The sequence of a murine cDNA encoding Ear-2, a nuclear orphan receptor

Gene, 142 (1994) 3133314 0 1994 Elsevier Science B.V. All rights reserved.

313

0378-l 119/94/$07.00

GENE 07853

The sequence of a murine cDNA encoding Ear-2, a nuclear orphan receptor* (Transcription

factor; mouse; steroid receptor superfamily; e&A related)

Kerry M. Barnharta,b,c and Pamela L. Mellona,b+ ‘Department of Reproductive Medicine; bDepartment of Neurosciences; and “Centerfor Molecular Genetics, Unioersity of California, San Diego, School of Medicine, La Jolla, CA 92093-0674, USA Received by C.M. Kane: 18 November

1993; Revised/Accepted:

3 December

1993; Received at publishers:

27 January

1994

SUMMARY

A 1643-bp cDNA that encodes the mouse Ear-2 orphan nuclear receptor has been isolated from a pituitary cell library. The predicted 389-aa mouse Ear-2 protein bears significant homology to the known human Ear-2 protein especially in the DNA-binding domain.

Members of the steroid/thyroid receptor superfamily are key regulators of gene expression which interact with their cognate DNA sequences as heterodimers, homodimers, or monomers (O’Malley and Conneely, 1992; Wilson et al., 1993). Several members of this family have been identified for which the ligands are unknown and these have been termed orphan receptors. These factors exhibit varied patterns of developmental and tissuespecific expression. Because of the crucial role these factors play in cellular differentiation and developmental processes, studies continue to focus on how these factors may regulate genes expressed in a tissue-restricted or inducible manner. During the course of our studies on the transcriptional regulation of the gene encoding the glycoprotein hormone a-subunit in pituitary gonadotropes, we have isolated the murine homologue of the human ear-2 @A-related) gene, a member of this family first isolated Correspondence to: Dr. P.L. Mellon, Department of Reproductive Medicine, 0674, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0674, USA. Tel. (1-619) 534-1312; Fax (1-619) 534-1438; e-mail: [email protected] *On request, the authors will supply detailed the conclusions reached in this Brief Note.

experimental

evidence for

Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, DNA complementary to RNA; ear-2, erbA related; Ear-2, ErbA related orphan receptor protein; ErbA, thyroid hormone receptor protein; nt, nucleotide(s). SSDI 0378-l

119(94)00064-Y

through its homology to ErbA, a thyroid hormone receptor (Miyajima et al., 1988). While its ligand remains unknown, the human protein has previously been shown to participate in the regulation of hepatic gene expression (Ladias et al., 1992). A murine clT3 pituitary gonadotrope (Windle et al., 1990) cDNA library was screened under conditions of relaxed stringency with a probe corresponding to the DNAbinding domain of the orphan receptors COUP-TF and ARP-1 (Wang et al., 1989). The positive clone containing the longest insert was purified and that insert cDNA fully sequenced. The 1643-bp insert contains an open reading frame from nt 114 to 1280, ending with three in-frame stop codons (Fig. 1). A consensus polyadenylation signal is located 19 nt upstream from the poly(A) tract. Northern analysis of various mouse tissues and cultured cell lines indicate the mRNA is widely expressed, albeit at differing levels (data not shown). Comparison of the cDNA to sequences contained in GenBank revealed a high degree of homology to the human ear-2 orphan receptor encoding sequence. A direct comparison of the 389-aa protein encoded by the murine cDNA to the 403-aa human Ear-2 protein is presented in Fig. 2. Perfect aa homology between the proteins is maintained throughout the DNA-binding domain (aa 56-121 of the murine factor). Homology between the

314 1 121 241 361 481 601 721 841 961 1081 1201 1321 1441 1561

Fig. 1. The nt sequence of mouse ear-2 cDNA. A mouse cDNA library was prepared from poly(A)+RNAs purified from ctT3-1 cells (Windle et al., 1990) using the vector hZapI1 (Stratagene, La Jolla, CA, USA). The sequence was obtained from a contiguous single clone by sequencing both strands using the dideoxy chain-termination method (Sanger et al., 1977). The largest open reading frame from nt 114 to 1280 is shown Noncoding sequences are shown in lower case. This sequence has been deposited in GenBank under accession No. L25674.

MOUSE

NANV'IGGWGGPGGDTNGVDKGSYPRATEDDSASPPGATSDAEPGDEER

HUNAN

NAMVI'SGWGGPGGDTNGVDKAGG-YPRAAEDDSASPPGAASDAEPGDEER 49

IIIIIIIIIIIIIIIIIlIIIII

III1

IllIIIIIII

50

IIIIIIIIII

PGLQVDCWCGDKSSGKHYGVFTCEGCKSFFKRTIFXNLSYTCRSNRECQ

100

IIIIlIIIIIIIIIIIIIIIIIIIIIIIIIIIIIlIIIIIIIIIIIIIIl

PGLQVECVVCGDKSSGKHYGVF'ICEGCKSFFKRTIRRNLSY'ICRS~Q IDQHHRN~QYCRLKKCFRVGNRKEAVQRGRIPHALPGP-AACS--PF+ IIIIIIIIIlIIIIIIIIIIIIIIIIIIIIIIII

Ill

II

I

99

149

DNVCELAARAV-STVEWARHAPFFPELPAADQVALLRLSWSELFVLNAAQ 235 IIIIIIII

IIIIII

IIIIIIII

IlIIIIIIIIII

248

AAVAVHTAPLLAAAGLHAAPbf+AERAVAFNDQVP.AFQEQ~KLGRLQVDA 285 IIIIIIIIIIIIIlIIIliIIIlIIIIIIIIIIIIIIlIIIIII II AALPLHTAPLLAAAGLHAAPMAAERAVAFMDQVRAFQEQVDKLGRLQVDS 298 AEYGCLKAIALFTPDACGLSDPAHVESLQEKAQVALTEYVRAQYPSQPQR 335 IIIIIIIIIIIIIIIIIIIIIIIlIIIIIIIIIIIIIIIIlIIIIIlIII

AEYGCLKAIALFTPDACGLSDPAHVESLQEKAQVALTEYVRAQYPSQPQR 348 FGRLLLRLPALRAVPASLISQLFFMRLVGKTPIETLIRDNLLSGSTFNWP 385 IIIlIIIIIIIIIIIIIIIIllIIIIIIIIIIIIIIIIIIIIIIIIIIII FGRLLLRLPALRAVPASLISQLFFNRLVGKTPIETLIRDMLLSGSTFNWP 398 YGSG

389

I I YGSG4

403

II

REFERENCES Ladias, J.A.A., Hadzopoulou-Cladaras, Cheng, J., Zannis, V. and Cladaras, of human apolipoprotein members of the steroid

M., Kardassis, D., Cardot, P., C.: Transcriptional regulation

genes ApoB, ApoCIII, and ApoAII by hormone receptor superfamily HNF-4,

ARP-1, EAR-2, and EAR-3. J. Biol. Chem. 267 (1992) 15849-15860. Miyajima, N., Kadowaki, Y., Fukushige, S., Shimizu, S., Semba, K., Yamanishi, Y., Matsubara, K., Toyoshima, K. and Yamamoto, T.: Identification of two novel members of erbA superfamily by molecular cloning:

the gene products

other. Nucleic

of the two are highly related

to each

Acids Res. 16 (1988) 11057-11074.

O’Malley, B.W., and Conneely, O.M.: Orphan receptors: in search of a unifying hypothesis for activation. Mol. Endocrinol. 6 (1992)

Fig. 2. Comparison of the deduced aa sequence of the mouse Ear-2 protein with the human protein. The aa sequences are presented in single letter. The human

obtained from other species. Such striking similarities suggest that the mouse protein may play a conserved role in the regulation of gene expression in mammals.

146

A'P-VEH----FTGPPVSELIAHVLRAEPYPAA-GRFG-GGGA---VLGI 186 IIIIIIIII IIll IIII I I I I II I I Illllll ALAAVASGGDLFffiQPVSELIAQLLRAEPYPAAAGRFGAGGGAAGAVLGI 199 IIIIllIII

letters.

I I I

IDQHHRN~QYCRLKKCFRVGMRKEAVQRGRIPHSLPGAVAASSGSPFGS

DNVCELAARLLFSTVEWARHG-FFPELPVADQVALLRMSW

in capital

Ear-2 sequence

is from Miyajima

et al. (1988).

N-terminal portions of the factors (typically low between distinct members of this family) is 95%. Adjacent and C-terminal to the DNA-binding domain is a region of decreased homology (74%), while the C-terminal region (149 aa, which includes the putative ligand-binding domain) retains a high (99%) degree of homology to the human protein. As yet, ear-2 cDNAs have not been

135991361. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Wang, L., Tsai, S.Y., Cook, R.G., Beattie, W.G., Tsai, M. and O’Malley, B.W.: COUP transcription factor is a member of the steroid receptor superfamily.

Nature

340 (1989) 163-166.

Wilson, T.E., Fahrner, T.J. and Milbrandt, J.: The orphan receptors NGFI-B and steroidogenic factor 1 establish monomer binding as a third paradigm of nuclear receptor-DNA interaction. Mol. Cell. Biol. 13 (1993) 5794-5804. Windle, J.J., Weiner, RI. and Mellon, P.L.: Cell lines of the pituitary gonadotrope lineage derived by targeted oncogenesis in transgenic mice. Mol. Endocrinol. 4 (1990) 5977603.