Group I Metabotropic Glutamate Receptors Bind to Protein Phosphatase 1C

Abstract

The modulation of neurotransmitter receptors by kinases and phosphatases represents a key mechanism in controlling synaptic signal transduction. However, molecular determinants involved in the specific targeting and interactions of these enzymes are largely unknown. Here, we identified both catalytic ␥-isoforms of protein phosphatase 1C (PP1␥1 and PP1␥2) as binding partners of the group I metabotropic glutamate receptors type 1a, 5a, and 5b in yeast cells and pull-down assays, using recombinant and native protein preparations. The tissue distribution of interacting proteins was compared, and protein phosphatase 1C was detected in dendrites of retinal bipolar cells expressing the respective interacting glutamate receptors. We mapped interacting domains within binding partners and identified five amino acids in the intracellular C termini of the metabotropic glutamate receptors type 1a, 5a, 5b, and 7b being both necessary and sufficient to bind protein phosphatase 1C. Furthermore, we show a dose-dependent competition of these C termini in binding the enzyme. Based on our data, we investigated the structure of the identified amino acids bound to protein phosphatase 1C by homology-based molecular modeling. In summary, these results provide a molecular description of the interaction between protein phosphatase 1C and metabotropic glutamate receptors and thereby increase our understanding of glutamatergic signal transduction.The correct targeting and localization of proteins to synaptic specializations represents an important biological mechanism to regulate neuronal excitability. Increasing evidence underlines the importance of macromolecular signaling complexes, where functionally related proteins such as ion channels, neurotransmitters receptors, kinases, and phosphatases are arranged in close vicinity and are physically anchored to the synaptic cytoskeleton. Therefore, identification and characterization of interactions between synaptically localized proteins adds substantially to our understanding of molecular mechanisms of neurotransmission.Receptors for glutamate are divided in ion channel-associated (ionotropic) receptors of the AMPA-, Kainate-, and NMDAtype and in G-protein coupled (metabotropic) receptors. Although ionotropic glutamate receptors mediate fast synaptic transmission, metabotropic glutamate receptors (mGluRs) 1 modulate neuronal excitability and development, synaptic plasticity, transmitter release, and memory function using a variety of intracellular second messenger systems (1). The eight known members of this protein family are sub-divided into three groups, based on sequence homology, associated second messenger systems, and pharmacological properties (2). mGluR1 and mGluR5 (group I) stimulate phospholipase C, are selectively activated by quisqualate, and are generally expressed perisynaptically at postsynaptic sites (3-7). mGluR2 and mGluR3 (group II) are negatively coupled to adenylyl cyclase and do not show a specific preference for pre-or postsynaptic neurons (8 -10). mGluR4, mGluR7, and...

Regulation of Metabotropic Glutamate Receptor 7 (mGluR7) Internalization and Surface Expression by Ser/Thr Protein Phosphatase 1

“…Therefore, 16 amino acids of mGluR7a and 23 amino acids of mGluR7b differ in their extreme C-terminal tails (31,32). Previous studies showed that PP1␥1 directly interacts with mGluR7b, but not mGluR7a, as determined using GST pulldown assays or yeast two-hybrid system interactions (33)(34)(35). This binding is mediated primarily by the N-terminal domain of PP1␥1 and the C-terminal region of mGluR7b (KSVTW; amino acids 911-915).…”

Dual neurotoxic and neuroprotective role of metabotropic glutamate receptor 1 in conditions of trophic deprivation – Possible role as a dependence receptor

“…In many cases the negative signaling is due to a caspase-mediated cleavage of the receptor C-terminal domain (CTD) which either releases a pro-apoptotic C-terminal receptor fragment (Bordeaux et al, 2000;Llambi et al, 2001), or exposes a pro-apoptotic region that presumably remains bound to the cell membrane (Mehlen et al, 1998;Forcet et al, 2001;Thibert et al, 2003). While a specific cleavage of the mGlu1 CDT has not been described, it should be pointed out that a sequence analysis of the long CTD of the mGlu1a splice variant reveals six putative caspase cleavage sites, as well as multiple sequences that may potentially interact with a variety of intracellular proteins including: seven in absentia homolog-1A (Siah-1A) and calmodulin (Ishikawa et al, 1999;Kammermeier and Ikeda, 2001), G-proteincoupled receptor kinases (Dale et al, 2000), alpha-tubulin (Ciruela et al, 1999), tamalin/ cytohesin complex (Kitano et al, 2002), homer proteins (Brakeman et al, 1997;Tu et al, 1999), protein phosphatase 1C (Croci et al, 2003), protein kinase C, regulators of G-protein signaling (RGS) proteins, Src-family protein tyrosine kinase and arrestins (Valenti et al, 2002;Hermans and Challiss, 2001). Therefore, the mGlu1 CTD is well equipped to participate and interfere in various intracellular signaling processes.…”

Band 4.1 proteins are expressed in the retina and interact with both isoforms of the metabotropic glutamate receptor type 8

“…Isolation of RNA and RT-PCR Total RNA was extracted from adult rat tissues, reverse transcribed and amplified as described (Croci et al 2003b) using primer pairs specific for the spectrin/actin binding domain (SAB) of 4.1B (sense nt1503-1523; antisense nt2313-2293) and the CTDs of 4.1B (sense nt3069-3087; antisense nt3424-3404), 41.G (sense nt2197-2217; antisense nt2646-2627), 4.1N (sense nt2133-2152; antisense nt2639-2618) and 4.1R (sense nt1959-1979; antisense nt2510-2491). Primers for the house keeping gene EF1a (sense nt1479-1497; antisense nt1770-1749) served as positive control.…”