The PDZ Scaffold NHERF-2 Interacts with mGluR5 and Regulates Receptor Activity

Abstract

The two members of the group I metabotropic glutamate receptor family, mGluR1 and mGluR5, both couple to G q to mediate rises in intracellular calcium. The alternatively spliced C termini (CT) of mGluRs 1 & 5 are known to be critical for regulating receptor activity and to terminate in motifs suggestive of potential interactions with PDZ domains. We therefore screened the CTs of both mGluR1a and mGluR5 against a PDZ domain proteomic array. Out of 96 PDZ domains examined, the domain that bound most strongly to mGluR5-CT was the second PDZ domain of the Na ؉ /H ؉ exchanger regulatory factor 2 (NHERF-2). This interaction was confirmed by reverse overlay, and a single point mutation to the mGluR5-CT was found to completely disrupt the interaction. Full-length mGluR5 robustly associated with full-length NHERF-2 in cells, as assessed by co-immunoprecipitation and confocal microscopy experiments. In contrast, mGluR1a was found to bind NHERF-2 in vitro with a weaker affinity than mGluR5, and furthermore mGluR1a did not detectably associate with NHERF-2 in a cellular context. Immunohistochemical experiments revealed that NHERF-2 and mGluR5 exhibit overlapping patterns of expression in mouse brain, being found most abundantly in astrocytic processes and postsynaptic neuronal elements. In functional experiments, the interaction of NHERF-2 with mGluR5 in cells was found to prolong mGluR5-mediated calcium mobilization and to also potentiate mGluR5-mediated cell death, whereas coexpression of mGluR1a with NHERF-2 had no evident effects on mGluR1a functional activity. These observations reveal that NHERF-2 can selectively modulate mGluR5 signaling, which may contribute to cell-specific regulation of mGluR5 activity.Many of the physiological effects of L-glutamate as a neurotransmitter are mediated via stimulation of metabotropic glutamate receptors (mGluRs), 2 which are considered to be important therapeutic targets in the potential treatment of a number of different diseases (1-3). The mGluRs are G protein-coupled receptors that regulate the physiology of neurons and glia through activation of various secondary messenger systems (4). Eight subtypes of mGluRs have been identified and classified into three groups (I, II, III) based on sequence homology, signaling pathways and pharmacological selectivity. The various mGluR subtypes are known to display differential regional and cellular distributions throughout the mammalian brain (4). Activation of group I mGluRs (mGluR1 and mGluR5) leads to signal transduction primarily through coupling to G q and phospholipase C, which then hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to form the second messenger inositol 1,4,5-phosphate (IP 3 ). IP 3 binding to its receptor in the membrane of the endoplasmic reticulum causes release of free Ca 2ϩ in the cytosol, which generates a signaling cascade. Group I mGluRs can also couple to other signaling pathways in a cell type-specific manner (5). Despite many similarities in their signaling pathways, mGluR1 and mGluR5 expressed in th...

ATP Induces Synaptic Gene Expressions in Cortical Neurons: Transduction and Transcription Control via P2Y1 Receptors

“…NHERF-2 is present postsynaptically in forebrain neurons in situ, as shown in the mouse cortex (Paquet et al, 2006), and we found P2Y 1 R also located there. NHERF-2 is presumed to contribute to the organization of P2Y 1 Rs at the membrane.…”

“…The PDZ-domain isolated from the PSD-95 sequence did not bind to that region of the P2Y 1 R in this system. Positive results from this screen have identified various PDZ-domain/receptor interactions, including NHERF-2/P2Y 1 R (Fam et al, 2005;Paquet et al, 2006). However, we considered whether another interaction might be made there for certain pairs such as PSD-95/P2Y 1 R. Thus, first, the full-length PSD-95 is known from crystallographic and other evidence to require a specific folding for a PDZ domain to bind a GPCR partner held within the PDS-95 tertiary structure (Kim and Sheng, 2004), and this may not always be attainable in an isolated PDZ domain/fusion protein screen.…”

WDR36 acts as a scaffold protein tethering a G-protein-coupled receptor, Gαq and phospholipase Cβ in a signalling complex

“…Choi et al showed that PAR-3 mediated physical interaction between PLCb1 and the bradykinin receptor, whereas NHERF2 did so between PLCb3 and the LPA2 receptor (Choi et al, 2010). The association of NHERF1 and/or NHERF2 with the parathyroid hormone 1 receptor (PTH1R) (Mahon and Segre, 2004;Sneddon et al, 2003;Wang et al, 2009;Wheeler et al, 2008), purinergic receptor (P2RY1) (Fam et al, 2005) and metabotropic glutamate receptor 5 (mGluR5) (Paquet et al, 2006), can enhance their PLCb-mediated signalling (Ritter and Hall, 2009). However, no evidence of Gaq involvement in the NHERF-promoted PLCb signalling was found.…”

Muscarinic-induced Recruitment of Plasma Membrane Ca2+-ATPase Involves PSD-95/Dlg/Zo-1-mediated Interactions

“…NHERF-2 also associates with metabotropic glutamine receptor 5 (mGluR5) to prolong intracellular Ca 2ϩ mobilization by mGluR5 and thereby promoting mGluR5-mediated Ca 2ϩ toxicity (49). As a scaffold, NHERF-2 has been shown to stabilize or tether membrane proteins in functionally distinct complexes particularly in polarized epithelial cells.…”