Phosphorylation and Feedback Regulation of Metabotropic Glutamate Receptor 1 by Calcium/Calmodulin-Dependent Protein Kinase II

Abstract

The metabotropic glutamate receptor 1 (mGluR1) is a Gαq protein-coupled receptor and is distributed in broad regions of the mammalian brain. As a key element in excitatory synaptic transmission, the receptor regulates a wide range of cellular and synaptic activities. In addition to regulating its targets, the receptor itself is believed to be actively regulated by intracellular signals, although underlying mechanisms are largely unknown. Here we found that a synapse- enriched protein kinase, Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα), directly binds to the intracellular C terminus (CT) of mGluR1a. This binding is augmented by Ca2+ in vitro. The direct interaction promotes CaMKIIα to phosphorylate mGluR1a at a specific threonine site (T871). In rat striatal neurons, the mGluR1 agonist triggers the receptor- associated phosphoinositide signaling pathway to induce Ca2+-dependent recruitment of CaMKIIα to mGluR1a-CT. This enables the kinase to inhibit the response of the receptor to subsequent agonist exposure. Our data identify an agonist-induced and Ca2+-dependent protein-protein interaction between a synaptic kinase and mGluR1, which constitutes a feedback loop facilitating desensitization of mGluR1a.

Actinin-4 Governs Dendritic Spine Dynamics and Promotes Their Remodeling by Metabotropic Glutamate Receptors

“…To explore this hypothesis, we first examined whether activation of group 1 mGluRs could induce CaMKII␤ autophosphorylation at Thr-287, triggered by Ca 2ϩ /Calmodulin binding. Group 1 mGluRs have been shown previously to induce autophosphorylation of CaMKII␣ in the striatum (46) and hippocampus (47), but their impact on CaMKII␤ is not known. Cortical neurons (DIV 13-17) were treated with DHPG (50 M) or vehicle for different times, and CaMKII␤ autophosphorylation was assessed by Western blot with anti-phospho-CaMKII antibodies that recognize Thr-287 in CaMKII␤.…”

Synaptic ERK2 Phosphorylates and Regulates Metabotropic Glutamate Receptor 1 In Vitro and in Neurons

“…Western blots were performed as described previously [22]. Briefly, sodium dodecyl sulfate (SDS) NuPAGE Bis-Tris 4–12 % gels (Invitrogen, Carlsbad, CA) were used to separate proteins.…”

“…This assay was carried out according to our previous work [20, 22]. Briefly, His-tagged active ERK2-FL with pT185/pY187 (Millipore, Billerica, MA; 20 ng) or His-tagged inactive ERK2-FL (Millipore; 20 ng) was equilibrated to binding buffer containing 200 mM NaCl, 0.2 % Triton X-100, 0.1 mg/ml bovine serum albumin (BSA), and 50 mM Tris, pH, 7.5.…”

“…We performed coimmunoprecipitation with rat brains as described previously [22]. After rats were anesthetized and decapitated, brains were removed.…”

“…Cellular IP 3 levels were measured in rat cerebellar slices using a HitHunter IP 3 Fluorescence Polarization Assay Kit from DiscoveRx (Fremont, CA) or a rat IP 3 ELISA Kit from CUSABIO (Wuhan, China) according to the manufacturer’s instructions [22, 27, 28]. …”