Host‐cell specific effects of the nicotinic acetylcholine receptor chaperone RIC‐3 revealed by a comparison of human and Drosophila RIC‐3 homologues

Abstract

RIC‐3 is a transmembrane protein which enhances maturation (folding and assembly) of neuronal nicotinic acetylcholine receptors (nAChRs). In this study, we report the cloning and characterisation of 11 alternatively spliced isoforms of Drosophila melanogaster RIC‐3 (DmRIC‐3). Heterologous expression studies of alternatively spliced DmRIC‐3 isoforms demonstrate that nAChR chaperone activity does not require a predicted coiled‐coil domain which is located entirely within exon 7. In contrast, isoforms containing an additional exon (exon 2), which is located within a proline‐rich N‐terminal region, have a greatly reduced ability to enhance nAChR maturation. The ability of DmRIC‐3 to influence nAChR maturation was examined in co‐expression studies with human α7 nAChRs and with hybrid nAChRs containing both Drosophila and rat nAChR subunits. When expressed in a Drosophila cell line, several of the DmRIC‐3 splice variants enhanced nAChR maturation to a significantly greater extent than observed with human RIC‐3. In contrast, when expressed in a human cell line, human RIC‐3 enhanced nAChR maturation more efficiently than DmRIC‐3. The cloning and characterisation of 11 alternatively spliced DmRIC‐3 isoforms has helped to identify domains influencing RIC‐3 chaperone activity. In addition, studies conducted in different expression systems suggest that additional host cell factors may modulate the chaperone activity of RIC‐3.

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

“…Since different cell lines may or may not express these accessory proteins, it is likely that the use of different cell lines has yielded different results regarding nAChR α9α10 functional expression. RT-PCR studies have provided evidence of a correlation between the endogenous expression of RIC-3 transcripts and the ability of cells to express functional α7 nAChRs (either endogenously or from heterologous expression of nAChRα7 cDNA) (Lansdell et al, 2008;Lansdell et al, 2005;Williams et al, 2005). For example, although tsA201 cells contain no RIC-3 mRNA, GH4C1 cells show detectable levels of endogenous RIC-3 mRNA as determined by RT-PCR (Lansdell et al, 2005).…”

Ric-3 chaperone-mediated stable cell-surface expression of the neuronal α7 nicotinic acetylcholine receptor in mammalian cells

“…The levels of cell-surface α7 AChRs in this new cell line increased from less than 1% to 20% (Figure 1). On the basis of previous reports [13,14,16,17] , we can surmise that Ric-3 facilitates the correct folding and assembly of α7 subunits at the ER. When the parental SHE-P1-hα7 cells were labeled with fluorescent αBTX, a punctate distribution reminiscent of the ER staining was observed throughout the cell ( Figure 1B).…”

“…There is evidence that AChR folding, assembly and trafficking are influenced by several chaperone proteins. Recent studies have shown that co-expression of α7 AChR and the chaperone RIC-3 facilitates the formation of functional homomeric AChRs in otherwise non-permissive cell types [13,14,16,17] . RIC-3 was originally identified in the nematode Caenorhabditis elegans as a protein encoded by the gene ric-3 (resistance to inhibitors of cholinesterase) and has subsequently been cloned and characterized for mammalian and insect species.…”

“…Ric-3, first discovered in Caenorhabditis elegans, is an endoplasmic reticulum-resident protein present in cells that endogenously express α7 AChR [13] . Ric-3 has been shown to increase α7 AChR heterologous expression in oocytes from Xenopus laevis and in mammalian cell lines such as HEK-293 [13][14][15][16][17] . According to these authors, Ric-3 is needed to attain the correct folding of the α7 AChR, and therefore to attain functional expression.…”

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

“…The shift to a lower temperature is necessary to allow appropriate folding of Torpedo nAChR subunit proteins prior to receptor assembly [115], presumably a consequence of nAChRs from species such as Torpedo being adapted to fold efficiently at temperatures lower than 37°C. Similarly, insect nAChR subunits, when expressed in mammalian cell lines, also require incubation at a temperature lower than 37°C for efficient subunit folding and assembly [96,116,117]. To an extent, these problems with insect nAChRs can be avoided by expression in insect cell lines [62,96,116,118], which are typically maintained at 20-25°C (although difficulties remain with the expression of 10 some invertebrate nAChRs, as will be discussed in more detail below).…”