Brain-derived neurotrophic factor (BDNF) has potent effects on neuronal survival and plasticity during development and after injury. In the nervous system, neurons are considered the major cellular source of BDNF. We demonstrate here that in addition, activated human T cells, B cells, and monocytes secrete bioactive BDNF in vitro. Notably, in T helper (Th)1- and Th2-type CD4+ T cell lines specific for myelin autoantigens such as myelin basic protein or myelin oligodendrocyte glycoprotein, BDNF production is increased upon antigen stimulation. The BDNF secreted by immune cells is bioactive, as it supports neuronal survival in vitro. Using anti-BDNF monoclonal antibody and polyclonal antiserum, BDNF immunoreactivity is demonstrable in inflammatory infiltrates in the brain of patients with acute disseminated encephalitis and multiple sclerosis. The results raise the possibility that in the nervous system, inflammatory infiltrates have a neuroprotective effect, which may limit the success of nonselective immunotherapies.

Neurotrophins bind to two structurally unrelated receptors, the trk tyrosine kinases and the neurotrophin receptor p75(NTR). Ligand activation of these two types of receptor can lead to opposite actions, in particular the prevention or activation of programmed cell death. Many cells co-express trk receptors and p75(NTR), and we found that p75(NTR) was co-precipitated with trkA, trkB and trkC in cells transfected with both receptor types. Co-precipitation of p75(NTR) was not observed with the epidermal growth factor receptor. Experiments with deletion constructs of trkB (the most abundant trk receptor in the brain) and p75(NTR) revealed that both the extracellular and intracellular domains of trkB and p75(NTR) contribute to the interaction. Blocking autophosphorylation of trkB substantially reduced the interactions between p75(NTR) and trkB constructs containing the intracellular, but not the extracellular, domains. We also found that co-expression of p75(NTR) with trkB resulted in a clear increase in the specificity of trkB activation by brain-derived neurotrophic factor, compared with neurotrophin-3 and neurotrophin-4/5. These results indicate a close proximity of the two neurotrophin receptors within cell membranes, and suggest that the signalling pathways they initiate may interact soon after their activation.

The .8-adrenergic receptor kinase (.BARK) is the prototypical member of the family of cytosolic kinases that phosphorylate guanine nucleotide binding-protein-coupled receptors and thereby trigger uncoupling between receptors and guanine nucleotide binding proteins. Herein we show that this kinase is subject to phosphorylation and regulation by protein kinase C (PKC). In cell lines stably expressing GIBadrenergic receptors, activation of these receptors by epinephrine resulted in an activation of cytosolic f3ARK. Similar data were obtained in 293 cells transiently coexpressing a1B-adrenergic receptors and 8ARK-1. Direct activation of PKC with phorbol esters in these cells caused not only an activation of cytosolic ,BARK-1 but also a translocation of .8ARK immunoreactivity from the cytosol to the membrane fraction. A PKC preparation purified from rat brain phosphorylated purified recombinant pARK-1 to a stoichiometry of 0.86 phosphate per 8ARK-1. This phosphorylation resulted in an increased activity of .8ARK-1 when membrane-bound rhodopsin served as its substrate but in no increase of its activity toward a soluble peptide substrate. The site of phosphorylation was mapped to the C terminus of f8ARK-1. We conclude that PKC activates DARK by enhancing its translocation to the plasma membrane.Signaling via guanine nucleotide binding (G)-protein-coupled receptors is initiated by binding of agonist ligands and is mediated by the activation of G proteins, which involves binding of GTP by the a subunit and its dissociation from the f,y-subunit complex (1, 2). Both the a subunit and the 13y-subunit complex can then regulate effector molecules and thereby produce intracellular signals (3,4). This signaling pathway is subject to a variety of regulatory mechanisms that alter the expression and the function of the proteins involved. One of the key regulatory processes is the desensitization of such receptors in response to prolonged or repeated agonist exposure. Various mechanisms contribute to receptor desensitization (5, 6). The most rapid and probably quantitatively the most important one (7,8) is triggered by phosphorylation of the receptors by members of the family of G-protein-coupled receptor kinases. Of the six members of this family cloned to date (9), the f3-adrenergic receptor kinase-1 (,3ARK-1; ref. 10) has been studied as the prototypical member that mediates receptor desensitization. Desensitization by this kinase occurs as a two-step process: (i) The kinase translocates from the cytosol to the plasma membrane (11) and phosphorylates several serine and threonine residues in the cytoplasmic portions of the receptors. (ii) Members of another family of cytosolic proteins, the arrestins, bind to the phosphorylated receptors and thereby impair the receptor-Gprotein interaction (12)(13)(14). Much interest has recently been concerned with the mode of translocation of P3ARK to the plasma membrane. Three anchoring points have been identiThe publication costs of this article were defrayed in part by page charge pa...

Binding of insulin to its receptor (IR) causes rapid autophosphorylation with concomitant activation of its tyrosine kinase which transmits the signal by phosphorylating cellular substrates. The IR activity is controlled by protein-tyrosine phosphatases, but those directly involved in regulating the insulin receptor and its signaling pathways have not yet been identified. Using baby hamster kidney cells overexpressing the IR and a novel insulin-based selection principle, we established stable cell lines with functionally coupled expression of the IR and protein-tyrosine phosphatases. The two closely related protein-tyrosine phosphatases ␣ and ⑀ were identified as negative regulators of IR tyrosine kinase.Insulin is an important regulator of different metabolic processes and plays a key role in the control of blood glucose. Defects related to its synthesis or signaling lead to diabetes mellitus. Binding of insulin to its receptor causes rapid autophosphorylation of several tyrosine residues in the intracellular part of the ␤-subunit. Three closely positioned tyrosine residues (the tyrosine 1150 domain) must all be phosphorylated to obtain full activity of the insulin receptor tyrosine kinase (IRTK) 1 which transmits the signal further downstream by tyrosine phosphorylation of other cellular substrates, including insulin receptor substrate-1 (IRS-1) (1-4). The structural basis for the function of the tyrosine triplet has been provided by recent x-ray crystallographic studies of IRTK that showed tyrosine 1150 to be autoinhibitory in its unphosphorylated state (5).Several studies clearly indicate that the activity of the autophosphorylated IRTK can be reversed by dephosphorylation in vitro (reviewed in Goldstein (6)) (7,8), with the triphosphorylated tyrosine 1150 domain being the most sensitive target for protein-tyrosine phosphatases (PTPs) as compared to the diand monophosphorylated forms (8). It is, therefore, tempting to speculate that this tyrosine triplet functions as a control switch of IRTK activity. Indeed, the IRTK appears to be tightly regulated by PTP-mediated dephosphorylation in vivo (9 -11). The intimate coupling of PTPs to the insulin signaling pathway is further evidenced by the finding that insulin differentially regulates PTP activity in rat hepatoma cells (12) and in livers from alloxan diabetic rats (13). However, little is known about the identity of the PTPs involved in IRTK regulation.To identify PTPs that negatively regulate the IRTK activity we developed a novel selection principle that allows establishment of stable cell lines with functionally coupled overexpression of IR and inhibitory PTPs. For this purpose we used a previously established baby hamster kidney cell line (BHK-IR) (14), which exhibits high levels of IR expression and responds to insulin stimulation with complete growth inhibition of adherent cells. PTPs that impede or block the insulin signal can consequently be identified by their capacity to restore cell growth. This effect was found to be induced through direct activ...

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This study examines the mechanisms by which the tyrosine kinase receptor TrkB is down-regulated following binding of brain-derived neurotrophic factor (BDNF). In primary cultures of cerebellar granule neurons, BDNF-induced reduction of TrkB receptors was largely prevented by the addition of specific proteasome inhibitors. HN10 cells, a neuronal cell line that can be readily transfected, also showed a marked down-regulation of cell surface TrkB following BDNF exposure. In addition, we observed that prolonged exposure to nerve growth factor of TrkA-transfected cells did not lead to the down-regulation seen with BDNF and TrkB. TrkA and TrkB chimeric molecules were therefore expressed in HN10 cells and tested for ligand-induced regulation. These experiments led to the conclusion that the motives responsible for down-regulation are contained in the cytoplasmic domain of TrkB, and a short sequence in the juxtamembrane domain of TrkB was identified that confers nerve growth factor-induced down-regulation when inserted into TrkA.

RNA-protein cross-links were introduced into E. coli 50S ribosomal subunits by treatment with 2-iminothiolane followed by mild ultraviolet irradiation. After partial digestion of the RNA, the cross-linked RNA-protein complexes were separated by our recently published three-step procedure. In cases where this separation was inadequate, a further purification step was introduced, involving affinity chromatography with antibodies to the ribosomal 50S proteins. Analysis of the isolated complexes enabled four new cross-link sites on the 23S RNA to be identified, as well as re-confirming several previously established sites. The new sites are as follows: Protein L2 is cross-linked within an oligonucleotide at positions 1818-1823 in the 23S RNA, protein L4 within positions 320-325, protein L24 within positions 99-107, and protein L27 within positions 2320-2323.

Purpose More than 15 years have passed since the first description of the unbound brain-to-plasma partition coefficient (Kp,uu,brain) by Prof. Margareta Hammarlund-Udenaes, which was enabled by advancements in experimental methodologies including cerebral microdialysis. Since then, growing knowledge and data continue to support the notion that the unbound (free) concentration of a drug at the site of action, such as the brain, is the driving force for pharmacological responses. Towards this end, Kp,uu,brain is the key parameter to obtain unbound brain concentrations from unbound plasma concentrations. Methods To understand the importance and impact of the Kp,uu,brain concept in contemporary drug discovery and development, a survey has been conducted amongst major pharmaceutical companies based in Europe and the USA. Here, we present the results from this survey which consisted of 47 questions addressing: 1) Background information of the companies, 2) Implementation, 3) Application areas, 4) Methodology, 5) Impact and 6) Future perspectives. Results and conclusions From the responses, it is clear that the majority of the companies (93%) has established a common understanding across disciplines of the concept and utility of Kp,uu,brain as compared to other parameters related to brain exposure. Adoption of the Kp,uu,brain concept has been mainly driven by individual scientists advocating its application in the various companies rather than by a top-down approach. Remarkably, 79% of all responders describe the portfolio impact of Kp,uu,brain implementation in their companies as ‘game-changing’. Although most companies (74%) consider the current toolbox for Kp,uu,brain assessment and its validation satisfactory for drug discovery and early development, areas of improvement and future research to better understand human brain pharmacokinetics/pharmacodynamics translation have been identified.