Differential Subcellular Localization of RIC-3 Isoforms and Their Role in Determining 5-HT3 Receptor Composition

Abstract

RIC-3 has been identified as a chaperone molecule involved in promoting the functional expression of nicotinic acetylcholine and 5-HT 3 receptors in mammalian cells. In this study, we examined the effects of RIC-3a (isoform a) and a truncated isoform (isoform d) on RIC-3 localization, mobility, and aggregation and its effect on 5-HT3 receptor composition in mammalian cells. Human RIC-3a possesses an amino-terminal signal sequence that targets it to the endoplasmic reticulum where it is distributed within the reticular network, often forming large diffuse "slicks" and bright "halo" structures. RIC-3a is highly mobile within and between these compartments. Despite the propensity for RIC-3a to aggregate, its expression enhances the level of surface 5-HT3A (homomeric) receptors. In contrast, RIC-3a exerts an inhibitory action on the surface expression of heteromeric 5-HT3A/B receptors. RIC-3d exhibits an altered subcellular distribution, being localized to the endoplasmic reticulum, large diffuse slicks, tubulo-vesicular structures, and the Golgi. Bidirectional trafficking between the endoplasmic reticulum and Golgi suggests that RIC-3d constitutively cycles between these two compartments. In support of the large coiledcoil domain of RIC-3a being responsible for protein aggregation, RIC-3d, lacking this cytoplasmic domain, does not aggregate or induce the formation of bright aggregates. Regardless of these differences, isoform d is still capable of enhancing homomeric, and inhibiting heteromeric, 5-HT3 receptor expression. Thus, both isoforms of RIC-3 play a role in determining 5-HT3 receptor composition.The ligand-gated ion channels are critical participants in cellular communication, playing a major role in synaptic transmission. The ligand-gated ion channels include receptors for acetylcholine (nACh) 3 , ␥-aminobutyric acid type A, serotonin (5-HT), and glycine (the cys-loop superfamily) and N-methyl-D-aspartic acid, kainate, and AMPA (glutamate receptors). A vast array of receptor-interacting proteins have been identified as participating in receptor trafficking and localization (1) and have led to dramatic advances in our knowledge of synaptic architecture and plasticity.Chaperone molecules play an important role in the intracellular transport of receptors. However, a fundamental question in neurobiology remains: How is receptor biogenesis orchestrated? Despite the fact that receptor composition determines receptor function and their pharmacological repertoire, little information is available regarding how this may be achieved. Specific assembly signals exist that can determine a preference for particular subunit partners (2). However, few selective chaperone molecules have been implicated in the process of receptor biogenesis. General chaperone proteins such as BiP, calnexin, calreticulin, and PDI do operate on the ligand-gated ion channels but offer no specificity. In contrast, molecules such as stargazin (3), PSD-95 (4), 14-3-3 (5), and RIC-3 (6) (7-12) are beginning to offer insight into protein-specific ch...

Mouse RIC-3, an Endoplasmic Reticulum Chaperone, Promotes Assembly of the α7 Acetylcholine Receptor through a Cytoplasmic Coiled-Coil Domain

“…Functional ␣7 nAChR is expressed poorly in transfected mammalian cell lines that lack endogenous ␣7 expression (Sweileh et al, 2000), but coexpression of RIC-3 dramatically boosts the levels of functional ␣7 (Castillo et al, 2005;Lansdell et al, 2005;Williams et al, 2005). Moreover, the extent of endogenous ␣7 expression is correlated with levels of RIC-3 (Williams et al, 2005), which appears to be localized largely in the ER (Halevi et al, 2002;Castillo et al, 2005;Cheng et al, 2007).…”

“…The lack of agreement has led to two different predictions for the transmembrane topology of the mammalian protein ( Fig. 1): RIC-3 either crosses the ER membrane twice with both N and C termini on the cytoplasmic side (Castelán et al, 2008); or has an N-terminal signal peptide and a single transmembrane segment with the N terminus on the lumenal side (Cheng et al, 2007).…”

Muscle-like nicotinic receptor accessory molecules in sensory hair cells of the inner ear

“…However, the precise mechanisms by which the hRIC-3 protein exerts its effects on nicotinic receptors are not known. It has been reported that the N-terminal and the C-terminal regions of hRIC-3 are involved in its action on α7 nAChRs (Ben-Ami et al, 2005) and 5HT3Rs (Cheng et al, 2007;Cheng et al, 2005). Because of the close sequence similarity between nAChRα7 and nAChR α9 subunits (42% identity at the amino acid level), we utilized in vitro and in vivo co-immunoprecipitation analyses and confirmed that α9 and hRIC-3 are able to co-associate directly in HEK293T cells and cochlear tissues.…”

“…Recent studies have identified a transmembrane protein (RIC-3), which is located within the endoplasmic reticulum and may chaperone nicotinic and serotonergic receptors (Cheng et al, 2007;Halevi et al, 2002;Halevi et al, 2003). Studies in Xenopus oocytes and mammalian cells demonstrate that co-expression of the human homologue (hRIC-3) can enhance functional expression of α7 nAChRs (Williams et al, 2005).…”

Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor

“…The orthologs of RIC-3 have been characterized in insects and vertebrates. They are proposed to act as chaperones that directly interact with various AChR or 5-HT 3A subunits and promote maturation and assembly of the receptors (28). The human hRIC-3 is able to enhance the expression of the C. elegans DES-2/DEG-3 AChR, demonstrating interspecies functionality (14).…”