Pharmacological and biochemical approaches were used to elucidate the involvement of growth factor signaling pathways mediating estrogen neuroprotection in primary cortical neurons after glutamate excitotoxicity. We addressed the activation of mitogen-activated protein kinase (MAPK) signaling pathways, which are activated by growth factors such as nerve growth factor (NGF). Inhibition of MAPK signaling with the MAPK kinase inhibitor PD98059 blocks both NGF and estrogen neuroprotection in these neurons. These results correlate with a rapid and sustained increase in MAPK activity within 30 min of estrogen exposure. The involvement of signaling molecules upstream from MAPK was also examined to determine whether activation of MAPK by estrogen is mediated by tyrosine kinase activity. Estrogen produces a rapid, transient activation of src-family tyrosine kinases and tyrosine phosphorylation of p21(ras)-guanine nucleotide activating protein. Effects of estrogen on neuroprotection, as well as rapid activation of tyrosine kinase and MAPK activity, are blocked by the anti-estrogen ICI 182,780. This provides evidence that activation of the MAPK pathway by estrogen participates in mediating neuroprotection via an estrogen receptor. These results describe a novel mechanism by which cytoplasmic actions of the estrogen receptor may activate the MAPK pathway, thus broadening the understanding of effects of estrogen in neurons.

The identification and functional characterization of proteins localized to synaptic vesicles has contributed significantly to our understanding of neurotransmission. Studies of synaptic vesicle protein interactions have both led to the identification of novel synaptic proteins and suggested hypotheses of protein function. Synaptic vesicle protein 2 (SV2), is an integral membrane glycoprotein present in all synaptic vesicles. There are two characterized isoforms, SV2A and SV2B. Despite their homology to transporter proteins, the function of the SV2s remains unknown. In an effort to determine SV2 function and identify cofactors required for SV2 activity, we examined the protein interactions of SV2 using a combination of cross-linking, immunoprecipitation, and recombinant protein affinity chromatography. We report that SV2 is part of a large protein complex that contains the synaptic vesicle protein synaptotagmin. The interaction between SV2 and synaptotagmin is direct, specific to SV2A, and inhibited by calcium with an EC 50 of approximately 10 M. Interaction is mediated by the cytoplasmic amino terminus of SV2A and the C2B domain of synaptotagmin. Our observations suggest a regulatory relationship between these two proteins.Formation and dissociation of protein complexes in the synapse mediate and regulate the events of the synaptic vesicle cycle. The identification of synaptic protein interactions has both suggested hypotheses for the role each protein plays in neurotransmission and has provided evidence for mechanistic models of synaptic vesicle docking, priming, and fusion (1).Synaptic vesicle protein 2 (SV2) 1 is a membrane glycoprotein present in all synaptic vesicles and regulated secretory vesicles of endocrine cells (2). Two isoforms of SV2, encoded by separate genes, have been characterized; . The SV2 cDNAs predict 12 transmembrane domain proteins that have significant sequence homology to the major facilitator family of transporters (3,4,7). This family of small molecule transporters includes the vesicular transporters of amines and acetylcholine (7). Based on this homology, the SV2s were initially hypothesized to be vesicular neurotransmitter transporters, with each isoform transporting a specific neurotransmitter. However, the expression of SV2A and SV2B does not correlate with neurotransmitter phenotype (8), therefore it is not likely that they serve this function.A current hypothesis of SV2 function is that it transports a constituent of synaptic vesicles other than neurotransmitters.However, attempts to demonstrate transport activity in SV2-expressing fibroblasts have been inconclusive, 2 perhaps due to the absence of a cofactor required for SV2 function. Alternatively, the structural similarity of transporters and channels (9, 10) suggests that SV2 is either a vesicular ion channel or a component of the proposed proteinaceous fusion pore which mediates neurotransmitter release.To distinguish between these hypotheses of SV2 function, and to identify potential cofactors or regulators of SV2 activit...

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Construction of artificial higher order protein complexes allows sampling of structural architectures and functional features not accessible by classical monomeric proteins. Here, we combine in silico modelling with expanded genetic code facilitated strain promoted azide-alkyne cycloaddition to construct artificial complexes that are structurally integrated protein dimers and demonstrate functional synergy. Using fluorescent proteins sfGFP and Venus as models, homodimers and heterodimers are constructed that switched ON once assembled and display enhanced spectral properties. Symmetrical crosslinks are found to be important for functional enhancement. The determined molecular structure of one artificial dimer shows that a new long-range polar network comprised mostly of organised water molecules links the two chromophores leading to activation and functional enhancement. Single molecule analysis reveals the dimer is more resistant to photobleaching spending longer times in the ON state. Thus, genetically encoded bioorthogonal chemistry can be used to generate truly integrated artificial protein complexes that enhance function.