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

Reversible and non-competitive antagonist profile of CPCCOEt at the human type 1 alpha metabotropic glutamate receptor

In transfected CHO cells expressing the human metabotropic glutamate receptor mGlu1 alpha, 7- hydroxyimino)cyclopropan[b]chromen-1a-carboxylic acid ethylester (CPCCOEt) was found to antagonize L-quisqualate-induced phosphoinositide hydrolysis in a non-competitive and reversible manner (apparent pK(i) value, 4.76 +/- 0.18: It = 3. This suggests that CPCCOEt antagonizes type Ir metabotropic glutamate receptor activation by interacting with a site distinct from the agonist binding site. (C) 1998 Elsevier Science Ltd. All rights reserved

Complex involvement of pertussis toxin-sensitive G proteins in the regulation of type 1 alpha metabotropic glutamate receptor signaling in baby hamster kidney cells

Previously, we demonstrated that the coupling of the metabotropic glutamate receptor mGlu1 alpha to phosphoinositide hydrolysis is enhanced by pertussis toxin (PTX) in stably transfected baby hamster kidney cells (BHK). Here, we show that the PTX effect on agonist-stimulated [H-3]inositol phosphate accumulation can be resolved into two components: an immediate increase in agonist potency, and a more slowly developing increase in the magnitude of the response observed at maximally effective agonist concentrations. Using G(q)/(11)alpha- and G(i/o)alpha-selective antibodies to immunoprecipitate [S-35]guanosine-5'-O-(3-thio)triphosphate-bound G alpha proteins, we also show that agonist stimulation of mGlu1a in BHK membranes increases specific [S-35]guanosine-5'-O-(3-thio)triphosphate binding to both G(q/11) and G(i/o) proteins. Preincubation of BHK-mGlu1 alpha with L-glutamate (300 mu M) results in a progressive loss (60% in 30 min) of L-quisqualate-induced [H-3]inositol phosphate accumulation (without a change in potency), providing evidence for agonist-induced receptor desensitization. Although such desensitization of mGlu receptor signaling was mimicked by a phorbol ester, agonist-induced phosphorylation of the receptor was not observed and protein kinase C inhibition by Ro 31-8220 did not prevent L-glutamate-mediated desensitization. In contrast, PTX treatment of the cells almost completely prevented L-glutamate-mediated desensitization. Together, these data provide evidence for a multifunctional coupling of mGlu1a to different types of G proteins, including PTX-sensitive G(i)-type G proteins. The latter are involved in the negative control of phospholipase C activity while also influencing the rate of desensitization of the mGlu1 alpha receptor

Inhibition of N-linked glycosylation of the human type 1 alpha metabotropic glutamate receptor by tunicamycin: effects on cell-surface receptor expression and function

The potential role of N-linked glycosylation of the human type 1 alpha metabotropic glutamate (mGlu1 alpha) receptor was studied in a recombinant, inducible expression system, where receptor expression was induced in the absence and presence of tunicamycin. In the absence of tunicamycin the mGlu1 alpha receptor appeared to be expressed, at least in part, as a dimer consisting of monomers of approx. 145 and 160 KDa relative molecular mass (M-r). In the presence of tunicamycin only a single monomeric protein could be detected approximating the M-r predicted for the human mGlu1 alpha receptor based on its primary amino acid sequence (130 KDa). Exposure to tunicamycin during receptor induction did not appear to affect the cell surface expression of the mGlu1 alpha receptor as determined immunocytochemically or using a cell-surface biotinylation strategy, but reduced agonist-stimulated phosphoinositide hydrolysis by approximately 50% compared to control cell populations. Our data suggest that non-N-glycosylated human mGlu1 alpha receptors can traffic to the cell surface and activate phospholipase C. (C) 1999 Elsevier Science Ltd. All rights reserved

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.</p

Nitric oxide is a volume transmitter regulating postsynaptic excitability at a glutamatergic synapse.

Neuronal nitric oxide synthase (nNOS) is broadly expressed in the brain and associated with synaptic plasticity through NMDAR -mediated calcium influx. However, its physiological activation and the mechanisms by which nitric oxide (NO) influences synaptic transmission have proved elusive. Here, we exploit the unique input-specificity of the calyx of Held to characterize NO modulation at this glutamatergic synapse in the auditory pathway. NO is generated in an activity-dependent manner by principle neurons receiving a calyceal synaptic input. It acts in the target and adjacent inactive neurons to modulate excitability and synaptic efficacy, inhibiting postsynaptic Kv3 potassium currents (via phosphorylation), reducing EPSCs and so increasing action potential duration and reducing the fidelity of transmission. We conclude that NO serves as a volume transmitter and slow dynamic modulator, integrating spontaneous and evoked neuronal firing, providing an index of global activity and regulating information transmission across a population of active and inactive neurons