Our goal is to understand the role of scaffold proteins in the organization of signal transduction. Scaffolds determine the outcome of signal transduction by controlling the location of cell surface receptors and connecting them to downstream effectors. This proposal focuses on post-synaptic glutamate signaling, which mediates excitatory neurotransmission. Glutamate receptor signaling pathways are organized by the membrane-associated guanylate kinases (MAGuKs). In excitatory neurons there are four MAGuKs (PSD-95, PSD-93, SAP102 and SAP97/Dlg). Existing biochemical data is in conflict with functional data regarding the specific role each protein plays in synaptic plasticity. This proposal investigates the molecular basis of MAGuK structure and specificity. We have a working reconstitution of receptor-scaffold interactions in the post-synapse to provide the missing quantitative data on MAGuK affinity and selectivity for glutamate receptors. Receptor cytoplasmic domains are attached to a planar phospholipid bilayer, creating a functionalized surface that mimics the postsynaptic membrane.
Aim 1 will test the hypothesis that functional differences between the four PSD-MAGuKs arises from differences in the kinetics of receptor binding. Single molecule fluorescence and simulations will solve the structure of all four, full-length MAGuKs by placing the known structures in context. We can watch individual binding events in real time to quantitate the MAGuK binding to both NMDA and AMPA receptors and also Stargazin.
Aim 2 will test the hypothesis that differences in MAGuK quaternary structure give rise to differences in binding affinity for receptors and other ligands.
Aim 3 is to confirm te structure of PSD-MAGuKs in vivo. Characterization of the GFP-tagged PSD-95 (and a live-cell FRET ruler) in vitro will form the basis for quantitative interpretation of live-cell FRET measurements. These studies are advancing towards a physical and kinetic description of PSD assembly. We strive to achieve a """"""""cellular structural biology"""""""" by reconstituting higher-order systems. This reconstitution will eventually serve as a platform to incorporate additional post synaptic components. These results will indicate how much of the variable signaling behavior in the synapse is attributable to the scaffold itself. Describing the molecular events in excitatory signaling is a fundamental challenge in neuroscience with direct relevance to brain development, memory and learning, and many neurological and neuropsychiatric disorders.
The MAGuK proteins are central players in neuronal signaling and have been implicated in brain development, memory and learning, and in diseases such as neurodegeneration following stroke, autism, epilepsy and schizophrenia. This proposal seeks to understand the molecular basis of MAGuK function by reconstituting this cellular signaling system and watching single molecules in action using video microscopy. Understanding the molecular basis of neuronal communication may provide a new avenue for the treatment of neurological and neuropsychiatric disorders.
|Yanez Orozco, Inna S; Mindlin, Frank A; Ma, Junyan et al. (2018) Identifying weak interdomain interactions that stabilize the supertertiary structure of the N-terminal tandem PDZ domains of PSD-95. Nat Commun 9:3724|
|Hellenkamp, Björn; Schmid, Sonja; Doroshenko, Olga et al. (2018) Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study. Nat Methods 15:669-676|
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|Pedersen, Søren W; Albertsen, Louise; Moran, Griffin E et al. (2017) Site-Specific Phosphorylation of PSD-95 PDZ Domains Reveals Fine-Tuned Regulation of Protein-Protein Interactions. ACS Chem Biol 12:2313-2323|
|McCann, James J; Choi, Ucheor B; Bowen, Mark E (2014) Reconstitution of multivalent PDZ domain binding to the scaffold protein PSD-95 reveals ternary-complex specificity of combinatorial inhibition. Structure 22:1458-66|
|Choi, Ucheor B; Kazi, Rashek; Stenzoski, Natalie et al. (2013) Modulating the intrinsic disorder in the cytoplasmic domain alters the biological activity of the N-methyl-D-aspartate-sensitive glutamate receptor. J Biol Chem 288:22506-15|
|Choi, Ucheor B; Weninger, Keith R; Bowen, Mark E (2012) Immobilization of proteins for single-molecule fluorescence resonance energy transfer measurements of conformation and dynamics. Methods Mol Biol 896:3-20|
|McCann, James J; Zheng, Liqiang; Rohrbeck, Daniel et al. (2012) Supertertiary structure of the synaptic MAGuK scaffold proteins is conserved. Proc Natl Acad Sci U S A 109:15775-80|
|Salussolia, Catherine L; Corrales, Alexandra; Talukder, Iehab et al. (2011) Interaction of the M4 segment with other transmembrane segments is required for surface expression of mammalian ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. J Biol Chem 286:40205-18|
|Choi, Ucheor B; Xiao, Shifeng; Wollmuth, Lonnie P et al. (2011) Effect of Src kinase phosphorylation on disordered C-terminal domain of N-methyl-D-aspartic acid (NMDA) receptor subunit GluN2B protein. J Biol Chem 286:29904-12|
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