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Translational control of mRNAs in dendrites is essential for certain forms of synaptic plasticity and learning and memory. CPEB is an RNA-binding protein that regulates local translation in dendrites. Here, we identify poly(A) polymerase Gld2, deadenylase PARN, and translation inhibitory factor neuroguidin (Ngd) as components of a dendritic CPEB-associated polyadenylation apparatus. Synaptic stimulation induces phosphorylation of CPEB, PARN expulsion from the ribonucleoprotein complex, and polyadenylation in dendrites. A screen for mRNAs whose polyadenylation is altered by Gld2 depletion identified >100 transcripts including one encoding NR2A, an NMDA receptor subunit. shRNA depletion studies demonstrate that Gld2 promotes and Ngd inhibits dendritic NR2A expression. Finally, shRNA-mediated depletion of Gld2 in vivo attenuates protein synthesis-dependent long-term potentiation (LTP) at hippocampal dentate gyrus synapses; conversely, Ngd depletion enhances LTP. These results identify a pivotal role for polyadenylation and the opposing effects of Gld2 and Ngd in hippocampal synaptic plasticity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Fragile X syndrome is a common form of cognitive deficit caused by the functional absence of fragile X mental retardation protein (FMRP), a dendritic RNA-binding protein that represses translation of specific messages. Although FMRP is phosphorylated in a group I metabotropic glutamate receptor (mGluR) activity-dependent manner following brief protein phosphatase 2A (PP2A)-mediated dephosphorylation, the kinase regulating FMRP function in neuronal protein synthesis is unclear. Here we identify ribosomal protein S6 kinase (S6K1) as a major FMRP kinase in the mouse hippocampus, finding that activity-dependent phosphorylation of FMRP by S6K1 requires signaling inputs from mammalian target of rapamycin (mTOR), ERK1/2, and PP2A. Further, the loss of hippocampal S6K1 and the subsequent absence of phospho-FMRP mimic FMRP loss in the increased expression of SAPAP3, a synapse-associated FMRP target mRNA. Together these data reveal a S6K1-PP2A signaling module regulating FMRP function and place FMRP phosphorylation in the mGluR-triggered signaling cascade required for protein-synthesis-dependent synaptic plasticity.

Fragile X syndrome is a common form of inherited mental retardation and is caused by loss of fragile X mental retardation protein (FMRP), a selective RNA-binding protein that influences the translation of target messages. Here, we identify protein phosphatase 2A (PP2A) as an FMRP phosphatase and report rapid FMRP dephosphorylation after immediate group I metabotropic glutamate receptor (mGluR) stimulation (<1 min) in neurons caused by enhanced PP2A enzymatic activity. In contrast, extended mGluR activation (1-5 min) resulted in mammalian target of rapamycin (mTOR)-mediated PP2A suppression and FMRP rephosphorylation. These activity-dependent changes in FMRP phosphorylation were also observed in dendrites and showed a temporal correlation with the translational profile of select FMRP target transcripts. Collectively, these data reveal an immediate-early signaling pathway linking group I mGluR activity to rapid FMRP phosphorylation dynamics mediated by mTOR and PP2A.

Fragile X syndrome (FXS), a common inherited form of mental retardation, is caused by the functional absence of the fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates the translation of specific mRNAs at synapses. Altered synaptic plasticity has been described in a mouse FXS model. However, the mechanism by which the loss of FMRP alters synaptic function, and subsequently causes the mental impairment, is unknown. Here, in cultured hippocampal neurons, we used siRNAs against Fmr1 to demonstrate that a reduction of FMRP in dendrites leads to an increase in internalization of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit, GluR1, in dendrites. This abnormal AMPAR trafficking was caused by spontaneous action potential-driven network activity without synaptic stimulation by an exogenous agonist and was rescued by 2-methyl-6-phenylethynyl-pyridine (MPEP), an mGluR5-specific inverse agonist. Because AMPAR internalization depends on local protein synthesis after mGluR5 stimulation, FMRP, a negative regulator of translation, may be viewed as a counterbalancing signal, wherein the absence of FMRP leads to an apparent excess of mGluR5 signaling in dendrites. Because AMPAR trafficking is a driving process for synaptic plasticity underlying learning and memory, our data suggest that hypersensitive AMPAR internalization in response to excess mGluR signaling may represent a principal cellular defect in FXS, which may be corrected by using mGluR antagonists.

Myosin IXb (Myo9b) was reported to be a single-headed, processive myosin. In its head domain it contains an N-terminal extension and a large loop 2 insertion that are specific for class IX myosins. We characterized the kinetic properties of purified, recombinant rat Myo9b, and we compared them with those of Myo9b mutants that had either the N-terminal extension or the loop 2 insertion deleted. Unlike other processive myosins, Myo9b exhibited a low affinity for ADP, and ADP release was not rate-limiting in the ATPase cycle. Myo9b is the first myosin for which ATP hydrolysis or an isomerization step after ATP binding is rate-limiting. Myo9b-ATP appeared to be in a conformation with a weak affinity for actin as determined by pyrene-actin fluorescence. However, in actin cosedimentation experiments, a subpopulation of Myo9b-ATP bound F-actin with a remarkably high affinity. Deletion of the N-terminal extension reduced actin affinity and increased the rate of nucleotide binding. Deletion of the loop 2 insertion reduced the actin affinity and altered the communication between actin and nucleotide-binding sites.

The extracellular domain (ECD) of the human follicle-stimulating hormone receptor (hFSHR) is believed to be the major determinant of hormone selectivity. Different discrete, discontinuous regions on the ECD of the hFSHR have been suggested to be crucial for hormone binding. However, the role of the ECD in signal transduction is not well understood. This study provides some insight into these aspects of the structure-function relationship of the ECD of hFSHR. Ten peptides were selected from the ECD on the basis of their ability to be surface oriented, synthesized by the solid-phase method using fluorenylmethyloxycarbonyl chemistry, purified and characterized. They were further studied for their ability to modulate both human follicle-stimulating hormone (hFSH)-FSHR binding and cAMP generation. Competitive inhibition studies showed that, of all the peptides studied, peptides 285-300 and 297-310 hFSHR were able to inhibit hFSH binding to FSHR. Both peptides function as weak competitive inhibitors of hFSH-FSHR binding. Peptides 285-300 hFSHR, 216-235 hFSHR, 184-195 hFSHR, 79-89 hFSHR and 15-31 hFSHR were observed to inhibit FSH-induced cAMP production. In summary, this study suggests that discrete, functional domains of the ECD have a role in hormone binding and signal transduction. Region 285-300 has been identified as a novel region crucial for both FSH binding and cAMP generation.