A coiled coil trigger site is essential for rapid binding of synaptobrevin to the SNARE acceptor complex.

Exocytosis from synaptic vesicles is driven by stepwise formation of a tight alpha-helical complex between the fusing membranes. The complex is composed of the three SNAREs: synaptobrevin 2, SNAP-25, and syntaxin 1a. An important step in complex formation is fast binding of vesicular synaptobrevin to the preformed syntaxin 1.SNAP-25 dimer. Exactly how this step relates to neurotransmitter release is not well understood. Here, we combined different approaches to gain insights into this reaction. Using computational methods, we identified a stretch in synaptobrevin 2 that may function as a coiled coil "trigger site." This site is also present in many synaptobrevin homologs functioning in other trafficking steps. Point mutations in this stretch inhibited binding to the syntaxin 1.SNAP-25 dimer and slowed fusion of liposomes. Moreover, the point mutations severely inhibited secretion from chromaffin cells. Altogether, this demonstrates that the trigger site in synaptobrevin is crucial for productive SNARE zippering.

Decreased binding of peptides-MHC class I (pMHC) multimeric complexes to CD8 affects their binding avidity for the TCR but does not significantly impact on pMHC/TCR dissociation rate.

The CD8 coreceptor plays a crucial role in both T cell development in the thymus and in the activation of mature T cells in response to Ag-specific stimulation. In this study we used soluble peptides-MHC class I (pMHC) multimeric complexes bearing mutations in the CD8 binding site that impair their binding to the MHC, together with altered peptide ligands, to assess the impact of CD8 on pMHC binding to the TCR. Our data support a model in which CD8 promotes the binding of TCR to pMHC. However, once the pMHC/TCR complex is formed, the TCR dominates the pMHC/TCR dissociation rates. As a consequence of these molecular interactions, under physiologic conditions CD8 plays a key role in complex formation, resulting in the enhancement of CD8 T cell functions whose specificity, however, is determined by the TCR.

Most residues on the floor of the antigen binding site of the class I MHC molecule H-2Kd influence peptide presentation.

A panel of 15 single alanine substitutions on the floor of the peptide binding groove of the murine class I histocompatibility molecule H-2Kd has been analyzed. All but two mutant molecules were expressed on the cell surface, and were tested for peptide binding and presentation to specific cytotoxic T lymphocytes. Eleven out of 13 mutant molecules appeared to be functionally altered. Five of the substituted residues were involved in the presentation of all peptides tested. Three participated in the presentation of certain peptides but not others. Three other residues participated in epitope formation through indirect interactions. Only two mutations had no detectable effect.

Comparison of [3H]tamsulosin and [3H]prazosin binding to wild-type and constitutively active alpha1B-adrenoceptors.

We have tested the hypothesis that smaller alpha1B-adrenoceptor labeling by [3H]tamsulosin compared to [3H]prazosin is related to differential recognition of agonist low affinity states. Paired saturation binding experiments with [3H]prazosin and [3H]tamsulosin were performed in membrane preparations from rat liver and Rat- fibroblasts stably transfected with wild-type hamster alpha1B-adrenoceptors or a constitutively active mutant thereof. In all three settings [3H]tamsulosin labeled significantly fewer alpha1B-adrenoceptors than [3H]prazosin. In noradrenaline competition binding experiments, the percentage of agonist low affinity sites was smallest for the constitutively active alpha1B-adrenoceptor but the percentage of agonist low affinity sites recognized by [3H]tamsulosin and [3H]prazosin did not differ significantly. We conclude that [3H]tamsulosin labels fewer alpha1B-adrenoceptors than [3H]prazosin but this is not fully explained by a poorer labeling of agonist low affinity sites.

Stimulation by leptin of 3H GDP binding to brown adipose tissue of fasted but not fed rats.

OBJECTIVES: To assess the effects of intracerebroventricular (i.c.v.) leptin administration on rats fed ad libitum or fasted on 3H GDP binding to brown adipose tissue (BAT). SUBJECTS: Groups of 5-6 ten-week-old male Wistar rats. EXPERIMENTAL DESIGN: An i.c.v. cannula was inserted and unilateral denervation of interscapular brown adipose tissue (BAT) was performed 5 d before each study. Thereafter, leptin was infused i.c.v. during 72 h while rats were fed ad libitum or fasted. Vehicle-infused, pair-fed or fasted rats were used as controls. MEASUREMENTS: 3H GDP binding to innervated and denervated BAT mitochondria. RESULTS: 3H GDP binding to innervated or denervated BAT of rats fed ab libitum compared to vehicle-infused, pair-fed rats was not increased by i.c.v. leptin. 3H GDP binding was lower in fasted than in fed rats, and the difference was larger in innervated than denervated BAT. I.c.v. leptin increased 3H GDP binding by 30% in innervated, and by 51% in denervated BAT (P < 0.05) in fasted rats. CONCLUSIONS: I.c.v. leptin does not increase 3H GDP binding to BAT of rats fed ad libitum compared to pair-fed (food-restricted) rats. In contrast, i.c.v. leptin produces a mild stimulation of 3H GDP binding to BAT of fasted rats. This effect is not mediated by the sympathetic nervous system, because it is observed in both innervated and denervated BAT. These results are compatible with the concept that, in fasting rats, the decrease in leptin secretion contributes to the reduction in 3H GDP binding to BAT mitochondria.

Binding of Lassa virus perturbs extracellular matrix-induced signal transduction via dystroglycan.

The arenavirus Lassa virus (LASV) causes a severe haemorrhagic fever with high mortality in man. The cellular receptor for LASV is dystroglycan (DG). DG is a ubiquitous receptor for extracellular matrix (ECM) proteins, which cooperates with β1 integrins to control cell-matrix interactions. Here, we investigated whether LASV binding to DG triggers signal transduction, mimicking the natural ligands. Engagement of DG by LASV resulted in the recruitment of the adaptor protein Grb2 and the protein kinase MEK1 by the cytoplasmic domain of DG without activating the MEK/ERK pathway, indicating assembly of an inactive signalling complex. LASV binding to cells however affected the activation of the MEK/ERK pathway via α6β1 integrins. The virus-induced perturbation of α6β1 integrin signalling critically depended on high-affinity LASV binding to DG and DG's cytoplasmic domain, indicating that LASV-receptor binding perturbed signalling cross-talk between DG and β1 integrins.

Archives parlementaires de 1787 à 1860 ; 52-61, 63-82. Convention nationale. Série 1 / Tome 53 / impr. par ordre du Sénat et de la Chambre des députés ; fondé par MM. Mavidal et E. Laurent ; continué́ par M. L. Lataste,... M. Louis Claveau,... M. Constant Pionnier,... [et al.]

Collection : Les archives de la Révolution française ; 3.1

Archives parlementaires de 1787 à 1860 ; 52-61, 63-82. Convention nationale. Série 1 / Tome 64 / impr. par ordre du Sénat et de la Chambre des députés ; fondé par MM. Mavidal et E. Laurent ; continué́ par M. L. Lataste,... M. Louis Claveau,... M. Constant Pionnier,... [et al.]

Collection : Les archives de la Révolution française ; 3.1

Archives parlementaires de 1787 à 1860 ; 52-61, 63-82. Convention nationale. Série 1 / Tome 66 / impr. par ordre du Sénat et de la Chambre des députés ; fondé par MM. Mavidal et E. Laurent ; continué́ par M. L. Lataste,... M. Louis Claveau,... M. Constant Pionnier,... [et al.]

Collection : Les archives de la Révolution française ; 3.1

Archives parlementaires de 1787 à 1860 ; 52-61, 63-82. Convention nationale. Série 1 / Tome 69 / impr. par ordre du Sénat et de la Chambre des députés ; fondé par MM. Mavidal et E. Laurent ; continué́ par M. L. Lataste,... M. Louis Claveau,... M. Constant Pionnier,... [et al.]

Collection : Les archives de la Révolution française ; 3.1

Archives parlementaires de 1787 à 1860 ; 52-61, 63-82. Convention nationale. Série 1 / Tome 71 / Partie 2 / impr. par ordre du Sénat et de la Chambre des députés ; fondé par MM. Mavidal et E. Laurent ; continué́ par M. L. Lataste,... M. Louis Claveau,... M. Constant Pionnier,... [et al.]

Collection : Les archives de la Révolution française ; 3.1

Archives parlementaires de 1787 à 1860 ; 52-61, 63-82. Convention nationale. Série 1 / Tome 58 / impr. par ordre du Sénat et de la Chambre des députés ; fondé par MM. Mavidal et E. Laurent ; continué́ par M. L. Lataste,... M. Louis Claveau,... M. Constant Pionnier,... [et al.]

Collection : Les archives de la Révolution française ; 3.1

Archives parlementaires de 1787 à 1860 ; 52-61, 63-82. Convention nationale. Série 1 / Tome 61 / impr. par ordre du Sénat et de la Chambre des députés ; fondé par MM. Mavidal et E. Laurent ; continué́ par M. L. Lataste,... M. Louis Claveau,... M. Constant Pionnier,... [et al.]

Collection : Les archives de la Révolution française ; 3.1

Archives parlementaires de 1787 à 1860 ; 52-61, 63-82. Convention nationale. Série 1 / Tome 55 / impr. par ordre du Sénat et de la Chambre des députés ; fondé par MM. Mavidal et E. Laurent ; continué́ par M. L. Lataste,... M. Louis Claveau,... M. Constant Pionnier,... [et al.]

Collection : Les archives de la Révolution française ; 3.1

Archives parlementaires de 1787 à 1860 ; 52-61, 63-82. Convention nationale. Série 1 / Tome 56 / impr. par ordre du Sénat et de la Chambre des députés ; fondé par MM. Mavidal et E. Laurent ; continué́ par M. L. Lataste,... M. Louis Claveau,... M. Constant Pionnier,... [et al.]

Collection : Les archives de la Révolution française ; 3.1