Structure and characterization of the gene encoding a mouse kappa3-related opioid receptor

Gene, 171 (1996) 255-260 0 1996 Elsevier Science B.V. All rights reserved.

255

0378-l 119/96/$15.00

GENE 09590

Structure and characterization related opioid receptor (KOR-3;

of the gene encoding a mouse kappa,-

gene structure; nucleotide sequence; transcription)

Ying-Xian Pana, Jin Xu” and Gavril W. Pasternaka,b “The Cotzias Laboratory

ofNeuro-Oncology,

Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA: and bDepartments of Neurology and

Neuroscience, and Pharmacology, Cornell University Medical College, New York, NY 10021, USA Received by J. Marmur:

14 August

1995; Accepted:

8 November

1995; Received at publishers:

21 December

1995

SUMMARY

A genomic clone comprising the entire cDNA sequence encoding a mouse kappa,-related opioid receptor (KOR-3) was isolated by screening a mouse genomic library with a radiolabeled mouse KOR-3 cDNA probe. Sequence analysis of the genomic clone indicates that the KOR-3 gene contains five exons separated by four introns. The transcription start point (tsp) of KOR-3 was mapped by primer extension analysis of RNAs synthesized either in vivo or in vitro. A TATA-box and several potential regulatory elements, including five GRE sites, four NF-El binding sites and one MRE site, are present in the 2 kb of 5’-flanking region. A putative poly(A) signal (AATAAA) is found in the 3’-flanking region.

INTRODUCTION

The kappa, opioid receptor (Price et al., 1989; Clark et al., 1989), corresponding to the Martin’s nalorphine, or ‘N’, receptor (Martin 1967), is easily distinguishable from the traditional mu, delta, and kappa, receptors both biochemically and pharmacologically (Pasternak 1993; Paul et al., 1990; Gistrak et al., 1990; Standifer et al., Correspondence Memorial

to: Dr. G.W.

Sloan-Kettering

Pasternak,

Cancer

Department

Center,

York, NY 10021, USA. Tel. (l-212)

of Neurology,

1275 York

639-7046;

Avenue,

Fax (l-212)

New

794-4332;

e-mail: [email protected] Abbreviations:

aa, amino acid(s); AMPA, m-amino-3-hydroxy-5-methyl-

isoxazole-4-propionic tary

receptor kilobase

acid; bp, base pair(s); cDNA,

DOR-I, gene (DNA,

to RNA,

RNA)

DNA complemen-

encoding

a delta

opioid

(DOR-1); GRE, glucocorticoid-responsive element(s); kb, or 1000 bp; KOR-I, gene (DNA, RNA) encoding a kappa,

opioid receptor (KOR-1); KOR-3, gene (DNA, kappa,-related opioid receptor (KOR-3); MOR-I,

RNA) encoding a gene (DNA, RNA)

encoding a mu opioid receptor (MOR-1); MRE, metal-responsive element(s); NF-El, erythroid-cell-specific nuclear factor; nt, nucleotide(s): PCR, polymerase transcription; region(s).

chain reaction;

tsp, transcription

SSDI 0378-1119(95)00890-X

rDNA, start

ribosomal point(s);

DNA; RT, reverse UTR,

untranslated

1994; Cheng et al., 1995). The cloning of the delta receptor (DOR-1)(Evans et al., 1992; Kieffer et al., 1992) quickly led to identification of mu (MOR-1)and kappa, (KOR-1)receptor clones with their anticipated pharmacological profiles after expression (Chen et al., 1993; Minami et al., 1993; Thompson et al., 1993; Reisine and Bell 1993; Wang et al., 1994b; Yasuda et al., 1993; Min et al., 1994; Bare et al., 1994; Zimprich et al., 1994). Using an RT-PCR approach based on DOR-1,we cloned a novel opioid receptor from a mouse cDNA library, KOR-3,(Pan et al., 1994, 1995; Uhl et al., 1994) which is the same as an orphan clone reported by several other laboratories at the same time (Chen et al., 1994; Keith et al., 1994; Uhl et al., 1994; Wick et al., 1994; Wang et al., 1994a; Mollereau et al., 1994; Lachowicz et al., 1995; Bunzow et al., 1994; Fukuda et al., 1994). The high homology of this clone to the traditional opioid receptors strongly suggests it belongs within the opioid receptor family. Additional evidence strongly implies that this clone is closely related to the kappa, opioid receptor. A monoclonal antibody generated against the kappa, receptor from a human neuroblastoma cell line (Brooks et al., 1995) recognizes the in vitro translation product

256

1 _~. I--_ Fig. 1. Sequencing

strategy

and structure

2

3

~~_ I- ~_

I

indicate

of the sequencing

with a synthetic

primer.

coding exons and open bars non-coding

V: EcoRV, X: XbaI, M: SmaI. The by upward

5

-1

in both directions The genomic

exons. Partial

6

1

8 kb

_~~~ mmm.m.-p!__L

of the KOR-3 gene. The genomic

into the SKII vector (see Section a) and sequenced length

4

clone (3-u) was isolated

with a primer-walking

clone is shown restriction

by screening

approach.

a mouse genomic

Each arrow represents

by the top heavy line and scaled by the bottom

sites are indicated

by the following:

library,

the direction

subcloned and the

light line. Solid bars

S: SacI, B: BarnHI, D: DraIII, R: EcoRI,

tsp is shown by + 1 and a bent arrow. The ATG start codon, TGA stop codon and poly(A) signal are indicated

arrows

of KOR-3 and at least seven different antisense oligodeoxynucleotides based upon the KOR-3 cDNA sequence down-regulate kappa, analgesia in mice (Pan et al., 1994; 1995; Uhl et al., 1994). However, KOR-3 is probably a splice variant of the kappa, receptor (Pan et al., 1995). In contrast to activity of the antisense oligodeoxynucleotides directed against the third and fourth exons of KOR-3, five of the six antisense probes targeting the second exon are inactive. Southern analysis indicates that a single-copy gene encodes the receptor (Pan et al., 1995) which is also supported by chromosomal mapping (Chen et al., 1994). To facilitate future studies of this putative opioid receptor clone, we have isolated and characterized the gene encoding the mouse KOR-3 receptor.

EXPERIMENTAL

AND DISCUSSION

(a) Isolation and sequencing of a mouse KOR-3 genomic clone

A 1.4-kb cDNA fragment of the mouse KOR-3 containing all of the coding region and part of the 3’ UTR was 32P-radiolabeled and used to screen a mouse genomic library in the hFIXI1 vector (Stratagene, La Jolla, CA, USA). Approximately 2 x lo6 plaques were hybridized at high stringency and nine clones were isolated. One of the clones (3-u) contained approximately 8.3 kb of genomic

DNA encompassing all of the KOR-3 cDNA sequence. Not1 sites were only located within the multiple cloning sites of the right and left arms of the hFIXI1 vector. A single 8.3-kb Not1 fragment from clone 3-u was subcloned into the Not1 site of Bluescript SKI1 (termed 3-a/SKIZ) and used for further sequence analysis. (b) Structural organization

of the KOR-3 gene

Exon-intron boundaries were determined by comparison of the nt sequence of the KOR-3 gene with its cDNA (Pan et al., 1995) (Figs. 1 and 2). The KOR-3 gene contains all the KOR-3 cDNA sequence with 100% identity. The KOR-3 gene consists of five exons divided by four introns (Fig. 1 and 2). All exon/intron splice sites are in agreement with the GT/AG rule (Breathnach and Chambon 1981). Exons 2, 3 and 4 contain the entire encoding region, while noncoding regions are included in exons 1,5 and parts of exons 2 and 4. Introns 1,2,3 and 4 are 458,2428,81 and 118 bp in length, respectively. The locations of intron 2 and 3 of the KOR-3 gene are similar to those of the corresponding introns of the mouse MOR-1 gene (Min et al., 1994) and KOR-1 gene (Yakovlev et al., 1995; Liu et al., 1995). The presence of the intron 2, the largest intron of the KOR-3 gene, provides the possibility of other alternative exons. At least four alternative splicing forms have been identified in this location (Y.-X.P., J.X. and

Fig. 2. The nt sequence

of the KOR-3

gene. The exon sequences

are shown in capital

letters, and the intron

and S’- and 3’-flanking

sequences

are

presented in lower-case letters. The tspdetermined by primer-extension analysis is indicated by underline and + 1. The nt sequence is numbered with respect to the tsp.The aa residues are numbered relative to the first Met. A stop codon (TGA) is indicated by an asterisk. The NF-El, GRE, MRE, TATA box, 5’-CAAAA; repeat and poly(A) signal are underlined. The sequence used for synthesizing the primer of primer underlined. The complete nt sequence has been submitted to the GenBank under the accession Nos. (U32925-U32935).

extension

analysis

is also

258 G.W.P., unpublished observations). It is noteworthy that sequence identical to portions of intron 3 also were obtained by screening a cDNA library (Pan et al., 1995) and by RT-PCR (data not shown), indicating that this intron occasionally may not be spliced out. Unlike a corresponding sequence in the rat encoding a 2%aa insertion (Uhl et al., 1994; Wang et al., 1994a), the mouse sequence contains a stop codon (TGA). If it is present in mature mRNA the stop codon will terminate the translation, although a suppressor tRNA possibly could read the stop codon to translate it through in some circumstances. Almost half of the KOR-3 cDNA clones isolated from the screening the cDNA library (Pan et al., 1995) contain the intron 4 sequence which is located in the 3’ UTR. The significance of this sequence is not clear. The existence of an alternative spliced exon 2 corresponding to the N-terminus was strongly suggested by in vivo antisense mapping studies (Pan et al., 1995). However we do not find an alternative exon in the genomic clones we have isolated, raising the possibility that this exon with its own promoter might be located 5’-upstream from the genomic clones we isolated. Efforts have been made towards isolating such an isoform of this receptor family from both cDNA and genomic DNA.

1234

CTAG T T T T T T T T T T'

I :: + Tfl T' T'

T T

T T T

T T G G

Fig. 3. Primer

extension

analysis.

Lanes: 1: Primer

extension

of RNA

extract; 2: Primer extension

of RNA

synthesized

with the HeLa nuclear

synthesized

with the HeLa nuclear extract in the presence of cc-amanitin

(8 pg/ml),

an inhibitor

of RNA polymerase

The putative tsp of the KOR-3 gene was determined by primer extension analysis of RNAs synthesized either in vivo, or in vitro with a HeLa nuclear extract (Promega, Madison, WI, USA). As shown in lane 3 of Fig. 3, primer extension of the total RNA extracted from mouse brain (in vivo synthesized RNA) gives a sharp band, which locates the tsp to a thymine residue within a poly(T) track, 8 bp upstream from exon 1. The same results were obtained using different source total RNA prepared from mouse brain (data not shown). Primer extension of the RNA synthesized from 2.8 kb of the 5’-genomic fragment of the KOR-3 gene with HeLa nuclear extract (in vitro synthesized RNA; lane 1 of Fig. 3) revealed a smear of bands that span 5-6 thymine residues and surround the tsp assigned by the in vivo synthesized RNA. The smear tsp given by in vitro synthesized RNA might be due to less precision in the transcriptional initiation in vitro. Here we arbitrarily assign the tsp based on the extension band obtained from in vivo synthesized RNA (Fig. 2). The location of the tsp was also supported by nuclease protection assay (data not shown). A variant TATA box (Xu et al., 1991) was found in 78 bp upstream from the tsp. Neither a CAAT box nor cap site was identified. Several potential regulatory elements, including five GRE sites (von der Ahe et al., 1985; Renkawitz et al., 1984; Cato et al., 1984), four NF-El

II; 3: Primer

extension

of

total RNA (15 1.18)extracted from mouse brain; 4: No RNA. The four sequencing ladders (C, T, A and G) were used as a chain-length marker to precisely map the tsp. Arrow and asterisks by using in vivo and in vitro synthesized

(c) Transcription start point (tsp) and 5’upstream region of the KOR-3 gene

C

\

indicate

the tsp determined

RNA, respectively.

Methods:

A primer S-CTTCTCACTCTAGCTTGGG, complementary to the nt position from 160 to 178 and located in exon 1, was 32P-labeled and used in the primer extension

reaction

sion kit protocol

Madison,

(Promega,

RNA was extracted genomic

5’-fragment

as described

of the KOR-3 from

appropriate

was subcloned

construct

5’UTRISKII.

-2000

template (Promega)

gene, including to +789),

exon 1 and part of

generated

by PCR with

into the EcoV site of the SKI1 to

The linearized

for the in vitro synthesis generated

sensitive run-off product were synthesized

total

Since the PstI site was present only in the SKI1

fragment.

which

brain

(Pan et al., 1995). A 2789-bp

vector, it was used to linearize the 5’UTR/SKII of the genomic

in the primer exten-

WI, USA). Mouse

previously

exon 2 (nt position primers

as described

construct

fragment

at the 3’ end

was then used as a

of RNA with a HeLa nuclear

approximately

0.8 kb

(data not shown). Unlabeled

under the same conditions

extract

of a-amanitinrun-off products

and were used in the primer

extension analysis. The genomic fragment was sequenced primer used for the primer extension. The extension

with the same products and

DNA

7 M urea-6%

sequencing

polyacrylamide

ladder

were analysed

gel (National

Diagnostic,

in the same Atlanta,

GA, USA).

binding sites (Wall et al., 1988) and one MRE site (Anderson et al., 1987), were identified in the 5’upstream from the tsp. In addition, a putative poly(A)-addition signal (AATAAA) was found in the 3’-flanking region. An repeat sequence (eleven 5’-CAAAA-3’) was found upstream from the TATA box in the 5’-flanking region. The significance of the sequence is not clear. However, similar repeat sequences were reported in the mouse et al., 1988), mouse (Braaten rDNAs c+2,3+.ialyltransferase cDNA (Sasaki et al., 1993; GenBank accession No. D28941) and in the mouse

259 AMPA receptor subunit gene (Kohler et al., 1994). Further studies will be directed towards identifying the detailed promoter region and studying the significance of these regulating elements in vivo. (d) Conclusions (I) Sequence analysis of the KOR-3 gene reveals five exons separated by four introns (2) The tsp of the KOR-3 gene was determined by primer extension analysis of RNAs synthesized either in vivo or in vitro. (3) A TATA-box and several potential regulatory elements are present in the 5’-flanking region while a putative poly(A)-addition signal is present in the 3’-flanking region

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