Marlin-1, a Novel RNA-binding Protein Associates with GABA Receptors

Abstract

GABA B receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Whereas heterodimerization between GABA B receptor GABA B R1 and GABA B R2 subunits is essential for functional expression, how neurons coordinate the assembly of these critical receptors remains to be established. Here we have identified Marlin-1, a novel GABA B receptor-binding protein that associates specifically with the GABA B R1 subunit in yeast, tissue culture cells, and neurons. Marlin-1 is expressed in the brain and exhibits a granular distribution in cultured hippocampal neurons. Marlin-1 binds different RNA species including the 3-untranslated regions of both the GABA B R1 and GABA B R2 mRNAs in vitro and also associates with RNA in cultured neurons. Inhibition of Marlin-1 expression via small RNA interference technology results in enhanced intracellular levels of the GABA B R2 receptor subunit without affecting the level of GABA B R1. Together our results suggest that Marlin-1 functions to regulate the cellular levels of GABA B R2 subunits, which may have significant effects on the production of functional GABA B receptor heterodimers. Therefore, our observations provide an added level of regulation for the control of GABA B receptor expression and for the efficacy of inhibitory synaptic transmission.It has become widely accepted that protein-protein interactions are responsible for the formation and maintenance of the signaling platforms that organize local transduction units (1). Protein associations have also been shown to modulate the synthesis, targeting, and stabilization of membrane receptors (2). The study of the spatial and temporal compartmentalization of G protein-coupled receptors (GPCRs) 1 is essential to understand the mechanisms for neuronal integration of multiple stimuli. The identification of signaling partners has been well documented for ion channels (3-5), but less is known about the proteins that assist GPCRs (6, 7). GABA B receptors mediate the slow and prolonged phase of synaptic inhibition (8) and, unlike other GPCRs, they require the formation of a heterodimer between GABA B R1 and GAB-A B R2 (9). These two subunits display high homology to metabotropic glutamate, Ca 2ϩ -sensing, vomeronasal, and putative pheromone receptors, and recombinant GABA B R1/GABA B R2 receptors mimic the effector-coupling and pharmacological properties of native receptors (10). Clinically, GABA B receptors have been implicated in depression, neuroprotection, and cognition, and the production of GABA B R1 knock-out mice has confirmed their role in epilepsy and pain (11,12). Furthermore, recent studies indicate that their activation may be beneficial in the treatment of withdrawal symptoms from addictive drugs (13). Given their critical role in synaptic transmission and their potential therapeutic implications, it is fundamental to understand all aspects of GABA B receptor signaling.The modulation of neuronal GABA B receptors is likely to occur at multiple levels rangi...

The Clustering of GABAAReceptor Subtypes at Inhibitory Synapses is Facilitated via the Direct Binding of Receptor α2 Subunits to Gephyrin

“…Neuronal transfection. Dissociated E18 hippocampal neurons were transfected by nucleofection according to the protocol of the manufacturer (Amaxa, Cologne, Germany) with 3 g of single subunit plasmid DNA, plated and maintained in culture for 18 -21 d (Couve et al, 2004;Kittler et al, 2004;Jacob et al, 2005).…”

Huntingtin-associated protein 1 regulates inhibitory synaptic transmission by modulating γ-aminobutyric acid type A receptor membrane trafficking

“…Cultures of cortical and hippocampal neurons were prepared as described (8,(17)(18)(19)(20). Nucleofection of cortical and hippocampal neurons (19) was carried out following the manufacturer's protocol (Amaxa, Gaithersburg, MD). Immunolocalization studies were carried out as described (8,17), using guinea pig ␣-GABA A R ␥2 subunit at 1:100 and ␣-HAP1 (1:100).…”

GABAB Receptor Association with the PDZ Scaffold Mupp1 Alters Receptor Stability and Function

“…Indeed, we previously reported that association of GABA B receptors with the GABA A receptor ␥2S subunit confers agonist-mediated endocytosis on GABA B receptors expressed in heterologous cells (16). Furthermore, GABA B receptors have also been shown to be regulated by interactions with several other protein partners, including the transcription factors cAMP-response element-binding protein 2 and ATF4 (17,18), the adaptor protein 14-3-3 (19), the RNAbinding protein Marlin-1 (20), and the coat protein I (21).…”