Conservation within the RIC-3 Gene Family

Abstract

In Caenorhabditis elegans, the ric-3 gene is required for the maturation of multiple nicotinic acetylcholine receptors (nAChRs), whereas other neurotransmittergated channels expressed within the same cells are unaffected by the presence of RIC-3. Here we show that RIC-3 is a member of a conserved gene family with representatives in both vertebrates and invertebrates. All members of this family have two transmembrane domains followed by a coiled-coil domain. Expression of the human ric-3 homolog, hric3, like the C. elegans ric-3, enhances C. elegans DEG-3/DES-2, rat α7, and human α7 nAChR-dependent whole-cell current amplitudes in Xenopus leavis oocytes, thus demonstrating functional conservation. However, hric3 also reduces human α4β2 and α3β4 nAChR-dependent whole-cell current amplitudes. Thus, hric3 shows differential effects on human nAChRs unlike the observed uniform effect of ric-3 on C. elegans nAChRs. Moreover, hric3 totally abolished currents evoked by 5-HT3 serotonin receptors, whereas it barely modified α1 glycine receptor currents. With this caveat, RIC-3 belongs to a conserved family of genes likely to regulate nAChR-mediated transmission throughout evolution. Analysis of transcripts encoded by the hric3 locus shows that it encodes for multiple transcripts, likely to produce multiple hric3 isoforms, and that hric3 is expressed in neurons and muscles, thus enabling its interactions with nAChRs in vivo. In Caenorhabditis elegans, the ric-3 gene is required for the maturation of multiple nicotinic acetylcholine receptors (nAChRs), whereas other neurotransmittergated channels expressed within the same cells are unaffected by the presence of RIC-3. Here we show that RIC-3 is a member of a conserved gene family with representatives in both vertebrates and invertebrates. All members of this family have two transmembrane domains followed by a coiled-coil domain. Expression of the human ric-3 homolog, hric3, like the C. elegans ric-3, enhances C. elegans DEG-3/DES-2, rat α7, and human α7 nAChR-dependent whole-cell current amplitudes in Xenopus leavis oocytes, thus demonstrating functional conservation. However, hric3 also reduces human α4β2 and α3β4 nAChR-dependent whole-cell current amplitudes. Thus, hric3 shows differential effects on human nAChRs unlike the observed uniform effect of ric-3 on C. elegans nAChRs. Moreover, hric3 totally abolished currents evoked by 5-HT3 serotonin receptors, whereas it barely modified α1 glycine receptor currents. With this caveat, RIC-3 belongs to a conserved family of genes likely to regulate nAChR-mediated transmission throughout evolution. Analysis of transcripts encoded by the hric3 locus shows that it encodes for multiple transcripts, likely to produce multiple hric3 isoforms, and that hric3 is expressed in neurons and muscles, thus enabling its interactions with nAChRs in vivo. Nicotinic acetylcholine receptors are widely expressed ligand-gated ion channels that mediate fast synaptic excitation and have additional roles including modulation of synaptic release (1Clementi F. Fornasari D. Gotti C. Eur. J. Pharmacol. 2000; 393: 3-10Crossref PubMed Scopus (109) Google Scholar). The nAChRs 1The abbreviations used are: Ach, acetylcholine; nAChR, nicotinic Ach receptor; 5-HT3, serotonin (5-hydroxytryptamine) 3; 5-HT3R, 5-HT3 receptor; EST, expressed sequence tag; Bgt, bungarotoxin; GluR3, glutamate receptor 3; GlyR, glycine receptor; h, human. are homomers or heteromers composed of five subunits. Each subunit traverses the membrane four times and is posttranslationally modified by both disulfide bond formation and glycosylation. Maturation of nAChRs, leading to production of fully assembled and functional receptors on the plasma membrane, is a complex, time-consuming, and poorly characterized process (2Merlie J.P. Lindstrom J. Cell. 1983; 34: 747-757Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 3Green W.N. J. Gen. Physiol. 1999; 113: 163-169Crossref PubMed Scopus (60) Google Scholar, 4Keller S.H. Taylor P. J. Gen. Physiol. 1999; 113: 171-176Crossref PubMed Scopus (36) Google Scholar). Recently, we identified a Caenorhabditis elegans gene, ric-3, likely to be an important player in the maturation of nAChRs. In C. elegans, RIC-3 is required for cholinergic transmission mediated by nAChRs in neurons and in muscles but not for synaptic transmission mediated by other ligand-gated ion channels, even when these are expressed within the same cells as the nAChRs. RIC-3 activity is required within the cells that express the nAChRs and is likely to affect the processes of receptor folding or assembly within the endoplasmic reticulum (5Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (185) Google Scholar). RIC-3 is a protein with two transmembrane domains followed by coiled-coil domains. When first identified, this protein showed no similarity to any characterized protein. Only one homolog was identified, the Drosophila CG9349 gene that is similar in both sequence and overall predicted structure. However, the demonstrated ability of RIC-3 to enhance whole-cell current amplitudes resulting from expression of both the C. elegans DEG-3/DES-2 and the rat α7 receptors in Xenopus oocytes suggested functional conservation of ric-3 in evolution (5Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (185) Google Scholar). Here we show that ric-3 belongs to a conserved gene family with both vertebrate and invertebrate representatives. We proceeded to characterize the human ric-3 homolog (hric3) and showed that, like ric-3, it is capable of enhancing nAChR-dependent whole-cell current amplitudes in oocytes. Moreover, this effect corresponds with enhanced surface expression of the α7 receptor. Therefore, the ric-3 family is a conserved family of genes regulating nAChR maturation. However, hric3 had diverse effects on co-expressed nAChRs and even on another ligand-gated channel from the same ligand-gated ion channel superfamily. Specifically, although it enhances whole-cell current amplitudes produced by the C. elegans DEG-3/DES-2 nAChR and rat and human α7 nAChRs, it inhibits the whole-cell current amplitudes produced by two other human nAChRs, α4β2 and α3β4, as well as a mouse 5-HT3 serotonin receptor. Thus, hric3 shows a high diversity in its effects on co-expressed receptors. In addition, the hric3 locus encodes multiple transcripts coding for different protein isoforms. These transcripts are expressed widely in both excitable and non-excitable tissues. Such isoforms, if functional, may interact with hRIC3 to modulate its activity. Sequence Analysis—ric-3 homologs were identified using the protein sequence of ric-3 as a probe in a standard tblastn search at www.ncbi.nlm.nih.gov/BLAST (6Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar). Sequences for ric-3 homologs were obtained from: Danio rerio sequence CA475611, Ostertagia ostertagi sequence BG734142, Drosophila melanogatser CG9349 encoded by FBg0034574, a reconstruction of two Xenopus leavis sequences, AW645559 and AW634327, the human clone AL520122 (clone FL1001, Invitrogen), which was sequenced in the Life Science Sequencing Facilities (Hebrew University) using internal primers (sequencing was done in both orientations (accession number AY326435)), and the mouse expressed sequence tags (EST) sequence BB642650 (the mouse sequence is a reconstruction of this EST sequence and the corresponding genomic sequence, a reconstruction made possible due to the strong conservation of hric3 and mouse ric3 nucleotide sequences). Sequence alignment was generated using Clustal_W analysis (McVector). Structure prediction analysis was done using programs on www.expasy.ch and psort.nib-b.ac.jp. Analysis of alternative hric3 transcripts and the resulting protein isoforms is based on sequences available in the databases, except clone BF680796 (IMAGE:4296897), which was sequenced in the Life Science Sequencing Facilities (Hebrew University) using internal primers (accession number AY326436). This cDNA is likely to be incomplete as it does not have a poly(A) sequence at the 3′-end. When a single base mismatch was found between different available versions of the same sequence, we chose the version that maintained continuity of the open reading frame. Heterologous Expression and Electrophysiology—A 1.2-kb XhoI-EcoRV fragment containing hric3 from AL520122 was cloned into pGEMH19, which contains Xenopus globin untranslated sequences. DEG-3, DES-2 (7Yassin L. Gillo B. Kahan T. Halevi S. Eshel M. Treinin M. Mol. Cell. Neurosci. 2001; 17: 589-599Crossref PubMed Scopus (64) Google Scholar), and GluR3(L507Y) (8Stern-Bach Y. Russo S. Neuman M. Rosenmund C. Neuron. 1998; 21: 907-918Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) clones were described previously (5Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (185) Google Scholar). DNAs of human neuronal nAChR subunits (α7, α3, α4, β2, and β4) (9Nelson M. Lindstrom J. J. Physiol. (Lond.). 1999; 516: 657-678Crossref Scopus (84) Google Scholar), human GlyR α1 (10Grenningloh G. Schmieden V. Schofield P.R. Seeburg P.H. Siddique T. Mohandas T.K. Becker C.M. Betz H. EMBO J. 1990; 9: 771-776Crossref PubMed Scopus (227) Google Scholar) subunit, and mouse 5-HT3R (11Uetz P. Abdelatty F. Villarroel A. Rappold G. Weiss B. Koenen M. FEBS Lett. 1994; 339: 302-306Crossref PubMed Scopus (37) Google Scholar) were inserted into the pSP64T vector (12Krieg P.A. Melton D.A. Nucleic Acids Res. 1984; 12: 7057-7070Crossref PubMed Scopus (1081) Google Scholar). In vitro transcribed and capped cRNAs were injected at final concentrations of 0.1–0.05 ng for DEG-3 and DES-2, 0.2 ng for GluR3(L507Y), and 5 ng for the mammalian nAChR subunits, except α7, GlyR α1, and 5-HT3R, which were used at 10 ng. Subunits of heteromeric receptors (DEG-3/DES-2, α4β2 and α3β4) were injected in an equimolar ratio. Optimal effects of hric3 on DEG-3/DES-2 expression were found at 1:5 hric3 to DEG-3/DES-2 concentrations, but for mammalian nAChR experiments, equal concentrations (5 ng in all cases) were injected. The final concentrations of subunits cRNAs were kept constant between injections with or without hric3 for each experiment. Injections and recordings were done as described in Treinin et al. (13Treinin M. Gillo B. Liebman L. Chalfie M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15492-15495Crossref PubMed Scopus (80) Google Scholar). The effects of hric3 co-expression on receptor-mediated whole-cell current amplitudes were assayed between the first and third day following injections. Errors are standard error of means, and significance was examined using the paired t test or the Mann-Whitney test. Specific surface expression of [125I]α-Bgt binding sites was tested with 5 nm [125I]α-Bgt as described (14García-Guzmán M. Sala F. Sala S. Campos-Caro A. Criado M. Biochemistry. 1994; 33: 15198-15203Crossref PubMed Scopus (54) Google Scholar). Briefly, oocytes were incubated with 5 nm [125I]α-Bgt for 1 h at room temperature. At the end of the incubation, unbound [125I]α-Bgt was removed, oocytes were washed, and bound radioactivity was counted. Non-specific binding was determined using non-inoculated oocytes. Northern and in Situ Analysis—Expression of hric3 was examined on human multitissue Northern blot number 7760-1 (Clontech), using random primed labeled cDNA probes for the entire hric3 open reading frame or the first two exons. For in situ analysis, we used 35S-labeled in vitro transcribed cRNA from a SKII– clone containing a 0.6-kb mouse ric3 fragment. This EcoRV-BamHI fragment is derived from clone BU705427 (IMAGE:6408852). Both the sense (T7) and the antisense (T3) cRNAs were used as probes on wax sections of mouse brain (15Wilkinson D.G. Bailes J.A. Champion J.E. McMahon A.P. Development. 1987; 99: 493-500PubMed Google Scholar). This analysis showed that the antisense probe produced a stronger and less uniform signal. A Conserved Family of ric-3-like Proteins—Blast searches of the available databases of ESTs have identified ric-3 homologs in a number of species, including the invertebrates O. ostertagi (a parasitic nematode) and D. melanogaster, and the vertebrates X. leavis, D. rerio (Zebrafish), Mus musculus, and Homo sapiens (Fig. 1). All sequences show high homology in the two membrane-spanning domains and in the first coiled-coil domain (Fig. 1). In addition, all members of the family have a proline-rich region (>10% prolines) in between the two transmembrane domains. For all homologs, structure prediction analysis suggest that both the N terminus and the terminus coiled-coil domain are Thus, all to the suggested structure and of the C. elegans ric-3, which the formation of a protein in the (5Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (185) Google Scholar). the structure is conserved within the RIC-3 Only the C. elegans ric-3 a domain and a coiled-coil the sequences of O. D. and X. leavis are we the that also for a coiled-coil domain for O. a are also found in the Drosophila ric-3, which sequences between the two transmembrane domains and between the transmembrane domain and the coiled-coil domain (Fig. A of characterize this gene we on the H. sapiens ric-3 homolog For this we obtained a sequence of one of the available a clone obtained from a This cDNA is coding for a open reading frame and a The corresponding genomic sequence on and is encoded by exons. A similar sequence of are with a similar genomic structure is found in M. on The sequence of hric3 to the structure prediction for the ric-3 family two transmembrane domains followed by a coiled-coil domain (Fig. Specifically, overall with the C. elegans ric-3 is and overall structure by a domain and one coiled-coil domain of the two found in ric-3 (5Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (185) Google Scholar). of ric3 shows similarity to C. elegans ric-3 in both sequence and predicted structure. However, sequence conservation does not functional conservation. for functional we examined the effects of co-expression with hric3 on the whole-cell current amplitudes produced by ion channels expressed in Xenopus oocytes. of concentrations of the C. elegans DEG-3 and DES-2 cRNAs to whole-cell currents oocytes, N with hric3 enhances these currents N of hric3 with does not the of whole-cell does expression of hric3 to or currents (Fig. Thus, like the C. elegans to enhance nAChR-dependent whole-cell current amplitudes. We have previously that C. elegans RIC-3 enhances whole-cell current amplitudes by the rat α7 neuronal was found in a expression of hric3 in neurons for of ric3 with α7 is Thus, hric3 is likely to be expressed in the same cells as α7, enabling interactions between the two in vivo. co-expression of hric3 with rat and human α7 to a in acetylcholine whole-cell current amplitudes (Fig. Moreover, of α7 nAChR expression on as by [125I]α-Bgt binding an of in binding sites in the presence of hric3 (Fig. its in in vivo. The in current is not with in current (Fig. additional of nAChR that also be for hRIC3 activity. Thus, it was to the effects of hric3 expression on two expressed neuronal nAChRs. of hric3 with both α4β2 and α3β4 produced of to and as with without hric3 (Fig. observed with α3β4 nAChRs were not by the presence of hric3 (Fig. and were also similar that was not due to a for These suggest of hRIC3 with these two heteromeric nAChRs. Such was not in C. elegans, all four nAChRs examined required RIC-3 for activity (5Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (185) Google Scholar). thus that from C. elegans in a for hRIC3 in cholinergic In these to the effect of hRIC3 on other ligand-gated receptors. Thus, co-expression of with 5-HT3 receptors produced of currents by serotonin (Fig. and another the α1 GlyR, showed no in whole-cell currents (Fig. and Expression of the hric3 expression of hric3 in we used a available multitissue Northern blot (Fig. a cDNA coding for the hric3 homolog, is in A probe from the first two of hric3 a in excitable and However, this same probe also additional transcripts that are expressed in non-excitable tissues. the of these transcripts, we EST and sequences available in the the human identified multiple sequences encoded by this Sequence protein is to the sequence of except for the of to the of a single at this a that may be to and alternative (Fig. M. E. 2002; PubMed Google Scholar). The of the for this alternative is the same as Thus, it is not which of these transcripts to the in and is for a coiled-coil domain (Fig. The expression of this is with the entire hric3 open reading frame not or the first two and the in the the same Thus, this may be or may with another is for the first membrane-spanning domain to the terminus (Fig. A of the same as is expressed in the The corresponding to ESTs and and is a of into the thus a protein coding for the two domains of this is not the available cDNA to be incomplete (Fig. isoforms encoded by the hric3 isoforms encoded by clones and both in with one the in or the presence in of a single that is an encoded by clone in coding for the coiled-coil domain an encoded by clone in coding for the first transmembrane domain to the N an encoded by clone coding for a protein. for this are the of the for this is In all transmembrane coiled-coil the of and sequences that are not found in The expression of the hric3 locus is not as have that mammalian for multiple transcripts and have suggested that of these transcripts are M. B. 2002; PubMed Scopus (36) Google Scholar). However, it is to on the roles of these isoforms, if functional, differential expression produce For the coiled-coil domain may with the coiled-coil for its activity. of ric3 in expression of hric3 transcripts is in and hric3 diverse effects on ligand-gated ion channel Thus, analysis of the of ric3 is important to its in For this we used a mouse the membrane domain and the coiled-coil for in situ analysis on mouse this we a from of the brain and a stronger from the region in the (Fig. A and the and the in the (Fig. and and the These with of high α7 expression E. X. Lindstrom J. Criado M. J. 1994; PubMed Scopus Google Scholar), enabling in between the two However, it is to that not all express high of as for the (Fig. A and we first identified ric-3 as coding for a protein with no similarity to any other characterized in the databases (5Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (185) Google Scholar), we show that ric-3 belongs to an conserved gene This conservation is also functional the human homolog hric3, like the C. elegans ric-3, enhances whole-cell current amplitudes for In C. elegans ric-3 is required for cholinergic transmission mediated by the neuronal DEG-3/DES-2, the the and the nAChRs. These receptors in subunit and Moreover, RIC-3 is capable of enhancing current amplitudes by the rat α7 nAChR in Xenopus oocytes. This of between different nAChRs that effects on to different receptor and likely to be conserved in evolution (5Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (185) Google Scholar). the human of ric-3, hric3, enhances the whole-cell current amplitudes by both the C. elegans DEG-3/DES-2 receptor and the human and rat α7 receptors. However, co-expression with the human α4β2 and α3β4 receptors to in whole-cell current that the between ric-3 homologs and members of the nAChR family was modified evolution. effects are also observed when the C. elegans ric-3 is used of hric3 not which may that within the mammalian nAChR family are for the different of the α3β4 and the α4β2 nAChR the of hric3 to to other members of the same ligand-gated receptor gene In this are also observed expression of the 5-HT3 receptors is totally by hric3, whereas no in expression of the α1 glycine receptor is These differential effects be in is that formation of a with hric3 by a conserved domain within all the receptor to of maturation if it is not followed by additional interactions of the receptor subunits with These additional produced with α7 nAChRs but not with α3β4 or α4β2 nAChRs, as well as with 5-HT3 receptors, are likely to be mediated by domains within the subunits. that formation of in to a conserved of hric3 with receptor subunits, on that interact with mammalian members of this gene superfamily. Both suggest that of receptor subunits with hric3 to a of maturation and a for regulating ligand-gated receptor Here it is important to that glutamate receptors, which are members of a different ligand-gated receptor and α1 GlyR, a of the nAChRs, are not by These suggest of the effects of hric3 on nAChRs and receptors, a that is similar to is in C. The RIC-3 family is a conserved gene family nAChRs maturation. The of these in nAChR maturation is In C. elegans, we showed that RIC-3 is found the on endoplasmic reticulum and not at We also showed that in its of the DEG-3 protein to the processes is (5Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (185) Google Scholar), a with the effects that we show on α7 surface These are with a for RIC-3 in folding or processes in the endoplasmic or in from the endoplasmic We show that ric-3 belongs to a conserved gene All members of this family are no homology to likely to nAChRs. within this family is to two transmembrane by a proline-rich and followed by a coiled-coil domain. Structure suggest that both the N terminus and the terminus coiled-coil are on this analysis, we suggest a for RIC-3 as receptors and for maturation. We also suggest that multiple domains within the RIC-3 homologs mediate interactions with of a domains have in interactions S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus (60) Google Scholar). The within the between the two transmembrane of which at conserved may a on which to The membrane-spanning domains may also mediate Here it is to that the transmembrane domain in RIC-3 homologs shows conservation is for the For within this domain are found a and a glycine that show conservation. the membrane-spanning domains of ligand-gated receptors are well conserved and may the conserved domain for RIC-3 Thus, interactions within the membrane may receptor subunits and the RIC-3 homologs, whereas other RIC-3 domains may be for additional interactions (the suggested and for interactions with thus a maturation Analysis of transcripts encoded by the hric3 locus shows multiple transcripts resulting from alternative and alternative These transcripts are expressed in including non-excitable At this the of this is are these additional transcripts produce functional that affect the that nAChRs. these transcripts produce functional is to nAChR maturation. these isoforms are as suggested by et al. M. B. 2002; PubMed Scopus (36) Google Scholar). is important to that expression of these transcripts in non-excitable and in is not for roles other of nAChRs, as expression and of nAChRs was in including non-excitable G. S. J. 2002; PubMed Scopus Google Scholar). In C. elegans, ric-3 is a player in cholinergic transmission mediated by nAChRs. In its cholinergic transmission in both muscles and neurons is Here we show that in the of hric3 is likely to be and However, the expression of the hric3 as well as the of on functional expression of hric3 and its isoforms, as to the of hric3 in nAChR-mediated transmission in With these in a likely of is that hric3 expression of α7 receptors, which are to on for expression W. J. J. S. Mol. Res. 2000; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). mouse brain high of ric3 transcripts are to express α7, thus enabling an between the two E. X. Lindstrom J. Criado M. J. 1994; PubMed Scopus Google Scholar). is also possible to that the differential interactions of hric3 with human ligand-gated ion channels are in to regulate expression of these receptors. The of L. M. and J. in the mammalian nAChRs experiments, of H. in the C. elegans nAChR experiments, of A. and in the in situ analysis, and of E. in is We J. M. and H. Betz for the human nAChRs, mouse 5-HT3 and human α1 GlyR