth muscle identified increased messenger RNA expression in asthma of NADPH oxidase (NOX) subtype 4. This NOX4 overexpression in asthma was supported by quantitative polymerase chain reaction, confirmed at the protein level. Airway smooth muscle from individuals with asthma exhibited increased agonist-induced contraction. This was abrogated by NOX4 small interfering RNA knockdown and the pharmacological inhibitors diphenyleneiodonium and apocynin. CONCLUSIONS: Our findings support a critical role for NOX4 overexpression in asthma in the promotion of oxidative stress and consequent airway smooth muscle hypercontractility. This implicates NOX4 as a potential novel target for asthma therapy.

GRK2 expression and catalytic activity are essential for vasoconstrictor/ERK-stimulated arterial smooth muscle proliferation.

Prolonged vasoconstrictor signalling found in hypertension, increases arterial contraction, and alters vessel architecture by stimulating arterial smooth muscle cell (ASMC) growth, underpinning the development of re-stenosis lesions and vascular remodelling. Vasoconstrictors interact with their cognate G protein coupled receptors activating a variety of signalling pathways to promote smooth muscle proliferation. Here, angiotensin II (AngII) and endothelin 1 (ET1), but not UTP stimulates ASMC proliferation. Moreover, siRNA-mediated depletion of endogenous GRK2 expression, or GRK2 inhibitors, compound 101 or paroxetine, prevented AngII and ET1-promoted ASMC growth. Depletion of GRK2 expression or inhibition of GRK2 activity ablated the prolonged phase of AngII and ET-stimulated ERK signalling, while enhancing and prolonging UTP-stimulated ERK signalling. Increased GRK2 expression enhanced and prolonged AngII and ET1-stimulated ERK signalling, but suppressed UTP-stimulated ERK signalling. In ASMC prepared from 6-week-old WKY and SHR, AngII and ET1-stimulated proliferation rates were similar, however, in cultures prepared from 12-week-old rats AngII and ET1-stimulated growth was enhanced in SHR-derived ASMC, which was reversed following depletion of GRK2 expression. Furthermore, in ASMC cultures isolated from 6-week-old WKY and SHR rats, AngII and ET1-stimulated ERK signals were similar, while in cultures from 12-week-old rats ERK signals were both enhanced and prolonged in SHR-derived ASMC, and were reversed to those seen in age-matched WKY-derived ASMC following pre-treatment of SHR-derived ASMC with compound 101. These data indicate that the presence of GRK2 and its catalytic activity are essential to enable pro-proliferative vasoconstrictors to promote growth via recruitment and activation of the ERK signalling pathway in ASMC.

Arrestins 2 and 3 differentially regulate ETA and P2Y2 receptor-mediated cell signaling and migration in arterial smooth muscle.

Overstimulation of endothelin type A (ET(A)) and nucleotide (P2Y) Gα(q)-coupled receptors in vascular smooth muscle causes vasoconstriction, hypertension, and, eventually, hypertrophy and vascular occlusion. G protein-coupled receptor kinases (GRKs) and arrestin proteins are sequentially recruited by agonist-occupied Gα(q)-coupled receptors to terminate phospholipase C signaling, preventing prolonged/inappropriate contractile signaling. However, these proteins also play roles in the regulation of several mitogen-activated protein kinase (MAPK) signaling cascades known to be essential for vascular remodeling. Here we investigated whether different arrestin isoforms regulate endothelin and nucleotide receptor MAPK signaling in rat aortic smooth muscle cells (ASMCs). When intracellular Ca(2+) levels were assessed in isolated ASMCs loaded with Ca(2+)-sensitive dyes, P2Y(2) and ET(A) receptor desensitization was attenuated by selective small-interfering (si)RNA-mediated depletion of G protein-coupled receptor kinase 2 (GRK2). Using similar siRNA techniques, knockdown of arrestin2 prevented P2Y(2) receptor desensitization and enhanced and prolonged p38 and ERK MAPK signals, while arrestin3 depletion was ineffective. Conversely, arrestin3 knockdown prevented ET(A) receptor desensitization and attenuated ET1-stimulated p38 and ERK signals, while arrestin2 depletion had no effect. Using Transwell assays to assess agonist-stimulated ASMC migration, we found that UTP-stimulated migration was markedly attenuated following arrestin2 depletion, while ET1-stimulated migration was attenuated following knockdown of either arrestin. These data highlight a differential arrestin-dependent regulation of ET(A) and P2Y(2) receptor-stimulated MAPK signaling. GRK2 and arrestin expression are essential for agonist-stimulated ASMC migration, which, as a key process in vascular remodeling, highlights the potential roles of GRK2 and arrestin proteins in the progression of vascular disease.

Quantitative analysis reveals multiple mechanisms of allosteric modulation of the mGlu5 receptor in rat astroglia.

Positive and negative allosteric modulators (PAMs and NAMs, respectively) of the type 5 metabotropic glutamate (mGlu5) receptor have demonstrable therapeutic potential in an array of neurological and psychiatric disorders. Here, we have used rat cortical astrocytes to investigate how PAMs and NAMs mediate their activity and reveal marked differences between PAMs with respect to their modulation of orthosteric agonist affinity and efficacy. Affinity cooperativity factors (α) were assessed using [(3)H]2-methyl-6-(phenylethynyl)-pyridine (MPEP)-PAM competition binding in the absence and presence of orthosteric agonist, whereas efficacy cooperativity factors (ß) were calculated from net affinity/efficacy cooperativity parameters (αß) obtained from analyses of the abilities of PAMs to potentiate [(3)H]inositol phosphate accumulation in astrocytes stimulated with a submaximal (EC(20)) concentration of orthosteric agonist. We report that whereas 3,3'-difluorobenzaldazine (DFB) and 3-cyano-N-(1,3-diphenyl-1H-prazol-5-yl)benzamide (CDPPB) primarily exert their allosteric modulatory effects through modifying the apparent orthosteric agonist affinity at the astrocyte mGlu5 receptor, the effects of S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidinl-1-yl}-methanone (ADX47273) are mediated primarily via efficacy-driven modulation. In [(3)H]MPEP-NAM competition binding assays, both MPEP and 2-(2-(3-methoxyphenyl)ethynyl)-5-methylpyridine (M-5MPEP) defined similar specific binding components, with affinities that were unaltered in the presence of orthosteric agonist, indicating wholly negative efficacy-driven modulations. It is noteworthy that whereas M-5MPEP only partially inhibited orthosteric agonist-stimulated [(3)H]inositol phosphate accumulation in astrocytes, it could completely suppress Ca(2+) oscillations stimulated by quisqualate or (S)-3,5-dihydroxyphenylglycine. In contrast, MPEP was fully inhibitory with respect to both functional responses. The finding that M-5MPEP has different functional effects depending on the endpoint measured is discussed as a possible example of permissive allosteric antagonism.

Early failure of N-methyl-D-aspartate receptors and deficient spine formation induced by reduction of regulatory heme in neurons.

An initial stage of many neurodegenerative processes is associated with compromised synaptic function and precedes synapse loss, neurite fragmentation, and neuronal death. We showed previously that deficiency of heme, regulating many proteins of pharmacological importance, causes neurodegeneration of primary cortical neurons via N-methyl-d-aspartate receptor (NMDAR)-dependent suppression of the extracellular signal-regulated kinase 1/2 pathway. Here, we asked whether the reduction of heme causes synaptic perturbation before neurite fragmentation in neuronal cultures and investigated molecular mechanisms of synaptic dysfunction in these cells. We showed the change in the NR2B subunit phosphorylation that correlates with compromised NMDAR function after the reduction of regulatory heme and a rapid rescue of NR2B phosphorylation and NMDAR function by exogenous heme. Electrophysiological recordings demonstrated diminished NMDAR currents and NMDAR-mediated calcium influx after 24 h of inhibition of heme synthesis. These effects were reversed by treatment with heme; however, inhibition of the Src family kinases abolished the rescue effect of heme on NMDA-evoked currents. Diminished NMDAR current and Ca(2+) influx resulted in suppressed cGMP production and impairment of spine formation. Exogenous heme exerted rescue effects on NR2B tyrosine phosphorylation and NMDA-evoked currents within minutes, suggesting direct interactions within the NMDAR complex. These synaptic changes after inhibition of heme synthesis occurred at this stage without apparent dysfunction of major hemoproteins. We conclude that regulatory heme is necessary in maintaining NR2B phosphorylation and NMDAR function. NMDAR failure occurs before neurite fragmentation and may be a causal factor in neurodegeneration; this could suggest a route for an early pharmacological intervention.

Alternative splicing of G protein-coupled receptors: physiology and pathophysiology.

The G protein-coupled receptors (GPCRs) are a superfamily of transmembrane receptors that have a broad distribution and can collectively recognise a diverse array of ligands. Activation or inhibition of GPCR signalling can affect many (patho)physiological processes, and consequently they are a major target for existing and emerging drug therapies. A common observation has been that the pharmacological, signalling and regulatory properties of GPCRs can differ in a cell- and tissue-specific manner. Such "phenotypic" diversity might be attributable to post-translational modifications and/or association of GPCRs with accessory proteins, however, post-transcriptional mechanisms are also likely to contribute. Although approximately 50% of GPCR genes are intronless, those that possess introns can undergo alternative splicing, generating GPCR subtype isoforms that may differ in their pharmacological, signalling and regulatory properties. In this review we shall highlight recent research into GPCR splice variation and discuss the potential consequences this might have for GPCR function in health and disease.

Effects of positive allosteric modulators on single-cell oscillatory Ca2+ signaling initiated by the type 5 metabotropic glutamate receptor.

Agonist stimulation of the type 5 metabotropic glutamate (mGlu5) receptor initiates robust oscillatory changes in cytosolic Ca2+ concentration ([Ca2+]i) in single cells by rapid, repeated cycles of phosphorylation/dephosphorylation of the mGlu5 receptor, involving protein kinase C and as-yet-unspecified protein phosphatase activities. An emergent property of this type of Ca2+ oscillation-generating mechanism (termed "dynamic uncoupling") is that once a threshold concentration has been reached to initiate the Ca2+ oscillation, its frequency is largely insensitive to further increases in orthosteric agonist concentration. Here, we report the effects of positive allosteric modulators (PAMs) on the patterns of single-cell Ca2+ signaling in recombinant and native mGlu5 receptor-expressing systems. In a Chinese hamster ovary cell-line (CHO-lac-mGlu5a), none of the mGlu5 receptor PAMs studied [3,3'-difluorobenzaldazine (DFB), N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) methyl]phenyl}-2-hydroxy-benzamide (CPPHA), 3-cyano-N-(1, 3-diphenyl-1H-prazol-5-yl)benzamide (CDPPB), S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidinl-1-yl}-methanone (ADX47273)], stimulated a Ca2+ response when applied alone, but each PAM concentration-dependently increased the frequency, without affecting the amplitude, of Ca2+ oscillations induced by glutamate or quisqualate. Therefore, PAMs can cause graded increases (and negative allosteric modulator-graded decreases) in the Ca2+ oscillation frequency stimulated by orthosteric agonist. Initial data in rat cerebrocortical astrocytes demonstrated that similar effects of PAMs could be observed in a native cell background, although at high orthosteric agonist concentrations, PAM addition could much more often be seen to drive rapid Ca2+ oscillations into peak-plateau responses. These data demonstrate that allosteric modulators can "tune" the Ca2+ oscillation frequency initiated by mGlu5 receptor activation, and this might allow pharmacological modification of the downstream processes (e.g., transcriptional regulation) that is unachievable through orthosteric ligand interactions.

Contrasting effects of allosteric and orthosteric agonists on m1 muscarinic acetylcholine receptor internalization and down-regulation.

A new class of subtype-selective muscarinic acetylcholine (mACh) receptor agonist that activates the receptor through interaction at a site distinct from the orthosteric acetylcholine binding site has been reported recently. Here, we have compared the effects of orthosteric (oxotremorine-M, arecoline, pilocarpine) and allosteric [4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine (AC-42); 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1)] agonists on M(1) mACh receptor internalization and down-regulation, as well as functional coupling in a Chinese hamster ovary (CHO) cell line. In contrast to full and partial orthosteric agonists, which cause significant receptor internalization and down-regulation, prolonged exposure to AC-42 did not significantly alter either cell-surface or total cellular M(1) mACh receptor expression. 77-LH-28-1, an AC-42 homolog, did cause some receptor internalization, but not down-regulation. The presence of atropine completely prevented the orthosteric agonist-induced adaptive changes in receptor populations; however, in contrast, the copresence of atropine and AC-42 significantly increased both cell-surface receptor and total M(1) mACh receptor expression. Maximal phosphoinositide hydrolysis responses to the partial agonist arecoline were similar in CHO-M(1) cells pretreated for 24 h with either AC-42 or vehicle; in contrast, these responses were markedly reduced when cells were pretreated with oxotremorine-M or pilocarpine. These data indicate that, whereas AC-42 binding to the M(1) mACh receptor can initiate signal transduction, the AC-42-liganded receptor is resistant to the usual mechanisms regulating receptor internalization and down-regulation. In addition, our data suggest unusual interactions between allosteric agonists and orthosteric antagonists to regulate cell-surface and total cellular receptor expression.

Selective regulation of H1 histamine receptor signaling by G protein-coupled receptor kinase 2 in uterine smooth muscle cells.

Histamine stimulates uterine contraction; however, little is known regarding the mechanism or regulation of uterine histamine receptor signaling. Here we investigated the regulation of Galpha(q/11)-coupled histamine receptor signaling in human myometrial smooth muscle cells using the inositol 1,4,5-trisphosphate biosensor pleckstrin homology domain of phospholipase-delta1 tagged to enhanced green fluorescent protein and the Ca(2+)-sensitive dye Fluo-4. Histamine addition caused concentration-dependent increases in inositol 1,4,5-trisphosphate and [Ca(2+)](i) in the ULTR human uterine smooth muscle cell line and primary human myometrial cells. These effects were completely inhibited by the H(1) histamine receptor antagonist, diphenhydramine, and were unaffected by the H(2) histamine receptor antagonist, cimetidine. ULTR and primary myometrial cells were transfected with either dominant-negative G protein-coupled receptor kinases (GRKs) or small interfering RNAs targeting specific GRKs to assess the roles of this protein kinase family in H(1) histamine receptor desensitization. Dominant-negative GRK2, but not GRK5 or GRK6, prevented H(1) histamine receptor desensitization. Similarly, transfection with short interfering RNAs (that each caused >70% depletion of the targeted GRK) for GRK2, but not GRK3 or GRK6, also prevented H(1) histamine receptor desensitization. Our data suggest that histamine stimulates phospholipase C-signaling in myometrial smooth muscle cells through H(1) histamine receptors and that GRK2 recruitment is a key mechanism in the regulation of H(1) histamine receptor signaling in human uterine smooth muscle. These data provide insights into the in situ regulation of this receptor subtype and may inform pathophysiological functioning in preterm labor and other conditions involving uterine smooth muscle dysregulation.