NID67, A Small Putative Membrane Protein,Is Preferentially Induced by NGF in PC12Pheochromocytoma Cells

Linda Vician,1–3 Abigail L. Silver,2 Robin Farias-Eisner,2,4 andHarvey R. Herschman1–3*1Department of Biological Chemistry, UCLA Center for the Health Sciences, Los Angeles, California2Molecular Biology Institute, UCLA Center for the Health Sciences, Los Angeles, California3Department of Molecular and Medical Pharmacology, UCLA Center for the Health Sciences, Los Angeles,California4Department of Obstetrics and Gynecology, UCLA Center for the Health Sciences, Los Angeles, California

In an effort to identify genes involved in neuronal differ-entiation, we have used representational difference anal-ysis (RDA) to clone cDNAs that are preferentially inducedby nerve growth factor (NGF) vs. epidermal growth factor(EGF) in PC12 pheochromocytoma cells. We now reportthe cloning of a previously unknown primary responsegene, NID67. In addition to a robust induction by NGFand FGF, both of which cause PC12 cells to differentiate,NID67 is strongly induced by forskolin, A23187 and ATP.EGF, TPA and KCl induce NID67 only weakly. NID67mRNA is most abundant in heart, ovary and adrenal.Modest levels are present in most brain regions, testis,thyroid, thymus, pituitary, kidney and intestine; littleNID67 is present in skeletal muscle and cerebellum. TheNID67 cDNA contains a 180 bp open reading frame(ORF) that encodes a 60 amino acid protein. The central29 amino acids are very hydrophobic and very likelycomprise a transmembrane domain. Mouse and humanNID67 cDNAs contain an ORF similar to NID67; the ratand human protein sequences are 85% identicalwhereas the rat and mouse sequences are 92% identical.In vitro transcription and translation reactions confirmedthat the ORF we identified produces a 6000 Da proteinproduct. Several small membrane proteins are similar toNID67; they contain a transmembrane domain and littlemore. All of these proteins participate in forming or reg-ulating ion channels. NID67 may play a similar role incellular physiology. J. Neurosci. Res. 64:108–120, 2001.© 2001 Wiley-Liss, Inc.

Key words: small membrane protein; neurotrophin; neuro-nal differentiation; primary response genes; immediate-early genes

During the development of an embryo, cells mustdetermine when to proliferate and when to differentiate.We use the PC12 cell model system to study the responseof neuronal precursors to a differentiation signal, NGF,relative to a mitogenic signal, EGF. Initially, NGF and

EGF elicit the induction of a strongly overlapping set ofprimary response genes in PC12 cells (Greenberg et al.,1985; Kujubu et al., 1987; Bartel et al., 1989). But within24 hr, serum-starved PC12 cells treated with NGF beginto differentiate (Rudkin et al., 1989), whereas thosetreated with EGF are stimulated to multiply (Huff andGuroff, 1979; Huff et al., 1981).

Many approaches have been applied to the search forthe genes responsible for the difference in PC12 cellresponse to NGF vs. EGF. Approaches ranged from cellgenetics (Green et al., 1986) to 2-dimensional gel electro-phoresis (Hondermarck et al., 1994). We reasoned thatthere might be important but undetected differences in thegenes induced by NGF and EGF. We have applied thepowerful subtractive procedure called cDNA representa-tional difference analysis (cDNA-RDA) to this problem.cDNA-RDA combines subtractive hybridization, kineticenrichment and the polymerase chain reaction (PCR) toselect and specifically amplify target sequences that areenriched in one population of cDNA relative to another(Lisitsyn et al., 1993; Hubank and Schatz, 1994; Braun etal., 1995).

Using cDNA-RDA, we previously cloned fourcDNAs preferentially induced by NGF relative to EGF(Vician et al., 1997). One of the advantages of RDA isthat, if certain sequences are already known to be prefer-entially induced, the researcher can avoid reselecting themby adding them to the driver cDNA. We refer to thisprocess as “doping the driver.” Using this technique, weshowed that the receptor for urokinase plasminogen acti-vator, UPAR, is preferentially induced by NGF vs. EGFin PC12 cells (Farias-Eisner et al., 2000). In the currentstudy, in addition to doping the driver, we used different

Contract grant sponsor: NIH; Contract grant number: NS-28660.

*Correspondence to: Harvey R. Herschman, Paul D. Boyer Hall, 611Charles E. Young Drive East, Los Angeles, CA 90095-1570.E-mail: hherschman@mednet.ucla.edu

Received 17 November 2000; Accepted 21 December 2000

Journal of Neuroscience Research 64:108–120 (2001)

© 2001 Wiley-Liss, Inc.

tester/driver ratios to determine whether changing theconditions of the RDA procedure will select for differentsequences. Using these modifications, we cloned thecDNA for a previously unknown small protein that we callNID67 (NGF-induced differentiation clone 67).

MATERIALS AND METHODS

Cells and Culture Conditions

Culture conditions, treatment of PC12 cells and RNApurification were carried out as previously reported (Vician etal., 1997). RNA from low density, serum-starved PC12 cellstreated for 45 min, 1 hr, 1.5 hr, 2 hr, 2.5 hr, 3 hr or 4 hr withNGF or EGF was purified and polyA1 selected twice usingPolyAttract (Promega, Madison, WI).

cDNA Preparation and RDA

Double stranded cDNA was synthesized and the driverwas doped with purified cDNA for the NGF-specific genes wehad previously cloned (Vician et al., 1997; Farias-Eisner et al.,2000). The cDNAs added were VGF and ARC (10 ng each),collagenase and plasminogen activator inhibitor (2.5 ng each)and transin (1 ng) per 1 mg of driver cDNA (EGF cDNA). Tomaintain an internal control, we did not add MKP3 cDNA tothe driver. The RDA reactions were carried out as previouslyreported (Vician et al., 1997), except for the changes in thedriver/tester ratios described in the Results section. Because wehad doped the driver for most of the easily selectable species, itwas not possible to predict how many rounds of PCR might berequired to amplify the products after each round of hybridiza-tion. We thus had to determine empirically the proper numberof rounds of PCR for each reaction. The PCR reaction thatprecedes the Mung Bean nuclease treatment was always run for10 rounds. After terminating the nuclease treatment, we set upseveral replicate PCR reactions for each sample. One reactionfor each sample was cycled whereas the others were stored onice. We periodically paused the PCR machine during a 72°extension step and determined the DNA concentration of eachreaction using the Hoechst dye assay (Labarca and Paigen, 1980)and a Hoefer Mini-Fluorometer equipped with a micro-cuvette(San Francisco, CA). This procedure was repeated until theamount of DNA in each reaction reached a maximum. TheHoechst dye reading would then drop to about half the maxi-mum, because single-stranded DNA yields half as much fluo-rescence as the same amount of double-stranded DNA. Byplotting these DNA readings, we could determine the numberof cycles possible before the generation of DNA began to leveloff. We then cycled the replicate reactions for the indicatednumber of cycles without interruption. The optimal number ofPCR cycles for the low ratio sample was 6–7 less than for thehigh ratio sample.

Cloning and Analysis of Final RDA Products

After the fifth round of RDA selection, the products weredirectly ligated into the pCR cloning vector (Invitrogen, Carls-bad, CA). Clones were screened as previously reported (Farias-Eisner et al., 2000).

Northern Blot Analysis

RNA was prepared from treated PC12 cells using theLiCl procedure as previously described (Vician et al., 1997).RNA from rat tissues was prepared using the method ofChomczynski and Sacchi (1987). RNA was subjected tonorthern analysis as previously described (Vician et al., 1995).Radioactive signals on the northern blots were quantifiedusing a Molecular Dynamics PhosphorImager 445 SI andImageQuant software (Sunnyvale, CA).

Isolation of Full-Length Clones and DNA Sequencing

A full-length cDNA library was prepared from thepolyA1 RNA isolated from PC12 cells treated with NGF forperiods between 45 min to 4 hr. The mRNA was converted tocDNA and adapters (containing EcoR I, Sal1 and Not I cutsites) were added using the Superscript II cDNA synthesis kitfrom Gibco BRL (Grand Island, NY) according to the manu-facturer’s directions. A non-directional library was prepared byligating the products into Lambda Zap II cut with EcoR I andin vitro packaged using a Gigapack Gold Kit (Stratagene, LaJolla, CA). The library was amplified, stored and screened bystandard methods (Sambrook et al., 1989). A nearly full-lengthplasmid clone of the human NID67 (I.M.A.G.E. Consortiumclone I.D. 510817) was purchased from Genome Systems Inc.(St. Louis, MO). A nearly full-length mouse NID67 clone wasisolated from a directional adult mouse brain cDNA library inLambda Zap II (provided by Dr. Cary Lai, The Scripps Re-search Institute, La Jolla, CA). The Lambda Zap clones wereconverted to plasmid form by in vivo excision using ExAssisthelper phage (Stratagene, La Jolla, CA). Plasmid DNA wasprepared using Qiagen plasmid preparation kits (Valencia, CA).The plasmids were sequenced using the Dye Terminator DNAsequencing kit from PE Applied Biosystems (WarringtonWA14SR, Great Britain) by primer walking. Sequencing prim-ers were designed using the Oligo 4.1 software (MolecularBiology Insights, Cascade, CO). DNA oligomers were pur-chased from Integrated DNA Technologies Inc., Coralville, IA).

In Vitro Synthesis of the NID67 Protein

The T7 TnT transcription/translation kit and dog pan-creatic, microsomal membranes were purchased from PromegaBiotech (Madison, WI). Translation grade L-[35S]-methionine(1175 Ci/mmol) was purchased from New England Nuclear(Boston, MA). Transcription/translation reactions were carriedout according to the manufacturer’s instructions.

The rat NID67 ORF, along with it’s 59 untranslatedregion and part of it’s 39 untranslated region was removed frompBluescript SK(2) using Sal1 (that cuts within the adapters usedto make the library) and Pml1 (that cuts at base 570 of theNID67 sequence before the first of the rapid degradation sites).This fragment was cloned into the Xho I and EcoR V sites ofpcDNA3.1(2) (Invitrogen, Carlsbad, CA) by standard methods.

The FLAG antigen site (Ford et al., 1991) was insertedinto the rat cDNA sequence by cutting the plasmid containingthe cDNA with either Bsm1 or Xcm1 (New England Biolabs,Beverly, MA). Four oligonucleotides encoding the FLAG se-quence, in both directions, plus the bases necessary to base pairwith the cut site overhangs and to maintain the same readingframe were purchased from Integrated DNA Technologies, Inc.

NGF Induces NID67 109

(Coralville, IA). The DNA oligomers were allowed to hybridizeto their reverse-complements and were ligated into the cutplasmid DNAs. Clones containing the FLAG inserts were se-lected using PCR analysis to detect the correct placement andorientation of the FLAG inserts. The sequence of the final cloneswas verified by sequencing.

The 35S-labeled products synthesized in vitro were ana-lyzed using the tricine-sodium dodecyl sulfate-polyacrylamidegel electrophoresis system of Schagger and von Jagow (1987).The gels consisted of two layers both with 3% cross-linker. Thestacking gel was 4% acrylamide and the separating gel was 16.5%acrylamide. After electrophoresis, the gels were fixed in 10%acetic acid, 40% isopropanol then impregnated with Amplify(Amersham Pharmacia Biotech, Arlington Heights, IL) anddried at 80°C under vacuum. The gel was then exposed to filmat 280°C.

RESULTSEffects of Varying the Driver/Tester Ratio on theOutcome of the RDA Procedure

Two RNA pools were used in this experiment.They consisted of polyA1 RNA from low density, serum-starved PC12 cells treated with either NGF (pool 1) orEGF (pool 2). RNAs from cultures treated with eachligand for various time intervals from 45 min to 4 hr werepooled, polyA1 selected twice and used to preparedouble-stranded cDNAs for the RDA procedure. SeveralcDNAs (VGF, ARC, collagenase, PAI-1 and transin), thathad been previously shown to be preferentially induced byNGF (Vician et al., 1997), were added to the driver (pool2), to prevent their selection during the subtraction. Wedid not add MKP3 cDNA to the driver, so that we wouldhave a preferentially NGF-induced cDNA to use as aninternal control.

Preliminary experiments suggested that changing theratio of driver to tester would change the final products ofthe RDA. To test this idea, we subtracted one sample atthe usual ratios for RDA (Vician et al., 1997) and onesample starting at a ten-fold lower ratio. At each newround of subtraction, the driver/tester ratios were in-creased in the two samples by the same factor. The ex-ception to this rule was that, in the fifth subtraction, theratio for the high ratio sample was not increased beyond5 3 106. The driver/tester ratios used to subtract the twoNGF minus EGF RDA samples at each RDA reaction areshown in Table I.

The final RDA samples were analyzed by gelelectrophoresis and Southern blotting. GAPDH, a con-stitutive gene, was effectively removed from both sam-ples by the fourth round of RDA. An amplicon fromUPAR was amplified in the high ratio sample. AnMKP3 amplicon was amplified in the low ratio sample.Virtually no UPAR remained in the final low ratiosample and virtually no MKP3 remained in the finalhigh ratio (Fig. 1). Thus, subtracting the same popula-tion of cDNAs using different ratios leads to the am-plification of different products.

A Previously Unknown cDNA That isPreferentially Induced by NGF vs. EGF in PC12Cells

After the fifth round of RDA selection, we clonedthe products by directly ligating them into the pCR IIcloning vector. Eighty random clones from the high ratiosample and 80 from the low ratio sample were selected.Redundant clones were eliminated by cross hybridization.The forty remaining clones were digested with EcoR1and analyzed on duplicate southern blots, using as probes32P-labeled cDNA synthesized from the starting mRNAsamples. Based on this southern analysis, twelve cloneswere chosen for further study. Probes to these inserts wereprepared and hybridized to northern blots containingpolyA1 RNA from control, NGF- and EGF-treatedPC12 cells. One of the clones from the low ratio samplecontained a Dpn II fragment that did not match anyknown sequence in the database. This clone has beendesignated NID67 for NGF Induced DifferentiationcDNA 67. NID67 mRNA is induced 2.4-fold in theNGF-induced pool but only 1.1-fold in the EGF-inducedpool (data not shown). We identified a full-length clone ofrat NID67 by screening a cDNA library prepared from theNGF-treated PC12 cell mRNA.

Time Course of NID67 Induction by NGFTo see how rapidly, to what extent, and for how

long NID67 remains induced, we prepared total RNAfrom PC12 cells stimulated with NGF or EGF for 0.5 hrto 8 hr. Northern blots were prepared from these RNAsamples and probed with the rat NID67 cDNA clone. Wealso probed this Northern blot with EGR1, to show thatthe NGF and EGF treatments were effective (Kujubu etal., 1987) and with VGF to show that the PC12 cells wereresponding in an NGF-specific manner (Salton et al.,1991). The signals were quantitated by phosphorimagingand normalized to GAPDH mRNA. NID67 message isstrongly induced by NGF, reaching 9.8 times the controllevel 4 hr after the addition of NGF (Fig. 2). EGF induc-tion of NID67 was only 2.6 times the control level. Weconclude that NID67 is preferentially induced by NGF inPC12 cells.

NID67 Is a Primary Response GeneTranscriptional responses for primary response genes

can be induced in response to stimulation by using pre-existing second messenger systems and transcription factors;

TABLE I. Hybridization Ratios Used in the cDNA-RDAProcedure

H1 H2 H3 H4 H5

Low ratio 10 100 5 3 103 5 3 104 5 3 106

High ratio 100 1000 5 3 104 5 3 105 5 3 106

The hybridization ratios used for each round of cDNA-RDA are shown interms of the mass excess of driver to tester (i.e., mg driver/mg tester) for the“low ratio” and “high ratio” samples. H1 refers to the first hybridization,H2 to the second hybridization, etc.

110 Vician et al.

no new proteins need to be synthesized (Herschman, 1991).To determine whether NID67 is a primary response to NGF,we tested whether NGF could induce NID67 mRNA in theabsence of protein synthesis. We compared untreated PC12cells with cells treated either with cycloheximide alone orwith both cycloheximide and NGF for 1.5 hr (Fig. 3). Forcomparison, we also analyzed VGF mRNA; VGF is a knownNGF primary response gene (Salton et al., 1991). Cyclohex-imide alone caused a small increase in NID67 message levels.The addition of NGF in the presence of cycloheximidecaused a further increase in the message levels, to 4-fold over

the control level; NID67 mRNA induction is a primaryresponse to NGF.

Induction of NID67 by Other LigandsWe next compared the effects other growth factors and

second messenger systems on NID67 mRNA levels. PC12cells were treated with various agents for either 45 min or3 hr and RNA was analyzed by Northern blotting (Fig. 4).NGF and FGF, both of which cause these cells to differen-tiate (Greene and Tischler, 1976; Togari et al., 1985) elicitedstronger induction of NID67 than EGF. TPA, which stim-

Fig. 1. RDA yields different products, depending on the driver/testerratios used. Amplicons (100 ng) from each RDA cycle of the low ratiosample and the high ratio sample were separated on an agarose gel andtransferred to a nylon membrane. The upper panel shows a photo of theethidium bromide stained DNA. Filters were hybridized to the probesas indicated in the figure. The exposure time for each panel is indicatedin its lower right corner. NL

20 is the starting amplicon population

derived from mRNA isolated from NGF-treated PC12 cells by 20rounds of PCR, using the LBgl adapters and primers. EL

20 is a similarpopulation of amplicons derived from EGF-treated PC12 cells. H1indicates the amplicons generated by the first RDA cycle of hybridiza-tion and amplification. H2–H5 similarly indicate the amplicons fromthe second through fifth rounds of RDA.

NGF Induces NID67 111

ulates Ca11-and phospholipid-dependent protein kinases(PKC), induced NID67 mRNA only weakly. Forskolin,which stimulates cAMP-dependent protein kinase (PKA),caused a very strong induction of NID67.

Application of 50 mM KCl depolarizes PC12 cells,which opens voltage-regulated Ca11 channels. In con-trast, the Ca11 ionophore, A23187, forms artificialCa11-selective channels that allow the ion to enter thecytoplasm from the external milieu (Pressman, 1976).

Finally, ATP, a neurotransmitter for PC12 cells, bindspurinergic P2X2 receptors and opens ligand-gatedCa11 channels, allowing Ca11 to enter the cytoplasmand stimulate neurotransmitter release (Michel et al.,1996). Both A23187 and ATP induced NID67 mRNAabout as well as NGF and FGF. In contrast, KCl in-duced little or no NID67 mRNA. EGR-1 mRNAinduction served as a positive control for both theligands and the cells.

Fig. 2. NGF preferentially induces NID67 mRNA in PC12 cells. A: Northern blot analysis ofNID67, VGF, EGR1 and GAPDH mRNA levels in low-density, serum-starved PC12 cells treated forthe indicated times (in hours) with NGF (50 ng/ml) or EGF (25 ng/ml). Each lane contains 8 mg oftotal RNA. B: Quantitative PhosphorImager analysis of the NGF and EGF induction of NID67, VGFand EGR1. NGF (closed diamonds) or EGF (open boxes). Data are normalized to GAPDH mRNAlevels.

112 Vician et al.

Tissue Distribution of NID67To determine whether NID67 expression is re-

stricted to neuronal cells, we hybridized the NID67cDNA to a panel of RNA samples from various rat tissues(Fig. 5). NID67 RNA is present in many tissues. It isparticularly abundant in heart, ovary and adrenal. All thebrain regions tested had moderate levels of NID67mRNA. Although very prevalent in heart muscle, NID67mRNA is absent from skeletal muscle.

Cloning and Sequencing of the NID67 cDNAWe used the initial amplicon cloned from the RDA-

selected population to identify longer clones of NID67 ina library prepared from NGF-induced PC12 cell mRNA.Two independent clones that differed at their 59 ends byonly one base were sequenced (Fig. 6). We found only ashort 180 bp open reading frame (ORF). To confirm thatthis ORF is the correct one, we asked whether it isconserved in other species. We searched the expressedsequence tag (EST) database for matches to the rat se-quence. Approximately 30 human ESTs, five mouse ESTsand one rat EST were found. We found no significant

matches to sequences from C. elegans, Drosophila melano-gaster or any other species. We obtained the longest humanclone available from Genome Systems and used the ratNID67 clone to identify a nearly full-length clone from amouse brain cDNA library. We sequenced these humanand mouse NID67 clones and compared them to the ratsequence (Fig. 7A). The proposed ORF begins at the firstATG and is surrounded by a good Kozak sequence(Kozak, 1991) in all three species. The sequence through-out the ORF is well conserved among all three species,and a TGA stop codon is present in the same position inall three species. The ORF we identified in the rat se-quence was the only ORF in common among the threesequences.

The NID67 cDNA sequences from all three speciescontain 8–10 occurrences of a sequence reported to targetthe mRNA for rapid degradation (underlined in Fig. 6)(Shaw and Kamen, 1986; Reeves et al., 1987). All threecDNA sequences also contain the consensus sequence forpolyA addition. In the rodent species, however, this site isapparently not used because it precedes the polyA by 80bases rather than the usual 10–30 bases (Zhao et al., 1999).In these two species a rare polyA addition signal, 18 basesbefore the polyA, seems to be the site that is used. Thissequence is used in less than 4% of mouse ESTs (Graber etal., 1999).

The amino acid sequences from the rat, mouse andhuman NID67 cDNAs are aligned in Figure 7B. Thisalignment shows the high degree of conservation. Allthree sequences are identical at 47 out of 60 residues(78%). The only substitution, out of the remaining 13

Fig. 3. NID67 is a primary response gene. Low-density serum-starvedPC12 cells were treated with NGF (50 ng/ml) in the presence ofcycloheximide (CHX, 10 mg/ml). The mRNA levels for untreatedcells (open bars), cells treated with CHX only (crosshatched bars) andcells treated with both NGF and CHX (solid bars) are shown. Eight mgof total RNA were subjected to northern analysis, using the probesindicated. The mRNA was quantitated as in Figure 2.

Fig. 4. NID67 is strongly induced in PC12 cells by NGF, FGF,forskolin, A23187 and ATP. PC12 cells were treated with NGF(50 ng/ml), EGF (20 ng/ml), bFGF (20 ng/ml), TPA (50 ng/ml),forskolin (50 mM), KCl (50 mM), A23187 (10 mM) or ATP (200 mM).Controls were treated with medium containing the same concentrationof ethanol used for TPA, forskolin and A23187. Cells were harvestedafter the times indicated. RNA was prepared and 10 mg were subjectedto Northern analysis.

NGF Induces NID67 113

residues, that is not conservative is the serine to valinesubstitution at position 7. Analysis of the predicted peptidesequence of NID67 reveals a stretch of 29 hydrophobicand uncharged residues (underlined in Figs. 6, 7B). A widerange of computer analyses predicts that this region islikely to be a transmembrane domain (Eisenberg et al.,1984; Sonnhammer et al., 1998), leaving only 19 aminoacids on one face of the membrane and 12 amino acids onthe other. The neural net method (Sonnhammer et al.,1998) predicts that the amino terminal of NID67 is exter-nal and the carboxyl terminal is internal. Analysis using theNetPhos 2.0 Protein Phosphorylation Prediction Server(Blom et al., 1999) predicts that the carboxyl terminalcontains a conserved, potential phosphorylation site forboth Ca11/calmodulin-dependent protein kinase (CaMII) and PKA at threonine 52 (Kennelly and Krebs, 1991;Kemp and Pearson, 1990). The NID67 protein is ex-tremely hydrophobic, having a grand average hydropathyvalue (GRAVY, Kyte and Doolittle, 1982) of 11.26 (rat),11.19 (mouse) or 11.41 (human).

In Vitro Synthesis of NID67 ProteinTo confirm that the proposed ORF is correct, we

transcribed and translated the full-length rat NID67cDNA in vitro. In these reactions, mRNA is synthesizedby T7 polymerase from plasmid DNA that contains a T7promoter upstream of the cDNA sequence. The transla-tional apparatus supplied by a reticulocyte lysate thensynthesizes the encoded protein. Reactions programmedwith the empty Bluescript vector produced a weak band ofabout 6300 Da. (Fig. 8A, lane labeled “pBS”). In reactionsprogrammed by Bluescript containing the entire NID67cDNA, the 6300 Da protein disappeared and was replacedby a 6000 Da protein (Fig. 8A, lane labeled “pBS/

NID67”). We next subcloned the 59 end of NID67 intopcDNA3.1. In this DNA construct, the mRNA producedcontains the normal 59 untranslated region (UTL) ofNID67, the NID67 ORF and 232 bp of the NID67 39UTL. The rest of the 39 untranslated region of the NID67sequence, which contains several rapid degradation signals,is replaced by the more stable bovine growth hormone 39UTL region. Somewhat more of the same size protein wasproduced in reactions programmed with this construct(lane labeled “pcDNA3.1/NID67”) but no protein wassynthesized in reactions programmed with this empty vec-tor (lane labeled “pcDNA3.1”). To prove that the appro-priate protein was being produced by the proposed ORF,we made 2 constructs containing the NID67 ORF inpcDNA3.1, but with 24 bp encoding the 8 amino acidFLAG tag, inserted in-frame within the proposed NID67ORF at 2 different positions. Each of these constructsproduced a slightly larger protein (lanes labeled “NID671FLAG1” and “NID671FLAG2”). These data demonstratethat the proposed 180 bp open reading frame is directing thesynthesis of the 6000 Da protein band.

Analysis of the predicted NID67 protein sequencesuggests that it is a membrane protein. Additional com-puter analysis using a program that predicts signal peptides,SignalP V1.1 (Nielson et al., 1997), suggested that NID67might have a signal sequence and that the most likelyposition for cleavage would be between amino acids 44and 45. To test whether NID67 might be cleaved by signalpeptidase or glycosylated, we translated it in the presenceof microsomes. Figure 8B shows that the addition of dogpancreatic membranes did not alter the apparent molecularweight of the NID67 protein (Fig 8B; “NID67”, “2” vs.“1” microsomal membranes). If the NID67 translated

Fig. 5. NID67 mRNA expression in various rat tissues. Total RNA was extracted from adult rattissues and 10 mg of each was subjected to Northern analysis. The filter was probed with the full-lengthNID67 probe and with a probe to the ribosomal protein S2.

114 Vician et al.

product were cut at the predicted position, both pep-tides would contain labeled methionines and should bevisible on the autoradiograph. Control reactionsshowed that these membranes could actively glycosylateyeast a-mating factor, transforming it from an 18.6 kDapeptide to a glycoprotein that runs at approximately 30kDa (Fig. 8B, “a-mating factor”, “2” vs. “1” micro-somal membranes) (Rothblatt and Meyer, 1986). Themicrosomal membranes also effectively removed thesignal peptide from b-lactamase, converting it from a

31.5 kDa precursor to its 28.9 kDa processed form (Fig.8B. “b-lactamase”, “2” vs. “1” microsomal mem-branes) (Sutcliffe, 1978). A decrease in translationalefficiency is frequently seen in these reactions whenmicrosomal membranes are added (Promega BiotechTechnical Manual 231). This may account for the re-duction in the amounts of NID67 and b-lactamase seenin the reactions to which microsomal membranes wereadded. We conclude that NID67 is neither cleaved by asignal peptidase nor glycosylated.

Fig. 6. Sequence of the full-length rat NID67 cDNA and predicted protein sequence of the NID67gene product. The amino acid sequence is indicated in single letter code below the nucleic acidsequence. The amino acids included in the putative transmembrane domain are underlined. Sites inthe 39 untranslated region that direct rapid degradation of the message are also underlined. The polyAaddition signal sequences are in bold type. GenBank accession number AF313411.

NGF Induces NID67 115

DISCUSSION

NGF-Specific Induction of Gene ExpressionMany of the same primary response genes are in-

duced after stimulation of different cell types by variousligands, leading to a wide range of cellular responses(Greenberg et al., 1985; Kujubu et al., 1987; Bartel et al.,1989). The obvious question is, “How does the specificityof the response arise?” To answer this question we havesearched for a cohort of genes that are specifically inducedin PC12 cells by NGF, which induces differentiation, butnot by EGF that stimulates mitosis. We previously usedcDNA-RDA to identify five cDNAs for messages that arepreferentially induced by NGF vs. EGF (Vician et al.,1997; Farias-Eisner et al., 2000). By “doping the driver”and by varying the RDA subtraction ratios, we have nowidentified a previously unknown, small hydrophobic pro-tein that is also preferentially induced by NGF.

Role of Small Membrane Proteins in MammalianCells

What could be the function of such a small, putativemembrane protein? NID67 shows no significant homol-ogy to any known protein in the databases. A number ofsmall membrane proteins have been reported in the liter-ature; we have looked for clues to NID67’s functionamong these proteins. The minK family (Kaczmarek andBlumenthal, 1997), the g subunit of the Na,K-ATPaseand proteins related to it (Minor et al., 1998; Arystarkhovaet al., 1999; Therien et al., 1999) and phospholamban(Simmerman and Jones, 1998), are examples from mam-malian systems. In each of these cases, small membraneproteins have important regulatory effects on ion channelproteins, functioning as regulators controlling large cellu-lar machines. We will describe two of these families.

NID67 is very highly expressed in heart muscle, butvirtually absent from skeletal muscle (Fig. 5). In heartmuscle, the repolarization of the cell after an action po-tential requires the exquisite regulation of two main po-tassium currents, a rapid and a slow current. The genesencoding the proteins that comprise these channels havebeen cloned and functionally characterized. The slowcomponent of the K1 current is believed to be due to achannel composed of the KVLQT1 gene product and theminK protein. KVLQT1 contains 6 transmembrane do-mains and is a member of the Shaker potassium channelfamily. The minK protein contains 126–130 amino acids,depending on the species, and, like NID67, has a singletransmembrane domain. Expressed alone, minK cannotform functional channels. If KVLQT1 is expressed alone inCOS cells, potassium channels that allow a small, rapidpotassium influx after depolarization can be detected. IfminK is co-expressed with KVLQT1, however, potassiumchannels are formed that open much more slowly and stayopen longer, allowing a much larger potassium current topass into the cell. These channels, composed of both theKVLQT1 and minK proteins, exhibit all the characteristicsof the slow potassium channels detected in heart muscle(Kaczmarek and Blumenthal, 1997).

Until recently, the HERG gene product was be-lieved to be responsible for the rapid potassium current incardiac muscle. Important differences, however, betweenthe channels formed by HERG subunits alone and thenative, rapid cardiac channels led Abbott et al., (1999) tosearch for minK-related partners for HERG. They havenow cloned 3 cDNAs (KCNE2–4) with homology tominK (KCNE1). One of these, MirP1 (KCNE2) interactswith HERG in the heart to form a channel with kineticsand pharmacology similar to the rapid, cardiac K1 current(Abbott et al., 1999). Another of these gene products,KCNE3, interacts with KVLQT1 in the intestine to forma potassium channel that is important for intestinal chlo-ride secretion and is regulated by cAMP (Schroeder et al.,2000). Thus the minK family is emerging as a family ofsmall, transmembrane proteins capable of modifying thegating characteristics of the much larger K1-channel pro-teins with which they interact.

Utilizing one ATP, Na,K-ATPase pumps three Na1

ions out of the cell for every two K1 ions pumped in, thusestablishing the electrochemical gradient necessary forfunctions such as excitability, regulation of cell volume,pH and fluid transport. In most tissues, Na,K-ATPase isbelieved to be composed of two subunits, the catalytic asubunit and the b subunit (Blanko and Mercer, 1998). Athird subunit, g, was discovered in 1978 by Forbush et al.(1978) but its role has remained elusive. The g subunit is49–97 amino acids long and has – like NID67 – a singletransmembrane domain (Minor et al., 1998). Recently,Therien et al., (1997) showed that the g subunit is specif-ically expressed in kidney medulla and that, in the pres-ence of the g subunit, the affinity of the Na,K-ATPase forATP is significantly increased (Therien et al., 1999). Arys-tarkhova et al. (1999) further showed that both a and gsubunits are strongly expressed in the distal convolutedtubules and weakly expressed in the proximal convolutedtubules, whereas only the a subunit is expressed in thecortical thick ascending limb. They also found that coex-pression of the g subunit decreases the affinities of theenzyme for both Na1 and K1. These observations areconsistent with the observation that the affinity of theNa,K-ATPase for Na1 is higher in the cortical thickascending limb (Barlet-Bas et al., 1994; Feraille et al.,

‹

Fig. 7. A: Comparison of the nucleic acid sequences of rat, mouse andhuman NID67. The three sequences were aligned using the ClustalWalgorithm (Thompson et al., 1994). Nucleic acid positions are indicatedon the left and right ends. The shaded boxes enclose identical bases; theconsensus sequences are indicated below. The ORF is underlined by adashed line. GenBank accession numbers AF313412 (mouse) andAF313413 (human). B: The amino acid sequence of NID67 is stronglyconserved among rat, mouse and human. Amino acids are given in thesingle letter code. The species is indicated to the right of each line andthe amino acid positions are indicated above the sequences. The aminoacids that vary from the consensus are boxed. The bar graph above theconsensus sequence indicates the percent agreement among the threesequences. The putative transmembrane domain is underlined by athick line.

116 Vician et al.

Figu

re7.

NGF Induces NID67 117

1994, 1995; Buffin-Meyer et al., 1996). The g subunitseems to be a critical modulator of the Na,K-ATPase inkidney, making it possible for the kidney to concentrateNa1 ion against a steep gradient.

Features of NID67NID67 is more strongly induced by NGF than by

EGF and is a primary response gene. The NID67 mRNAis likely to be quite unstable, because it contains manyrapid degradation sites indicated in Figure 6 (Shaw andKamen, 1986; Reeves et al., 1987). It is quite common forthe products of primary response genes to be unstable(Herschman, 1991). They often need to be made rapidly,perform their function and then be rapidly cleared fromthe cell. The predicted instability of the NID67 message isconsistent with it being a primary response gene.

The 1.5 kb message encodes a very small proteinonly 60 amino acids long. Initially we thought it might bea neuropeptide targeted for secretion. Although the pro-tein contains a putative signal sequence, however, it is notcleaved by active microsomes in vitro (Fig. 8B). Theputative signal sequence may direct NID67 to the cellsurface but not be removed. Additional computer analysisindicates that the 19 amino acids at the N-terminal areprobably on the outside of the cell, whereas the 12 aminoacids at the C-terminal are likely on the inside of the cell.The cellular location and topology of NID67 remain to beshown experimentally.

This protein is very hydrophobic; it has a calculatedGRAVY value of 11.2–1.4. The grand average hydrop-athy, or GRAVY value, as defined by Kyte and Doolittle(1982), expresses how hydrophobic a protein is. TheGRAVY value is calculated by adding and subtracting thehydrophobicity values for all the amino acids in the pro-tein. When the GRAVY values for all the proteins in theSWISS-PROT database from E. coli, B. subtilis or S.cerevisiae are calculated, they range from 11.7 (very hy-drophobic) to 22.2 (very hydrophilic) (Wilkins et al.,1998). In all these organisms, however, most of the pro-teins are in the range of 11.0 to 21.5. With a GRAVYvalue in excess of 11.2, NID67 is extremely hydrophobic.It is even more hydrophobic than any of the small mam-malian membrane proteins mentioned above. Of thatgroup, only phospholamban has a GRAVY value in thepositive range (10.835).

The human NID67 sequence is contained in UniGenecluster Hs. 29444. This cluster contains the sequence taggedsite dbSTS 1727 that has been mapped by radiation hybridmapping to position 583.13 (cR3000) in the GeneMap ‘99/Genebridge4 map of human chromosome 5. NID67 is in the152.8–157.6 cM interval, which corresponds to 5q32. Thisinterval contains the CD74 antigen, which is the invariant,gamma polypeptide of the class II major histocompatibilitycomplex. It also contains Nef-associated factor 1 (NAF1) andpossibly annexin VI (p68) (Schuler et al., 1996; Deloukas etal., 1998). The human NID67 gene sequence is contained infour chromosome 5 genomic clones that are currently beingsequenced by the DOE Joint Genome Institute. The se-quences of these clones suggest that the NID67 gene consists

Fig. 8. Transcription and translation of NID67 cDNA produces a 6000Da protein that is not processed by microsomal membrane enzyme sys-tems. A. DNA constructs were added to transcription/translation reactionscontaining 35S-methionine. After incubation, the reaction products wereseparated on a 16.5 % acrylamide-Tris/Tricine-SDS gel and the gel wasprocessed for fluorography. pBS is the pBluescript II SK(2) vector with noinsert. pBS/NID67 is the original full-length rat NID67 cDNA in pBlue-script SK II (2) isolated from the Lambda Zap library. pcDNA3.1 indicatesthe empty pcDNA3.1(2) vector. pcDNA3.1/NID67 is a construct con-taining the 59 end of the rat NID67sequence, including the putative ORF.NID671FLAG1 is the same construct but with the FLAG sequenceinserted “in frame” within the putative ORF at the Xcm I site.NID671FLAG2 is a similar pcDNA3.1 construct but with the FLAGsequence inserted at the Bsm I site. B: Microsomal membranes were addedto transcription/translation reactions as indicated by “1” or “2”. TheNID67 reactions contained the pcDNA3.1/NID67 DNA construct. Thea-mating factor reactions contained RNA encoding the yeast a-matingfactor and the b-lactamase reactions contained RNA encoding E. colib-lactamase. The b-lactamase lanes are from the same film but werescanned at a lower exposure.

118 Vician et al.

of two exons, separated by a 16.5 Kb intron. The intronoccurs in the 59 untranslated region at base 149, just 12 bpbefore the beginning of the ORF (GenBank accession num-bers AC010309.5, AC008643.4, AC011408.5, andAC010441.4).

CONCLUSIONSThe use of cDNA-RDA, along with doping of the

driver and varying the subtraction ratios, made the discov-ery of this previously unknown cDNA possible. Becausethere are no known proteins in the databases with signif-icant homology to NID67, we have tried to find proteinswith a similar structure in the literature. All of the smallmembrane proteins from mammalian systems that we havefound in the literature that resemble NID67 are ion chan-nel subunits involved in the regulation of the channels. Itis well known that NGF increases the number of Na1

channels in PC12 cells during the differentiation process(Amy and Bennett, 1983; Boonstra et al., 1981; Reed andEngland, 1986; Rudy et al., 1987). The role of NID67remains to be identified.

ACKNOWLEDGMENTSWe thank Dr. Cary Lai for supplying the mouse

brain cDNA library, Ray Basconcillo and Arthur Cata-pang for excellent technical assistance and the members ofthe Herschman group for helpful discussions.

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