Monoaminylation of Fibrinogen and Glia-Derived Proteins: Indication for Similar Mechanisms in Posttranslational Protein Modification in Blood and Brain.

Distinct proteins have been demonstrated to be posttranslationally modified by covalent transamidation of serotonin (5-hydropxytryptamin) to glutamine residues of the target proteins. This process is mediated by transglutaminase (TGase) and has been termed "serotonylation." It has also been shown that other biogenic amines, including the neurotransmitters dopamine and norepinephrine, can substitute for serotonin, implying a more general mechanism of "monoaminylation" for this kind of protein modification. Here we transamidated the autofluorescent monoamine monodansylcadaverine (MDC) to purified plasma fibrinogen and to proteins from a primary glia cell culture. Electrophoretic separation of MDC-conjugated proteins followed by mass spectrometry identified three fibrinogen subunits (Aα, Bß, γ), a homomeric Aα2 dimer, and adducts of >250 kDa molecular weight, as well as several glial proteins. TGase-mediated MDC incorporation was strongly reduced by serotonin, underlining the general mechanism of monoaminylation.

Transglutaminase-mediated transamidation of serotonin, dopamine and noradrenaline to fibronectin: evidence for a general mechanism of monoaminylation.

The activity of some small GTPases is regulated by covalent transamidation of serotonin (5-hydropxytryptamien) to glutamine residues of the enzymes. This process is mediated by transglutaminase (TGase) and is termed "serotonylation". In addition, serotonylation of neural proteins and proteins of the extracellular matrix such as fibronectin has been demonstrated. Here we show that the catecholamines dopamine (DA) and noradrenaline (NA) inhibit serotonylation of fibronectin and that DA and NA themselves can be selectively transamidated into fibronectin by TGase. All three biogenic monoamines also block TGase-mediated transamidation of another monoamine, monodansylacadaverine, into fibronectin, suggesting a general mechanism of TGase-mediated "monoaminylation".

Serotonin--more than a neurotransmitter: transglutaminase-mediated serotonylation of C6 glioma cells and fibronectin.

In the central nervous system serotonin plays important roles as a neurotransmitter as well as during neuronal development and in synaptogenesis. Outside the central nervous system, serotonin is covalently transamidated to procoagulant proteins involved in blood clotting. This process is mediated by transglutaminases and named "serotonylation". Serotonylated proteins then tightly bind to specific serotonin binding sites on fibrinogen and thrombospondin to form stable extracellular multivalent complexes needed for thrombus formation. Here, we have investigated whether transglutaminases can also covalently incorporate extracellular serotonin to neural proteins and whether this might affect extracellular protein expression. Our data reveal that recombinant transglutaminase specifically transamidates [(3)H]-serotonin to cell-surface proteins from C6 glioma cells and the extracellular matrix protein fibronectin. Serotonylation of [(3)H]-serotonin was inhibited by the transglutaminase inhibitor cystamine and unlabelled serotonin. Transglutaminase-mediated transamidation of unlabelled serotonin to C6 cells induced an aggregation of extracellular protein matrices adjacent to and between single cells. Transglutaminase also transamidated the autofluorescent serotonin analogue 5,7-dihydroxytryptamine and monodansylcadaverine (MDC) into living C6 glioma cells. Electrophoretic separation of MDC-labelled C6 cells identified several distinct fluorescent proteins one of which was fibronectin.

Ex vivo and in vivo evaluation of [18F]PR04.MZ in rodents: a selective dopamine transporter imaging agent.

N-4-Fluorobut-2-yn-1-yl-2beta-carbomethoxy-3beta-phenyltropane (PR04.MZ) has been developed as dopamine transporter (DAT) ligand for molecular imaging. It contains a terminally fluorinated, conformationally constrained nitrogen substituent that is well suited for the introduction of fluorine-18. The present report describes the pharmacological characterisation of [18F]PR04.MZ. The ligand shows an IC50 value of 2 nM against human DAT, whereas the IC50 value against human serotonin transporter and human noradrenalin transporter are lower (110 nM and 22 nM, respectively). Furthermore, its ex vivo organ distribution, its binding profile in the rat brain and reversibility of binding were examined. A muPET study illuminates a fast kinetic profile and specific binding to rat DAT.

Synthesis and monoamine uptake inhibition of conformationally constrained 2beta-carbomethoxy-3beta-phenyl tropanes.

A series of 2beta-carbomethoxy-3beta-phenyl tropanes with conformationally constrained nitrogen substituents were synthesized as potential selective dopamine transporter ligands. These novel compounds were examined for their monoamine uptake inhibition potency at the human dopamine transporter (hDAT), the human serotonin transporter (hSERT) and the human noradrenalin transporter (hNET), stably expressed in human embryonic kidney cells (HEK). A SAR-study was conducted to determine the contribution of extended, 4-fluorinated, conformationally constrained C4 chains at the tropane nitrogen to human monoamine transporter affinity and selectivity.

Inhibition of serotonin transport by (+)McN5652 is noncompetitive.

INTRODUCTION: Imaging of the serotonergic innervation of the brain using positron emission tomography (PET) with the serotonin transporter (SERT) ligand [11C] (+)McN5652 might be affected by serotonin in the synaptic cleft if there is relevant interaction between [11C] (+)McN5652 and serotonin at the SERT. The aim of the present study therefore was to pharmacologically characterize the interaction of [11C] (+)McN5652 and serotonin at the SERT. METHODS: In vitro saturation analyses of [3H]serotonin uptake into HEK293 cells stably expressing the human SERT were performed in the absence and presence of unlabelled (+)McN5652. Data were evaluated assuming Michaelis-Menten kinetics. RESULTS: Unlabelled (+)McN5652 significantly reduced the maximal rate of serotonin transport V(max) of SERT without affecting the Michaelis-Menten constant K(M). CONCLUSIONS: This finding indicates that (+)McN5652 inhibits serotonin transport through the SERT in a noncompetitive manner. This might suggest that [11C] (+)McN5652 PET is not significantly affected by endogenous serotonin.

DASB -in vitro binding characteristics on human recombinant monoamine transporters with regard to its potential as positron emission tomography (PET) tracer.

The efficiency of serotonergic signal transduction is controlled by the density of serotonegic synapses and by the activity of the serotonin transporter (SERT), which selectively clears the synaptic cleft of the neurotransmitter. SERT is located in axons, where it is concentrated in varicosities and terminal boutons and thus is an exquisite marker for serotonergic synapses. This finding has been taken advantage of for neuroimaging serotonergic synaptic contact sites. Previous positron emission tomography (PET) and single photon emission computed tomography (SPECT) studies were often carried out using radioligands that bind with high affinity to SERTs in the brainstem but also exhibit high affinity for dopamine and norepinephrine transporters and therefore did not allow quantification of serotonergic innervations in brain regions also containing dopaminergic or noradrenergic terminals. In order to visualize SERT availability more selectively, in recent years new tracers have been developed, one of which is [11C]DASB (N,N-dimethyl-2-2-amino-4-cyanophenylthiobenzylamine). Here, we have performed a detailed pharmacological characterization of unlabelled as well as radioactive DASB on recombinant human monoamine transporter proteins. Our results show that DASB selectively binds to SERT with high affinity (KD = 3.5 nm) to a site distinct from the serotonin (5-HT) recognition/translocation site. 5-HT inhibits DASB binding to SERT with more than one order of magnitude lower affinity than that of DASB binding (IC50 = 82.4 nm). These findings suggest DASB to be a highly selective PET tracer to visualize the density of serotonergic synapses in human brain.

Functional coupling of serotonin and noradrenaline transporters.

Re-uptake of the neurotransmitters serotonin and noradrenaline out of the synaptic cleft is mediated by selective transporter proteins, the serotonin transporter and the noradrenaline transporter respectively. Both are integral membrane proteins that are have a high degree of homology and represent members of a larger neurotransmitter transporter superfamily. Several studies have indicated that the serotonin transporter has an an oligomeric structure. To determine whether monoamine transporters can also function in oligomeric structures in situ, we constructed a concatenate consisting of one molecule of serotonin transporter covalently linked to one molecule of noradrenaline transporter. Heterologous expression of this hybrid construct allowed us to analyse the function, i.e. transport activity, and the structure, i.e. the molecular weight of the total construct and of its single components, at the same time. We showed that serotonin-noradrenaline transporter fusion proteins are fully active and exhibit the pharmacological profile of both their individual components. These findings support the hypothesis that monoamine transporters are expressed and may function as oligomeric proteins composed of non-interacting monomers.