ERK regulation of phosphodiesterase 4 enhances dopamine-stimulated AMPA receptor membrane insertion

Abstract

AMPA-type glutamate receptor (AMPAR) trafficking is essential for modulating synaptic transmission strength. Prior studies that have characterized signaling pathways underlying AMPAR trafficking have identified the cAMP/PKA-mediated phosphorylation of GluA1, an AMPAR subunit, as a key step in the membrane insertion of AMPAR. Inhibition of ERK impairs AMPAR membrane insertion, but the mechanism by which ERK exerts its effect is unknown. Dopamine, an activator of both PKA and ERK, induces AMPAR insertion, but the relationship between the two protein kinases in the process is not understood. We used a combination of computational modeling and live cell imaging to determine the relationship between ERK and PKA in AMPAR insertion. We developed a dynamical model to study the effects of phosphodiesterase 4 (PDE4), a cAMP phosphodiesterase that is phosphorylated and inhibited by ERK, on the membrane insertion of AMPAR. The model predicted that PKA could be a downstream effector of ERK in regulating AMPAR insertion. We experimentally tested the model predictions and found that dopamine-induced ERK phosphorylates and inhibits PDE4. This regulation results in increased cAMP levels and PKA-mediated phosphorylation of DARPP-32 and GluA1, leading to increased GluA1 trafficking to the membrane. These findings provide unique insight into an unanticipated network topology in which ERK uses PDE4 to regulate PKA output during dopamine signaling. The combination of dynamical models and experiments has helped us unravel the complex interactions between two protein kinase pathways in regulating a fundamental molecular process underlying synaptic plasticity.T he strength of synaptic transmission depends on the number of AMPA-type glutamate receptors (AMPARs) localized to the synaptic membrane. The regulated trafficking of AMPARs in and out of the postsynaptic membrane controls the number of synaptic AMPARs and is thought to underlie synaptic plasticity (1). AMPARs are composed of four subunits (GluA1-4), which assemble as homo-or hetero-tetramers to mediate excitatory transmissions in the brain. There are a number of intracellular pathways that regulate signal-initiated trafficking of GluA1-containing AMPARs. For instance, PKA and PKG, the cyclic nucleotide-activated kinases, phosphorylate GluA1 at S845 (2, 3). Phosphorylation of S845 is required for GluA1 synaptic insertion because mutation to A845 prevents GluA1 exocytosis (4). Dopamine, a modulatory neurotransmitter that increases cAMP/ PKA levels, promotes GluA1 phosphorylation at S845 and AMPAR insertion into the plasma membrane (3, 5, 6). Additional signaling pathways influence this process, but the role they play in dopamine-mediated AMPAR trafficking is not known. ERK, a downstream effector of dopamine, promotes AMPAR membrane insertion even though ERK does not directly phosphorylate GluA1 (7,8). The objective of this study was to identify the mechanism by which ERK regulates dopamine-mediated GluA1 membrane insertion. Based on our observation that ERK inhibition decreases dop...

Cross-regulation of Phosphodiesterase 1 and Phosphodiesterase 2 Activities Controls Dopamine-mediated Striatal α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Trafficking

“…Live-cell Imaging-Primary MSNs were obtained as described previously (39). To monitor GluA1 trafficking, MSNs (days in vitro 7-16) were transfected with PCI-SEP-GluR1.…”

“…Biotinylated proteins were eluted from streptavidin beads with radioimmune precipitation assay buffer containing 1ϫ SDS-PAGE reducing sample buffer with DTT at 95°C for 5 min. Eluted proteins were resolved by SDS-PAGE as described previously (39,73). Nitrocellulose membranes were incubated with anti-GluA1 N-terminal antibody (MAB2263, EMD Millipore).…”

“…Dynamical Modeling-We expanded our previous computational model of dopamine-induced AMPAR trafficking (39) by adding the following reactions: basal production of cGMP by soluble guanylyl cyclase from GTP, degradation of cAMP and cGMP by PDE1, allosteric activation of PDE2 by cGMP, and degradation of cAMP and cGMP by PDE2. The model was initially developed in VCell and was exported to MATLAB (Mathworks) for ease of computing and optimization.…”

“…In other words, given the affinity of cGMP for the allosteric site on PDE2, could biologically relevant changes to cGMP levels affect PDE2 activity sufficiently to have an impact on GluA1 trafficking? To answer this, we developed a dynamic model of PDE2 regulation of AMPAR trafficking in D1 MSNs by building upon our previous model (39). The novel connectivity (consisting of a detailed representation of PDE2 and its regulation by cGMP and PDE1 activity) is depicted in Fig.…”

“…The complexity of cyclic nucleotide signaling makes it difficult to intuit the effect each PDEs has on observed AMPAR trafficking. Here we use a systems pharmacology approach, extending our previous computational model of dopamine signaling and AMPAR trafficking to investigate the role of cGMP on dopamine-induced AMPAR trafficking (39). We present a novel mechanism of how cGMP may be controlling striatal AMPAR trafficking in response to DA via the cross-regulation of PDEs.…”