Metabotropic glutamate receptors (mGluRs) are a family of class 3 G protein-coupled receptors with important functions in the regulation of synaptic transmission, plasticity, neuronal excitability, and the regulation sensory transduction [1
]. The eight mGluRs (mGluR1-8) and their splice variants have been divided into three groups (I-III) based on sequence homology, pharmacology and G protein coupling profiles [3
]. Group II (mGluR2 and 3) and III (mGluR4, and 6 through 8) couple negatively to adenylate cyclase through activation of the Gi/o
family of G proteins [8
]. Group I mGluRs (mGluR1 and 5) couple to multiple G proteins in native and heterologous systems, and have been observed to couple to Gq/11
]. mGluRs appear to be important mediators of synaptic transmission in the brain. The group I mGluRs typically are located postsynaptically (as well as in other compartments of the cell) [16
] where they regulate activity of ionotropic receptors and are necessary in some forms of synaptic plasticity [19
]. The association of mGluRs with scaffolding proteins such as Homer (group I; [20
]) and PICK-1 (group III; [21
]) is important in regulating mGluR localization and function [21
]. In addition to those proteins involved in scaffolding, other proteins have been shown to associate with mGluRs. For example, group I and group III mGluRs are both known to bind Calmodulin (CaM) [25
]. In the case of group III mGluRs, the interaction with CaM is important in regulating the efficacy of G protein activation [27
]. CaM binding to both group I and group III mGluRs can be inhibited by serine/threonine phosphorylation at specific sites [25
]. Therefore, it is of interest in terms of mGluR function and localization to understand the roles of proteins which interact with these receptors.
The Seven in Absentia (SinA) protein was discovered in Drosophila as a mediator of photoreceptor development [29
]. Subsequently, SinA was shown to participate in the ubiquitin/proteasome pathway [30
]. The presence of a RING finger domain in the N-terminus of the mammalian homologue of SinA, "Siah", suggests it may be involved in targeting specific proteins for degradation in the proteasome as a ubiquitin ligase [32
]. Two mammalian genes encoding Siah, Siah1 and 2, are known. These proteins have been implicated in many processes including cancer [33
], apoptosis [34
] and cell cycle regulation [35
]. More recently, an interaction between Siah1a and the C-terminal tail of group I mGluRs was demonstrated [36
]. This interaction was specific for group I mGluRs, as it was not observed in the analogous sequences from group II (mGluR2) or group III (mGluR7) receptors. Due to the novelty of the mammalian homolog of Seven in Absentia (Siah), little information is available regarding its tissue distribution or subcellular localization in mammalian cells. To date the functional consequences of Siah1a association with group I mGluRs are unknown.
To assess the role of Siah1a binding, specific group I mGluRs were heterologously expressed in sympathetic neurons from the rat superior cervical ganglion (SCG), which do not natively express mGluRs [37
]. The functional role of Siah1a was assessed by examining calcium current inhibition mediated by the expressed mGluRs in the absence and presence of heterologous Siah1a expression. In addition, the surface expression and subcellular distribution of group I mGluRs was examined with and without Siah1a using various fluorescence techniques. Finally, the effect of CaM overexpression on Siah1a function was assessed.