The ric-3 gene is required for maturation of nicotinic acetylcholine receptors in Caenorhabditis elegans. The human homolog of RIC-3, hRIC-3, enhances expression of ␣7 nicotinic receptors in Xenopus laevis oocytes, whereas it totally abolishes expression of ␣4␤2 nicotinic and 5-HT 3 serotonergic receptors. Both the N-terminal region of hRIC-3, which contains two transmembrane segments, and the C-terminal region are needed for these differential effects. hRIC-3 inhibits receptor expression by hindering export of mature receptors to the cell membrane. By using chimeric proteins made of ␣7 and 5-HT 3 receptors, we have shown that the presence of an extracellular isoleucine close to the first transmembrane receptor fragment is responsible for the transport arrest induced by hRIC-3. Enhancement of ␣7 receptor expression occurs, at least, at two levels: by increasing the number of mature receptors and facilitating its transport to the membrane. Certain amino acids of a putative amphipathic helix present at the large cytoplasmic region of the ␣7 subunit are required for these actions. Therefore, hRIC-3 can act as a specific regulator of receptor expression at different levels.

The RIC‐3 protein acts as a regulator of acetylcholine nicotinic receptor (nAChR) expression. In Xenopus laevis oocytes the human RIC‐3 (hRIC‐3) protein enhances expression of α7 receptors and abolishes expression of α4β2 receptors. In vitro translation of hRIC‐3 evidenced its membrane insertion but not the role as signal peptide of its first transmembrane domain (TMD). When the TMDs of hRIC‐3 were substituted, its effects on nAChR expression were attenuated. A certain linker length between the TMDs was also needed for α7 expression enhancement but not for α4β2 inhibition. A combination of increased α7 receptor steady state levels, facilitated transport and reduced receptor internalization appears to be responsible for the increase in α7 membrane expression induced by hRIC‐3. Antibodies against hRIC‐3 showed its expression in SH‐SY5Y and PC12 cells and its induction upon differentiation. Immunohistochemistry demonstrated the presence of RIC‐3 in rat brain localized, in general, in places where α7 nAChRs were found.

We studied the role of the a-helix present at the N-terminus of nicotinic acetylcholine receptor (nAChR) subunits in the expression of functional channels. Deletion of this motif in a7 subunits abolished expression of nAChRs at the membrane of Xenopus oocytes. The same effect was observed upon substitution by homologous motifs of other ligand-gated receptors. When residues from Gln4 to Tyr15 were individually mutated to proline, receptor expression strongly decreased or was totally abolished. Equivalent substitutions to alanine were less harmful, suggesting that proline-induced break of the ahelix is responsible for the low expression. Steady-state levels of wild-type and mutant subunits were similar but the formation of pentameric receptors was impaired in the latter. In addition, those mutants that reached the membrane showed a slightly increased internalization rate. Expression of a7 nAChRs in neuroblastoma cells confirmed that mutant subunits, although stable, were unable to reach the cell membrane. Analogous mutations in heteromeric nAChRs (a3b4 and a4b2) and 5-HT 3A receptors also abolished their expression at the membrane. We conclude that the N-terminal a-helix of nAChRs is an important requirement for receptor assembly and, therefore, for membrane expression.

Using a yeast two-hybrid screening we report the isolation of a novel human protein, hCRELD2b, that interacts specifically with the large cytoplasmic regions of human nicotinic acetylcholine receptor (nAChR) a4 and b2 subunits, both in yeast cells and in vitro. This interaction is not detected with nAChR a7 and a3 subunits. The hCRELD2 gene encodes for multiple transcripts, likely to produce multiple protein isoforms. A previously reported one has been renamed as CRELD2a. Isoforms a and b are expressed in all tissues examined and have the same N-terminal and central regions but alternative C-terminal regions. Both isoforms interact with the a4 subunit. Within this subunit the interaction was localized to the N-terminal region of the large cytoplasmic loop. The CRELD2b protein is present at the endoplasmic reticulum where colocalized with a4b2 nAChRs upon cell transfection. Immunohistochemistry experiments demonstrated the presence of CRELD2 in the rat brain at sites where a4b2 receptors have been previously detected. Labeling was restricted to neuronal perikarya. Finally, CRELD2 decreases the functional expression and impairs membrane transport of a4b2 nAChRs in Xenopus leavis oocytes, without affecting a3b4 and a7 nAChR expression. These results suggest that CRELD2 can act as a specific regulator of a4b2 nAChR expression.

Neurotransmitter-gated receptors are assembled in the endoplasmic reticulum and transported to the cell surface through a process that might be of central importance to regulate the efficacy of synaptic transmission (Kneussel and Betz, 2000; Kittler and Moss, 2003). This process is relatively inefficient- what may be the consequence of tight quality controls that guarantee the functional competence of the final product. For this purpose, specific proteins involved in assembly and trafficking of receptors might be required (Keller and Taylor, 1999; Millar, 2003; Wanamaker et al., 2003). The RIC-3 protein could be one of them, as mutations in the ric-3 gene affect maturation of nicotinic acetylcholine receptors (nAChRs) in Caenorhabditis elegans (Halevi et al., 2002). Moreover, the human homolog hRIC-3 showed differential effects when coexpressed with several ligand-gated receptors (Halevi et al., 2003). Thus, it enhanced alpha7 nAChR expression while inhibiting expression of other nAChR subtypes (alpha4beta2 and alpha3beta4) and 5-HT3 serotonin receptors (5-HT3Rs). These opposite effects suggested that the RIC-3 protein might play a key role in the biogenesis of some ligand-gated receptors and prompted us to investigate how it performs its action. Here, we show that the RIC-3 protein acts as a barrier for some receptors like alpha4beta2 nAChRs and 5-HT3Rs, stopping the traffic of mature receptors to the membrane. In contrast, the inefficient transport of alpha7 nAChRs is enhanced by RIC-3 in a process in which certain amino acids at the amphipathic helix located at the C-terminal region of the large cytoplasmic domain are involved.

The ␣9 subunit is a component of the neuronal nicotinic acetylcholine receptor gene superfamily that is expressed in very restricted locations. The promoter of the human gene has been analyzed in the human neuroblastoma SH-SY5Y, where ␣9 subunit expression was detected, and in C2C12 cells that do not express ␣9. A proximal promoter region (from ؊322 to ؉113) showed maximal transcriptional activity in SH-SY5Y cells, whereas its activity in C1C12 cells was much lower. Two elements unusually located at the 5-noncoding region exhibited opposite roles. A negative element located between ؉15 and ؉48 appears to be cell-specific because it was effective in C2C12 but not in SH-SY5Y cells, where it was counterbalanced by the presence of the promoter region 5 to the initiation site. An activating element located between ؉66 and ؉79 and formed by two adjacent Sox boxes increased the activity of the ␣9 promoter about 4-fold and was even able to activate other promoters. This element interacts with Sox proteins, probably through a cooperative mechanism in which the two Sox boxes are necessary. We propose that the Sox complex provides an initial scaffold that facilitates the recruiting of the transcriptional machinery responsible for ␣9 subunit expression. Neuronal nicotinic acetylcholine receptors (nAChRs)1 are members of a supergene family of ion channels gated by neurotransmitters (1). They are pentameric oligomers composed of related subunits, which are commonly classified as agonistbinding (designated ␣2-␣10) and structural (␤2-␤4) subunits. Unlike some nAChR subunits that have a relatively broad expression, the ␣9 subunit has been found only in very restricted areas such as the pituitary pars tuberalis, the olfactory epithelium, and the cochlea (2, 3). This limited expression could be the consequence of tight mechanisms of transcriptional regulation, which might be of great interest in understanding how the regional and developmental expression of neuronal nAChRs is controlled at the transcriptional level (see Ref. 4 for a review). For this reason, here we have analyzed the human ␣9 promoter, finding that two cis-elements unusually located at the 5Ј-noncoding region of ␣9 transcripts control in opposite ways the basal transcriptional activity of the ␣9 subunit gene. EXPERIMENTAL PROCEDURESIsolation and Analysis of the 5Ј-Flanking Sequence of the ␣9 Subunit-The human ␣9 coding sequence was obtained by PCR from a human pituitary cDNA library (Clontech, Heidelberg, Germany) by using the information contained in the GenBank TM sequence AJ243342. A fragment from the 5Ј-end was used to screen a human genomic library constructed in EMBL-3 SP6/T7 (Clontech) and tested as described previously (5). A bacteriophage clone was purified and characterized. It contained ϳ4,600 bp of 5Ј-flanking region and at least the first exon.5Ј-RACE Analysis of 5Ј mRNA Ends-The 5Ј-end of ␣9 mRNA was mapped by 5Ј-RACE, as primer extension and RNase protection methods did not yield satisfactory results. For this purpose the Marathon Ready cDNA system ...

Acetylcholine-evoked currents of the receptor chimera a7-5HT 3A V201 expressed in Xenopus oocytes are strikingly small when compared to the amount of a-bungarotoxin binding sites detected at the oocyte membrane. Since the chimeric receptor is made of the extracellular N-terminal region of the rat a7 nicotinic acetylcholine receptor and the C-terminal region of the mouse 5-HT 3A receptor, which includes the ion channel, we hypothesized that communication between these two regions was not optimal. Here, we show that mutating to aspartate several adjacent positions in the M2-M3 extracellular linker increases current amplitudes to different extents, thus confirming the important role of this region on receptor gating.

Recently, we have shown that the a-helix present at the Ntermini of a7 nicotinic acetylcholine receptors plays a crucial role in their biogenesis. Structural data suggest that this helix interacts with the loop linking b-strands b2 and b3 (loop 3). We studied the role of this loop as well as its interaction with the helix in membrane receptor expression. Residues from Asp62 to Val75 in loop 3 were mutated. Mutations of conserved amino acids, such as Asp62, Leu65 and Trp67 abolished membrane receptor expression in Xenopus oocytes. Others mutations, at residues Asn68, Ala69, Ser70, Tyr72, Gly74, and Val 75 were less harmful although still produced significant expression decreases. Steady state levels of wild-type and mutant a7 receptors (L65A, W67A, and Y72A) were similar but the formation of pentameric receptors was impaired in the latter (W67A). Mutation of critical residues in subunits of heteromeric nicotinic acetylcholine receptors (a3b4) also abolished their membrane expression. Complementarity between the helix and loop 3 was evidenced by studying the expression of chimeric a7 receptors in which these domains were substituted by homologous sequences from other subunits. We conclude that loop 3 and its docking to the a-helix is an important requirement for receptor assembly.