Activation of Group I metabotropic glutamate receptors (mGluRs) activates signaling cascades, resulting in calcium release from intracellular stores, ERK1/2 activation, and long term changes in synaptic activity that are implicated in learning, memory, and neurodegenerative diseases. As such, elucidating the molecular mechanisms underlying Group I mGluR signaling is important for understanding physiological responses initiated by the activation of these receptors. In the current study, we identify the multifunctional scaffolding protein spinophilin as a novel Group I mGluR-interacting protein. We demonstrate that spinophilin interacts with the C-terminal tail and second intracellular loop of Group I mGluRs. Furthermore, we show that interaction of spinophilin with Group I mGluRs attenuates receptor endocytosis and phosphorylation of ERK1/2, an effect that is dependent upon the interaction of spinophilin with the C-terminal PDZ binding motif encoded by Group I mGluRs. Spinophilin knock-out results in enhanced mGluR5 endocytosis as well as increased ERK1/2, AKT, and Ca 2؉ signaling in primary cortical neurons. In addition, the loss of spinophilin expression results in impaired mGluR5-stimulated LTD. Our results indicate that spinophilin plays an important role in regulating the activity of Group I mGluRs as well as their influence on synaptic activity.Glutamate is the main excitatory neurotransmitter in the central nervous system. The actions of glutamate are mediated via two types of receptors: ionotropic glutamate receptors, which are ligand-gated cation channels, and metabotropic glutamate receptors, which are G protein-coupled receptors (GPCRs) 2 (1, 2). Activation of mGluRs produces long term changes in synaptic plasticity, including long term potentiation and long term depression (LTD) (3, 4). Consequently, mGluRs have been implicated in both memory and learning as well as neurodegenerative disorders, such as Alzheimer and Parkinson diseases (5). The mechanism by which Group I mGluR signaling contributes to neurodegenerative disease remains unclear. However, genetic deletion of mGluR5 has been shown to improve cognitive performance and reduce Alzheimer disease-like pathology in an APPswe/PS1⌬E9 mouse model of Alzheimer disease (6). Multiple protein partners have been shown to interact with Group I mGluRs to alter their signaling and trafficking (7,8). Recently, via a tandem affinity purification proteomic screen with the mGluR1a C-terminal tail, our laboratory has discovered a novel Group I mGluR-interacting protein, protein phosphatase 1, regulatory subunit 9B (spinophilin/neurabin II). Spinophilin is a multifunctional synaptic scaffolding protein that is compartmentalized to the heads of dendritic spines and interacts with protein phosphatases (PP1␣ and -␥) and F-actin (9 -11). It also encodes three putative Src homology 3 domains, a GPCR-binding domain, a PDZ (PSD95/Disc Large/zona occludens) domain, three coiled-coiled domains, and a potential leucine/isoleucine zipper motif (12).Spinophilin is recruited...