Biomedical subjects

Ryan T Terry-Lorenzo

Publications and source records attributed to Ryan T Terry-Lorenzo.

5 recordsLinked to original sources

PubMed2006

Transsynaptic signaling by postsynaptic synapse-associated protein 97.

The molecular mechanisms by which postsynaptic modifications lead to precisely coordinated changes in presynaptic structure and function are primarily unknown. To address this issue, we examined the presynaptic consequences of postsynaptic expression of members of the membrane-associated guanylate kinase family of synaptic scaffolding proteins. Postsynaptic expression of synapse-associated protein 97 (SAP97) increased presynaptic protein content and active zone size to a greater extent than comparable amounts of postsynaptic PSD-95 (postsynaptic density-95) or SAP102. In addition, postsynaptic expression of SAP97 enhanced presynaptic function, as measured by increased FM4-64 dye uptake. The structural presynaptic effects of postsynaptic SAP97 required ligand binding through two of its PDZ (PSD-95/Discs large/zona occludens-1) domains as well as intact N-terminal and guanylate kinase domains. Expression of SAP97 recruited a complex of additional postsynaptic proteins to synapses including glutamate receptor 1, Shank1a, SPAR (spine-associated RapGAP), and proSAP2. Furthermore, inhibition of several different transsynaptic signaling proteins including cadherins, integrins, and EphB receptor/ephrinB significantly reduced the presynaptic growth caused by postsynaptic SAP97. These results suggest that SAP97 may play a central role in the coordinated growth of synapses during development and plasticity by recruiting a complex of postsynaptic proteins that enhances presynaptic terminal growth and function via multiple transsynaptic molecular interactions.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Neurabin/protein phosphatase-1 complex regulates dendritic spine morphogenesis and maturation.

The majority of excitatory synapses in the mammalian brain form on filopodia and spines, actin-rich membrane protrusions present on neuronal dendrites. The biochemical events that induce filopodia and remodel these structures into dendritic spines remain poorly understood. Here, we show that the neuronal actin- and protein phosphatase-1-binding protein, neurabin-I, promotes filopodia in neurons and nonneuronal cells. Neurabin-I actin-binding domain bundled F-actin, promoted filopodia, and delayed the maturation of dendritic spines in cultured hippocampal neurons. In contrast, dimerization of neurabin-I via C-terminal coiled-coil domains and association of protein phosphatase-1 (PP1) with neurabin-I through a canonical KIXF motif inhibited filopodia. Furthermore, the expression of a neurabin-I polypeptide unable to bind PP1 delayed the maturation of neuronal filopodia into spines, reduced the synaptic targeting of AMPA-type glutamate (GluR1) receptors, and decreased AMPA receptor-mediated synaptic transmission. Reduction of endogenous neurabin levels by interference RNA (RNAi)-mediated knockdown also inhibited the surface expression of GluR1 receptors. Together, our studies suggested that disrupting the functions of a cytoskeletal neurabin/PP1 complex enhanced filopodia and impaired surface GluR1 expression in hippocampal neurons, thereby hindering the morphological and functional maturation of dendritic spines.

Actins↗

PubMed2002

Neurabins recruit protein phosphatase-1 and inhibitor-2 to the actin cytoskeleton.

Inhibitor-2 (I-2) bound protein phosphatase-1 (PP1) and several PP1-binding proteins from rat brain extracts, including the actin-binding proteins, neurabin I and neurabin II. Neurabins from rat brain lysates were sedimented by I-2 and its structural homologue, I-4. The central domain of both neurabins bound PP1 and I-2, and mutation of a conserved PP1-binding motif abolished neurabin binding to both proteins. Microcystin-LR, a PP1 inhibitor, also attenuated I-2 binding to neurabins. Immunoprecipitation of neurabin I established its association with PP1 and I-2 in HEK293T cells and suggested that PP1 mediated I-2 binding to neurabins. The C terminus of I-2, although not required for PP1 binding, facilitated PP1 recruitment by neurabins, which also targeted I-2 to polymerized F-actin. Mutations that attenuated PP1 binding to I-2 and neurabin I suggested distinct and overlapping sites for these two proteins on the PP1 catalytic subunit. Immunocytochemistry in epithelial cells and cultured hippocampal neurons showed that endogenous neurabin II and I-2 colocalized at actin-rich structures, consistent with the ability of neurabins to target the PP1.I-2 complex to actin cytoskeleton and regulate cell morphology.

Actins↗

PubMed2002

The neuronal actin-binding proteins, neurabin I and neurabin II, recruit specific isoforms of protein phosphatase-1 catalytic subunits.

Neurabins are protein phosphatase-1 (PP1) targeting subunits that are highly concentrated in dendritic spines and post-synaptic densities. Immunoprecipitation of neurabin I and neurabin II/spinophilin from rat brain extracts sedimented PP1gamma1 and PP1alpha but not PP1beta. In vitro studies showed that recombinant peptides representing central regions of neurabins also preferentially bound PP1gamma1 and PP1alpha from brain extracts and associated poorly with PP1beta. Analysis of PP1 binding to chimeric neurabins suggested that sequences flanking a conserved PP1-binding motif altered their selectivity for PP1beta and their activity as regulators of PP1 in vitro. Assays using recombinant PP1 catalytic subunits and a chimera of PP1 and protein phosphatase-2A indicated that the C-terminal sequences unique to the PP1 isoforms contributed to their recognition by neurabins. Collectively, the results from several different in vitro assays established the rank order of PP1 isoform selection by neurabins to be PP1gamma1 > PP1alpha > PP1beta. This PP1 isoform selectivity was confirmed by immunoprecipitation of neurabin I and II from brain extracts from wild type and mutant PP1gamma null mice. In the absence of PP1gamma1, both neurabins showed enhanced association with PP1alpha but not PP1beta. These studies identified some of the structural determinants in PP1 and neurabins that together contribute to preferential targeting of PP1gamma1 and PP1alpha to the mammalian synapse.

Actins↗

PubMed2002

Targeting protein phosphatase 1 (PP1) to the actin cytoskeleton: the neurabin I/PP1 complex regulates cell morphology.

Neurabin I, a neuronal actin-binding protein, binds protein phosphatase 1 (PP1) and p70 ribosomal S6 protein kinase (p70S6K), both proteins implicated in cytoskeletal dynamics. We expressed wild-type and mutant neurabins fused to green fluorescent protein in Cos7, HEK293, and hippocampal neurons. Biochemical and cellular studies showed that an N-terminal F-actin-binding domain dictated neurabin I localization at actin cytoskeleton and promoted disassembly of stress fibers. Deletion of the C-terminal coiled-coil and sterile alpha motif domains abolished neurabin I dimerization and induced filopodium extension. Immune complex assays showed that neurabin I recruited an active PP1 via a PP1-docking sequence,(457)KIKF(460). Mutation of the PP1-binding motif or PP1 inhibition by okadaic acid and calyculin A abolished filopodia and restored stress fibers in cells expressing neurabin I. In vitro and in vivo studies suggested that the actin-binding domain attenuated protein kinase A (PKA) phosphorylation of neurabin I. Modification of a major PKA site, serine-461, impaired PP1 binding. Finally, p70S6K was excluded from neurabin I/PP1 complexes and required the displacement of PP1 for recruitment to neurabin I. These studies provided new insights into the assembly and regulation of a neurabin I/PP1 complex that controls actin rearrangement to promote spine development in mammalian neurons.

Actins↗