Vol.
177,
No.
June
28,
1991
3, 1991
BIOCHEMICAL
AND
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RESEARCH
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ANALYSIS INSULIN-LIKE
OF THE HUMAN GROWTH
FACTOR
PROMOTER
1113-1120
TYPE I RECEPTOR
REGION
David W. Cooke’*, Laura A. Banker-t’, Charles T. Roberts, Jr?, Derek LeRoith’, ‘Department
and Samuel J. Casella’
of Pediatrics, Johns Hopkins University
Medicine,
CMSC 3-110, Baltimore,
2Diabetes Branch, Building
School of
MD 21205
10, Room 8S-243, National
Institutes
of Health, Bethesda, MD 20892 Received
May 7, 1991
SUMMARY: We isolated genomic fragments containing the 5’ region of the human type I insulin-like growth factor receptor gene. A unique transcription start site was identified, defining a 1038 bp 5’untranslated region. No TATA or CCAAT elements were identified in the proximal 480 nucleotides of 5’-flanking region. The region surrounding the transcription start site was similar to a recently described “initiator” sequence. The 5’-flanking and S-untranslated regions were highly GC-rich, with numerous potential Spl binding sites. A potential AP-2 binding site was identified in the 5’-flanking region and a potential thyroid response element was identified in the 5’untranslated region. The 5’ region of the human gene was very similar to that of the rat gene, with conservation of many 0 1991Academic Press. Inc. of the potential regulatory elements.
The
type
heterotetramer
I insulin-like
growth
consisting of two extracellular
domain and two intracellular
receptor
(IGFR)
is a disulfide-linked
(a) subunits that contain the ligand binding
(a) subunits that contain tyrosine kinase activity (1). The
receptor is homologous
to the insulin receptor and other members of the tyrosine kinase
family (1). Insulin-like
growth factor I (IGF-I)
although in some circumstances IGF-II IGFR.
l
factor
(2) and insulin (3) may also stimulate
IGF-I mediates the growth-promoting
Corresponding
is the primary ligand of the type I IGFR, the type I
properties of growth hormone (4). IGF-I and
author.
Abbreviations: IGFR, insulin-like growth factor receptor; IGF-I, insulin-like I; IGF-II, insulin-like growth factor II; AP-2, activator protein 2.
1113
growth factor
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in anv form reserved.
Vol.
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the homologous
IGF-II
synthesized in multiple
BIOCHEMICAL
circulate
AND
BIOPHYSICAL
in serum in relatively
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high concentration
and are
tissues (4), suggesting that they act through both endocrine and
paracrine or autocrine mechanisms. The response of a given cell type to the IGFs may depend on the number receptors at the cell surface. The rat type I IGPR gene is developmentally tissue-specific manner (5). Furthermore, hormone
a number of factors, including
(6), estrogen (7), thyroid hormone
IGFR expression.
Finally, the proximal
recently characterized
regulated in a
follicle-stimulating
(8), and phorbol esters (9), stimulate
promoter
of
type I
region of the rat type I IGFR gene was
(lo), and was found to show similarities
to promoters
regulated genes. These findings all support the hypothesis that type I IGFR
of highly
expression is
carefully regulated. As a first step in defining the regulatory
elements that control the expression of the
human type I IGFR gene, we have isolated and characterized genomic fragments containing the 5’-flanking nucleotide
region of the human type I IGFR
gene.
sequence of the human type I IGFR promoter
In this study, we present the region, define the transcription
start site, and compare the regulatory regions of the human and rat type I IGFR genes.
MATERIALS
AND METHODS
A human chromosome 15 genomic library (#57737, American Type Culture Collection, Rockville, MD) was screened using an oligonucleotide encoding amino acid residues 155-170 of the type I IGFR Q subunit as published by Ullrich et al. (1). A 1.7-kb Hind III fragment (R737P2) was isolated that contained 538 bp of exonic sequence encoding amino acid residues 3-183 of the type I IGFR flanked by intronic sequences. The genomic clone R737P2 was then used to isolate a 1.6-kb cDNA (IGFRa) from a human foreskin fibroblast Xgtll library (kindly provided by Drs. Frank French and David Joseph, Laboratories for Reproductive Biology, University of N. Carolina at Chapel Hill). Clone IGFRa contained a 310-bp 5’-untranslated region that extended upstream from the published sequence of Ullrich et al (1). A fragment of IGFRa containing the sequence 5’ to the defined exon/intron boundary was then used to isolate a 3.5-kb Hind III genomic clone (R737P3) from the human genomic library (ATCC #57737); this clone contained the 5’-flanking region and first exon of the type I IGFR gene. After restriction mapping of the cloned inserts, the appropriate fragments were subcloned into pTZ vectors (U.S. Biochemical Corp., Cleveland, OH) for sequencing by the dideoxynucleotide chain termination method using modified T7 polymerase (Sequenase, U.S. Biochemical Corp., Cleveland, OH). The sequence was confirmed by sequencing both strands of the clones. Total RNA was extracted from freshly frozen human placenta by homogenization in guanidinium thiocyanate and pelleting through a cesium chloride cushion (11). Polyadenylated (poly (A)+) RNA was then isolated by afhnity chromatography on an oligo(dT) cellulose column. The flow-through of the oligo(dT) colunm was retained as a control (poly (A)‘ RNA). A 17-base oligonucleotide complementary to nucleotides 56 to 72 of the 5’-untranslated region of the human type I IGFR gene was end-labeled with 32P and purified on an 8% polyacrylamide/8M urea gel. Approximately 2 x lo6 dpm of the 1114
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AND
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labeled probe were hybridized with 24 bg poly (A)+ RNA, 660 pg poly (A)- RNA (flowthrough of the oligo (dT) column), or 100 pg tRNA (Sigma, St. Louis, MO) for 15 hrs at 30°C. After ethanol precipitation, the samples were resuspended in 20 ~1 reverse transcriptase buffer containing 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl,, 10 mM dithiotreitol, 1 U/cl1 RNA@ and 1 PM of each dNTP. Moloney Mmine Leukemia Virus reverse transcriptase (5OU, Bethesda Research Laboratories, Gaithersburg, MD) was added and the reaction was incubated for 2 hours at 37’C. The reaction was terminated by the addition of 1 ~1 of 0.5M EDTA and 5 pg of RNAase (Sigma, St. Louis, MO). After 30 minutes at 37°C the extended products were extracted with phenol:chloroform, ethanolprecipitated, and resolved on an 8% polyacrylamide/8M urea gel. Radiolabeled antisense RNA probes were transcribed from SmaI-BarnHI, BarnHI-Sac1 and SacI-BspIvIII genomic fragments. Together, these fragments were complementary to 128 bases of 5’-flanking region and the entire 1038 bases of 5’-untranslated region, RNase protection assays were performed as previously described (12) using RNA prepared from human placenta to protect the riboprobes. RESULTS
AND DISCUSSION
We have isolated genomic clones that contain the S-flanking
region and first two
exons of the human type I IGFR gene. The first two introns of the human type I IGFR gene, predicted by comparing
the genomic clones with the cDNA sequence, interrupt
codons for amino acids 2 (isoleucine) IGFR.
Therefore,
and 184 (methionine)
the
of the (Ysubunit of the type I
these first two introns are located in positions homologous
to the first
two introns of the insulin receptor (13) and the recently identified
insulin receptor-related
receptor (14). Clone R737P3 extends 1518 bp 5’ to the translation
start site. To define the
Figure 1. Mapping of the 5’ end of the human type I IGFR mRNA by primer extension. Poly (A)+ RNA from human placenta was analyzed, with poly (A)- human placenta and yeast tRNA as controls. The three right hand lanes contain the primer-extended cDNAs obtained from the indicated RNA sample. The sequence ladder was obtained by using the primer extension oligonucleotide as a DNA sequencing primer on the appropriate template. Therefore, the size of the extended product indicates the transcription start site (arrow). 1115
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BIOCHEMICAL
3, 1991
point of transcription
initiation
a 17-base oligonucleotide
complementary
to transcription
BIOPHYSICAL
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we analyzed human placenta RNA by primer extension using
region of the human type I IGFR corresponding
AND
to nucleotides
56 to 72 of the S-untranslated
gene. The resulting product was 72 bp in length (Fig. l), initiation
at nucleotide
1 (Fig. 2). An RNase protection
assay, using an antisense RNA probe that was generated from a genomic sequence that -480
tttcaagaaccggggaaacgcgctttccagccgcgctgttgttgttttcaatgaacctct
-420
cccagccccgcactccccacccacccctcccctctcctgcccacccctcccctctcctgc
-360
ccacccctcccctgcctagcctttccctggctacccacccct~cccc~ccgagaccggac
-300
cggcgg=ggggg==ttgtttttggagtc~=gggg=gggcgcgtgcgggtggccg
-240
gcgcagtgcggtggnggcgggagcgggtgggacgcgggtgggacgcgcgcgtgtctctgtgtgcgcgcggg
-180
aggcggtnagecagat~e~ece~cgcctcgcagtctcgcgccccacgcccgggctc
-120
cggttttttgcgcgcgccggcctgggccgggccctcggcgcgccgctgctcggcggtggc
-60
cgctcgagtgtgcgagcgggcgcgtgtgtgccagggcgccgcgcgcgcga~ 1 AGTGTGTGGCAGCGGCGGCGGCGGCGCGGCGAGGCTGGGGCTCTTGTTTACCAGCATTAA
61
CTCGCTGAGCGGAGGGAAAAAA
CCCGAGGAGGAGCGAGCGCACCAGGCGAAC
121
TCGAGAGAGGCGGGAGAGCGAGAGGGACGCCGCCAGCGAGCCTGCCCACGGCCGGCGCTC
181
GCAGACCCTCGGCCCCGCTCCCCGGATCCCCCCGCGCCCTCCACGCCCCTCCCGCGCGGG
241
GGCAGCTCCACGGCGCGCCTCGCCTCGGCTGTGACCTTCAGCGAGCCGGAGCCCCCGCGC -----------
301
AGAGCAGGCGGCGGCGGGCGGGGGCCGGGCGGGGGCCGGCGCGGGGCGGGCGGCGGCGCA
361
GAGCCGGGCGGCGCGGCGGGAGTGCTGAGCCGGCGGGCCGGCCCGCCGCTTTGTGTGTGT
421
CCTGGATTTGGGAAGGAGCTCGCGGCGGCGGCGGCGCTGAGGGAGGAGGCGGCGGCGAGC
481
GGAGCCAGGAGGAGGAGGAGGAGGAGGGGGAGCCGCTCATTCATTTTGACTCCGCGTTTC
541
TGCCCCTCGCCGGCCTCGCCTGTGACCCGGACTTCGGGGCGATCTTGCG~CTGCGTCGC
601
GCCCTCCCGCGGCGGAAGCTCGGGCGTCCGGCCGCCTCCCGCGCGCCAGGGCCGGGCTTG
661
TTTTTCCTCGCCTAGGCAGATTTGGGCTTTGCCCCCTTTCTTTGCAGTTTTCCCCCCTTC
721
CTGCCTCTCCGGGTTTGATGGAGGCCGACGACGCCGACAGCCCGCCCCGGCGCGCCT ---------~------_
781
CGGGTTCCCGACTCCGCCGAGCCCTGGGCCGCTGCTGCCGGCGCTGAGGGGCCGCCCCGC ------------------
841
GCCGCCCGCCCCGTCCGCGCACCCGGAGGGCCCCGGCGGCGGCCCTTCGGAGTATTGTTT
901
CCTTCGCCCTTGTTTTTGGAGGGGGAGCGAAGACTGACTGAGTTTGAGACTTGTTTCCTTTCAT
961
TTCCTTTTTTTCTTTTCTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTGAG~GGGGATT
1021
TCATCCCAAATAAAAGGATCTGGCTCCGGAGGAGGGTCCCCGACCTCGCTGTGG
1081
GGGCTCCTGTTTCTCTCCGCCGCGCTCTCGCTCTGGCCGACGAGTGGAG~
Figure 2. Nucleotide sequence of the 5’ region of the human type I IGPR gene. Nucleotide 1 corresponds to the trauscription start site. The ATG codon that initiates translation is underlined, beginniug at nucleotide 1039. The sequence ends at the end of the first exon. Potential Spl binding sites are underlined. A potential AP-2 binding site is underlined by a thick line (nucleotides -136 to -129). The Ynitiatoi’ sequence surroundiug the transcription start site is underlined with a double hue. A potential thyroid response element is underlined with a thick dashed line (266 to 277). The open reading frame in the S-uutrauslated region is underlined with a dashed line. 1116
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No.
3, 1991
BIOCHEMICAL
AND
extended 128 bases upstream from this initiation confirmed the location of the transcription that were complementary Y-untranslated
BIOPHYSICAL
RESEARCH
site, produced a single protected band that
start site at nucleotide 1. Antisense RNA probes
to nucleotides
204 to 1056 (covering the remainder
region) were fully protected, confirming the single transcription
(data not shown). Thus, the R737P3 clone contains 480 nucleotides and a 1038-bp S-untranslated 5’-flanking
COMMUNICATIONS
region.
No TATA
or CCAAT
of the
initiation
of 5’-flanking
site region
elements are found in the
region, despite the fact that there is a single specific transcription
initiation
site.
However, the region between nucleotides -6 to + 11 is very similar to the recently described “initiator” element
sequence that directs specific transcription
initiation
in the absence of a TATA
(15). Both the S-flanking
and S-untranslated
regions are highly GC-rich, with 75% and
68% GC content each. There are numerous potential The type I IGFR promoter
Spl binding sites (16) in both regions.
is therefore similar to the promoters of many housekeeping
cellular growth control genes that lack TATA
elements and are GC-rich,
insulin receptor (17), EGF receptor (18), and NGF receptor (19) promoters. sites have been shown to interact with the “initiator” transcription
including
sequence to direct high levels of initiation
element (20). A potential binding site for the transcription
AI’-2 is found at nucleotides
the
Spl binding
from a single start site (IS), and may be necessary for transcription
in genes lacking a TATA
and
-136 to -129 in the 5’-flanking
factor
region. The AP-2 element can
mediate both cyclic AMP and phorbol ester control of transcription
(21,22), and its presence
in the type I IGFR gene may explain previous observations that cyclic Ah4P (6) and phorbol esters (9) stimulate type I IGFR expression, and this may be the site through which folliclestimulating
hormone regulates IGFR
the 5’-untranslated response element
expression (6). The region from bases 266 to 277 in
region includes one-half of the palindromic and is similar
sequence of the thyroid
to thyroid response elements found in the rat growth
hormone gene (23) and the human TSH a gene (24). The presence of this potential response elements supports the observation that thyroid hormone expression (S), and suggests a mechanism whereby this regulation
thyroid
regulates type I IGFR can occur at the level of
transcription. The type I IGFR gene is unusual in that the 5’-untranslated Interestingly,
many of the rare genes that have long S-untranslated
involved in the regulation
of cellular proliferation,
be examined,
regions seem to be
including growth factors and many of the
cellular oncogenes (25). The role of these long 5’-untranslated translation
region is extremely long.
regions is just beginning to
but there is growing evidence that they are involved in the regulation
of
(26-28). Kozak (27) and others (28-30) have shown that secondary structures in 1117
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177,
No.
BIOCHEMICAL
3, 1991
HUEII-I Rat Human Rat Human
AND
BIOPHYSICAL
RESEARCH
ttttcaagaaccggggaaacgcgctttccagccgcgctgttgttgttttc :: : : ::: tt--------ccggg--.--------------------------------
-432 -405
aatgaacctctcccagccccgcactccccgcccacccctcccctctcctg : : :: :: :::::::::::: ------------------ccgcactccccgcccaccgcgcccg------cccacccctcccctctcctgcccacccctcccctgcctagcctttccctg :: ::: ::: ::::
-382 : ::: -379 -332 ::::
Rat
--cagcccgccc-----.-gccc----------tgcc--tg==--g~=--------
Human
gctacccacccctgccccgccgagaccggaccggcggcgggggcattgtt :: ::::: : : :: ::: :: : :::::: gc------ccccttggccaccgagtcccg-ccggcgcccggggcattgtt
Rat Human Rat Human Rat Human Rat Human Rat Human Rat Human Rat Human Rat
COMMUNICATIONS
-358 -282
: :: : : ::::::: -316
ttt-ggagtcgggcgggaggggagggcgcgtgcgggt--ggccggcgcag : : : : : :::: ::::: : : : : :: :: ::: : :: ttttggagtcctgcgggtggggagggtgcggacagggccggccggcgcag
-235 ::::::::::: -266
tgcgg-tgggggcgggagcgggtgggacgcgcgcgtgtctctgtgtgcgc ::::: ::::::::: :::: :::: : : : : :::::: aagcg--tggg-cgcgcgcgtggctcagtgtgcgc tgcgggtgegagCgg gcgggaggcggtggggcgggagat--gggggcggcgcctcgcagtctcg..,.... . . . : ::: : :: :::: :::::::: gcgggggccggcgcgggtggaggcgc~tgcctcctgggcccgg
:::
-186 :::
:::::::: -219 -139
:::::
: : :: -169
--gt--------------------cgccccacgcccgggctccg----::::::::::: :::::: ctccccacgcccgcgctccgtccgcacgtccctgcgatcccgaactccgg
-11-J -119
-tttttgcgcgcgccg-gcctgggccgggccctcggcggcgcgccgctgctcg :: ::: : : :: : : ::::: : : :::::::::::::: ctcttggcgactgccgagtc-gggcccggccctcggcgcgccgggactcg gcggtggccgctcg-agtgtgcgagcgggcgcgtgtgcgcgggccagggc : : : : : : : : :: : :::::::: : : : : : : : : ::::::::: gctgtagccgcttggagtgtgcgcgcgggcacgtgtgcgcggccccgaga
-69 :::: -70 -20 ::
: : -20
gccgcgcgcgc-gagcccccAGTGTGTGGC :: ::: ::: :::::::::::::::::: gcg-CgcgcgtagaecccccAGTGTGTGGC
10 10
+ Transcription start site
Figure 3. Comparison of the S-flanking regions of the rat and human type I IGFR genes. Nudeotide 1 corresponds to the transcription initiation site (arrow). Homology is shown as dots between identical nucleotides, the dashes represent gaps inserted to a.IIow optimal alignment. There is 75% overall homology. Conserved potential Spl binding sites are underlined, the potential conserved AP-2 site is underlined with a thick line (nucleotides 136 to -129 in the human gene), and the “initiator” sequence is underlined with a double line.
the S-untranslated inhibit
translation.
region with stability in the range of AG = -60 k&/mole Computer
analysis of the S’-untranslated
can severely
region of the human type I
IGFR gene (FOLD, (31)) reveals the potential for significant secondary structures including three hairpin
throughout
structures with free energies greater than AG = -60 kcal/mole the S-untranslated
region.
Another 1118
located
unusual feature that is found in the
Vol.
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3, 1991
S-untranslated
BIOCHEMICAL
AND
region of the type I IGFR
frame upstream of the actual translation at nucleotide translation
741 with an ATG initiator
mRNAs,
RESEARCH
COMMUNICATIONS
gene is the presence of a short open reading
start site.
This 84-bp open reading frame begins
codon that conforms to the requirements
(26) and terminates
of a strong
prior to the major coding sequence.
occurs in a small subset of cellular mRNAs oncogene
BIOPHYSICAL
as well as mRNAs
that again includes a remarkable for other genes requiring
carefully
This motif number of regulated
expression (32). The presence of a short upstream open reading frame may regulate gene expression by reducing translation observed for the rat ornithine
of the downstream open reading frame (26), as has been
decarboxylase gene (28).
The 5’-region of the human type I IGFR gene is very similar to that of the rat IGFR gene (10). There is 75% homology in the 5’-flanking in the 5’-untranslated conservation
regions.
to this high overall homology, there is marked
of sequence at many of the potential
Many of the potential 5’-untranslated potential
In addition
Spl binding
regions.
regions (Fig. 3), and 85% homology regulatory
sites are conserved in both the S-flanking
The potential
AP-2 binding site in the 5’-flanking
thyroid response element in the 5’untranslated
is almost
complete
transcription
initiation
homology
over a large segment
site, including
In summary, characterization human type I IGFR
sites found in these genes. region and the
region are also conserved. There of the region
the putative “initiator” of the nucleotide
and the
surrounding
the
sequence.
sequence of the 5’-region
of the
gene has provided further evidence that this gene is highly regulated.
Prior in vitro and in viva studies have shown that type I IGFR
expression is stimulated
by
a number of factors (6-9) and is regulated during development
in a tissue-specific manner
(5). The present work identifies a number of potential sites and mechanisms through which this regulation
may be achieved. Many of these regulatory elements are conserved between
the human and the rat genes, further supporting expression the type I IGFR gene is important
Acknowledements: We thank Haim Sierra for her technical assistance. Fellowship (DWC), the American (Cl’& Jr.), the Genentech Clinical (SJC).
the hypothesis that regulation
in the control of hormonally
of the
mediated growth.
Werner for his helpful discussions and Maria de la Luz This work was supported by the Harriet Lane Pediatric Diabetes Association Washington, D.C. area affiliate Scholar Award (SJC), and NIDDK grant R29 DK38542
REFERENCES 1.
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Conover, CA., Rosenfeld, R.G. and Hintz, R.L. (1987) J. Cell. Physiol. 133, 560-566. Van Wyk, J.J., Graves, DC., Case&, S.J., and Jacobs, S. (1985) J. CIin. Endo. Metab. 61, 639-643. Daughaday, W.H. and Rotwein, P. (1989) Endocr. Rev. 10, 68-91. Werner, H., Woloschak, M., Ada.mo, M., Shen-Orr, Z., Roberts, C.T., Jr. and LeRoith, D. (1989) Proc. Natl. Acad. Sci. USA. 86, 7451-7455. Ada&i, E.Y., Resnick, C.E., Svoboda, M.E. and Van Wyk, J.J. (1986) J. Biol. Chem. 261, 3923-3926. Ghahary, A. and Murphy, L.J. (1989) Endocrinology 125,597-604. Matsuo, K., Yamashita, S., Niwa, M., Kurihara, M., Harakawa, S., Izumi, M., Nagataki, S. and Melmed, S. (1990) Endocrinology 126,550-554. Ota, A, Shen-Orr, Z., Roberts, C.T., Jr. and LeRoith, D. (1989) Mol. Brain Res. 6, 69-76. Werner, H., Stannard, B., Bach, M-A., LeRoith, D. and Roberts, CT., Jr. (1990) Biochem. Biophys. Res. Common. 169, 1021-1027. Sambrook, J., Fritsch, E.F. and Maniatis, T. h4oZecuZurdoning: a laboratory manual, Cold Spring Harbor:Cold Spring Harbor Laboratory Press, 1989. Ed. 2 Lowe, W.L., Jr., Roberts, C.T., Jr., La&y, S.R. and LeRoith, D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8946-8950. Seino, S., Seino, M., Nishi, S. and Bell, G.I. (1989) Proc. NatI. Acad. Sci. U.S.A. 86, 114-118. Shier, P. and Watt, V.M. (1989) J. Biol. Chem. 264, 14605-14608. Smale, S.T. and Baltimore, D. (1989) Cell 57, 103-113. Mitchell, P.J. and Tjian, R. (1989) Science 245, 371-378. Mamula, P.W., Wong, KY., Maddux, BA, McDonald, A.R. and Goldfine, I.D. (1988) Diabetes. 37, 1241-1246. Ishii, S., Xu, Y.H., Stratton, R.H., Roe, B.A., Merlino, G.T. and Pastan, I. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 49204924. Sehgal, A., Patil, N. and Chao, M. (1988) Mol. Cell. Biol. 8, 3160-3167. Means, A.L. and Far&am, P.J. (1990) Mol. Cell. Biol. 10, 653-661. Imagawa, M., Chiu, R. and Karin, M. (1987) Cell 51,251-260. Roesler, W.J., Vandenbark, G.R. and Hanson, R.W. (1988) J. Biol. Chem. 263, 9063-9066. Glass, C.K., France, R., Weinberger, C., Albert, V.R., Evans, R.M. and Rosenfeld, M.G. (1987) Nature 329, 738-741. Chatterjee, V.K., Lee, J.K., Rentoumis, A. and Jameson, J.L. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9114-9118. Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8148. Kozak, M. (1989) J. Cell Biol. 108, 229-241. Kozak, M. (1989) Mol. Cell. Biol. 9, 5134-5142. Manzella, J.M. and Blackshear, P.J. (1990) J. Biol. Chem. 265, 11817-11822. Grens, A. and Scheffler, I.E. (1990) J. Biol. Chem. 265, 11810-11816. Rao, C.D., Pech, M., Robbins, KC. and Aaronson, S.A. (1988) Mol. Cell. Biol. 8, 284-292. Zuker, M. and Stiegler, P. (1981) Nucleic Acids Res. 9, 133-148. Kozak, M. (1986) Cell 47,481483.
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ANALYSIS INSULIN-LIKE
OF THE HUMAN GROWTH
FACTOR
PROMOTER
1113-1120
TYPE I RECEPTOR
REGION
David W. Cooke’*, Laura A. Banker-t’, Charles T. Roberts, Jr?, Derek LeRoith’, ‘Department
and Samuel J. Casella’
of Pediatrics, Johns Hopkins University
Medicine,
CMSC 3-110, Baltimore,
2Diabetes Branch, Building
School of
MD 21205
10, Room 8S-243, National
Institutes
of Health, Bethesda, MD 20892 Received
May 7, 1991
SUMMARY: We isolated genomic fragments containing the 5’ region of the human type I insulin-like growth factor receptor gene. A unique transcription start site was identified, defining a 1038 bp 5’untranslated region. No TATA or CCAAT elements were identified in the proximal 480 nucleotides of 5’-flanking region. The region surrounding the transcription start site was similar to a recently described “initiator” sequence. The 5’-flanking and S-untranslated regions were highly GC-rich, with numerous potential Spl binding sites. A potential AP-2 binding site was identified in the 5’-flanking region and a potential thyroid response element was identified in the 5’untranslated region. The 5’ region of the human gene was very similar to that of the rat gene, with conservation of many 0 1991Academic Press. Inc. of the potential regulatory elements.
The
type
heterotetramer
I insulin-like
growth
consisting of two extracellular
domain and two intracellular
receptor
(IGFR)
is a disulfide-linked
(a) subunits that contain the ligand binding
(a) subunits that contain tyrosine kinase activity (1). The
receptor is homologous
to the insulin receptor and other members of the tyrosine kinase
family (1). Insulin-like
growth factor I (IGF-I)
although in some circumstances IGF-II IGFR.
l
factor
(2) and insulin (3) may also stimulate
IGF-I mediates the growth-promoting
Corresponding
is the primary ligand of the type I IGFR, the type I
properties of growth hormone (4). IGF-I and
author.
Abbreviations: IGFR, insulin-like growth factor receptor; IGF-I, insulin-like I; IGF-II, insulin-like growth factor II; AP-2, activator protein 2.
1113
growth factor
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in anv form reserved.
Vol.
177,
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the homologous
IGF-II
synthesized in multiple
BIOCHEMICAL
circulate
AND
BIOPHYSICAL
in serum in relatively
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high concentration
and are
tissues (4), suggesting that they act through both endocrine and
paracrine or autocrine mechanisms. The response of a given cell type to the IGFs may depend on the number receptors at the cell surface. The rat type I IGPR gene is developmentally tissue-specific manner (5). Furthermore, hormone
a number of factors, including
(6), estrogen (7), thyroid hormone
IGFR expression.
Finally, the proximal
recently characterized
regulated in a
follicle-stimulating
(8), and phorbol esters (9), stimulate
promoter
of
type I
region of the rat type I IGFR gene was
(lo), and was found to show similarities
to promoters
regulated genes. These findings all support the hypothesis that type I IGFR
of highly
expression is
carefully regulated. As a first step in defining the regulatory
elements that control the expression of the
human type I IGFR gene, we have isolated and characterized genomic fragments containing the 5’-flanking nucleotide
region of the human type I IGFR
gene.
sequence of the human type I IGFR promoter
In this study, we present the region, define the transcription
start site, and compare the regulatory regions of the human and rat type I IGFR genes.
MATERIALS
AND METHODS
A human chromosome 15 genomic library (#57737, American Type Culture Collection, Rockville, MD) was screened using an oligonucleotide encoding amino acid residues 155-170 of the type I IGFR Q subunit as published by Ullrich et al. (1). A 1.7-kb Hind III fragment (R737P2) was isolated that contained 538 bp of exonic sequence encoding amino acid residues 3-183 of the type I IGFR flanked by intronic sequences. The genomic clone R737P2 was then used to isolate a 1.6-kb cDNA (IGFRa) from a human foreskin fibroblast Xgtll library (kindly provided by Drs. Frank French and David Joseph, Laboratories for Reproductive Biology, University of N. Carolina at Chapel Hill). Clone IGFRa contained a 310-bp 5’-untranslated region that extended upstream from the published sequence of Ullrich et al (1). A fragment of IGFRa containing the sequence 5’ to the defined exon/intron boundary was then used to isolate a 3.5-kb Hind III genomic clone (R737P3) from the human genomic library (ATCC #57737); this clone contained the 5’-flanking region and first exon of the type I IGFR gene. After restriction mapping of the cloned inserts, the appropriate fragments were subcloned into pTZ vectors (U.S. Biochemical Corp., Cleveland, OH) for sequencing by the dideoxynucleotide chain termination method using modified T7 polymerase (Sequenase, U.S. Biochemical Corp., Cleveland, OH). The sequence was confirmed by sequencing both strands of the clones. Total RNA was extracted from freshly frozen human placenta by homogenization in guanidinium thiocyanate and pelleting through a cesium chloride cushion (11). Polyadenylated (poly (A)+) RNA was then isolated by afhnity chromatography on an oligo(dT) cellulose column. The flow-through of the oligo(dT) colunm was retained as a control (poly (A)‘ RNA). A 17-base oligonucleotide complementary to nucleotides 56 to 72 of the 5’-untranslated region of the human type I IGFR gene was end-labeled with 32P and purified on an 8% polyacrylamide/8M urea gel. Approximately 2 x lo6 dpm of the 1114
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AND
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labeled probe were hybridized with 24 bg poly (A)+ RNA, 660 pg poly (A)- RNA (flowthrough of the oligo (dT) column), or 100 pg tRNA (Sigma, St. Louis, MO) for 15 hrs at 30°C. After ethanol precipitation, the samples were resuspended in 20 ~1 reverse transcriptase buffer containing 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl,, 10 mM dithiotreitol, 1 U/cl1 RNA@ and 1 PM of each dNTP. Moloney Mmine Leukemia Virus reverse transcriptase (5OU, Bethesda Research Laboratories, Gaithersburg, MD) was added and the reaction was incubated for 2 hours at 37’C. The reaction was terminated by the addition of 1 ~1 of 0.5M EDTA and 5 pg of RNAase (Sigma, St. Louis, MO). After 30 minutes at 37°C the extended products were extracted with phenol:chloroform, ethanolprecipitated, and resolved on an 8% polyacrylamide/8M urea gel. Radiolabeled antisense RNA probes were transcribed from SmaI-BarnHI, BarnHI-Sac1 and SacI-BspIvIII genomic fragments. Together, these fragments were complementary to 128 bases of 5’-flanking region and the entire 1038 bases of 5’-untranslated region, RNase protection assays were performed as previously described (12) using RNA prepared from human placenta to protect the riboprobes. RESULTS
AND DISCUSSION
We have isolated genomic clones that contain the S-flanking
region and first two
exons of the human type I IGFR gene. The first two introns of the human type I IGFR gene, predicted by comparing
the genomic clones with the cDNA sequence, interrupt
codons for amino acids 2 (isoleucine) IGFR.
Therefore,
and 184 (methionine)
the
of the (Ysubunit of the type I
these first two introns are located in positions homologous
to the first
two introns of the insulin receptor (13) and the recently identified
insulin receptor-related
receptor (14). Clone R737P3 extends 1518 bp 5’ to the translation
start site. To define the
Figure 1. Mapping of the 5’ end of the human type I IGFR mRNA by primer extension. Poly (A)+ RNA from human placenta was analyzed, with poly (A)- human placenta and yeast tRNA as controls. The three right hand lanes contain the primer-extended cDNAs obtained from the indicated RNA sample. The sequence ladder was obtained by using the primer extension oligonucleotide as a DNA sequencing primer on the appropriate template. Therefore, the size of the extended product indicates the transcription start site (arrow). 1115
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point of transcription
initiation
a 17-base oligonucleotide
complementary
to transcription
BIOPHYSICAL
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we analyzed human placenta RNA by primer extension using
region of the human type I IGFR corresponding
AND
to nucleotides
56 to 72 of the S-untranslated
gene. The resulting product was 72 bp in length (Fig. l), initiation
at nucleotide
1 (Fig. 2). An RNase protection
assay, using an antisense RNA probe that was generated from a genomic sequence that -480
tttcaagaaccggggaaacgcgctttccagccgcgctgttgttgttttcaatgaacctct
-420
cccagccccgcactccccacccacccctcccctctcctgcccacccctcccctctcctgc
-360
ccacccctcccctgcctagcctttccctggctacccacccct~cccc~ccgagaccggac
-300
cggcgg=ggggg==ttgtttttggagtc~=gggg=gggcgcgtgcgggtggccg
-240
gcgcagtgcggtggnggcgggagcgggtgggacgcgggtgggacgcgcgcgtgtctctgtgtgcgcgcggg
-180
aggcggtnagecagat~e~ece~cgcctcgcagtctcgcgccccacgcccgggctc
-120
cggttttttgcgcgcgccggcctgggccgggccctcggcgcgccgctgctcggcggtggc
-60
cgctcgagtgtgcgagcgggcgcgtgtgtgccagggcgccgcgcgcgcga~ 1 AGTGTGTGGCAGCGGCGGCGGCGGCGCGGCGAGGCTGGGGCTCTTGTTTACCAGCATTAA
61
CTCGCTGAGCGGAGGGAAAAAA
CCCGAGGAGGAGCGAGCGCACCAGGCGAAC
121
TCGAGAGAGGCGGGAGAGCGAGAGGGACGCCGCCAGCGAGCCTGCCCACGGCCGGCGCTC
181
GCAGACCCTCGGCCCCGCTCCCCGGATCCCCCCGCGCCCTCCACGCCCCTCCCGCGCGGG
241
GGCAGCTCCACGGCGCGCCTCGCCTCGGCTGTGACCTTCAGCGAGCCGGAGCCCCCGCGC -----------
301
AGAGCAGGCGGCGGCGGGCGGGGGCCGGGCGGGGGCCGGCGCGGGGCGGGCGGCGGCGCA
361
GAGCCGGGCGGCGCGGCGGGAGTGCTGAGCCGGCGGGCCGGCCCGCCGCTTTGTGTGTGT
421
CCTGGATTTGGGAAGGAGCTCGCGGCGGCGGCGGCGCTGAGGGAGGAGGCGGCGGCGAGC
481
GGAGCCAGGAGGAGGAGGAGGAGGAGGGGGAGCCGCTCATTCATTTTGACTCCGCGTTTC
541
TGCCCCTCGCCGGCCTCGCCTGTGACCCGGACTTCGGGGCGATCTTGCG~CTGCGTCGC
601
GCCCTCCCGCGGCGGAAGCTCGGGCGTCCGGCCGCCTCCCGCGCGCCAGGGCCGGGCTTG
661
TTTTTCCTCGCCTAGGCAGATTTGGGCTTTGCCCCCTTTCTTTGCAGTTTTCCCCCCTTC
721
CTGCCTCTCCGGGTTTGATGGAGGCCGACGACGCCGACAGCCCGCCCCGGCGCGCCT ---------~------_
781
CGGGTTCCCGACTCCGCCGAGCCCTGGGCCGCTGCTGCCGGCGCTGAGGGGCCGCCCCGC ------------------
841
GCCGCCCGCCCCGTCCGCGCACCCGGAGGGCCCCGGCGGCGGCCCTTCGGAGTATTGTTT
901
CCTTCGCCCTTGTTTTTGGAGGGGGAGCGAAGACTGACTGAGTTTGAGACTTGTTTCCTTTCAT
961
TTCCTTTTTTTCTTTTCTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTGAG~GGGGATT
1021
TCATCCCAAATAAAAGGATCTGGCTCCGGAGGAGGGTCCCCGACCTCGCTGTGG
1081
GGGCTCCTGTTTCTCTCCGCCGCGCTCTCGCTCTGGCCGACGAGTGGAG~
Figure 2. Nucleotide sequence of the 5’ region of the human type I IGPR gene. Nucleotide 1 corresponds to the trauscription start site. The ATG codon that initiates translation is underlined, beginniug at nucleotide 1039. The sequence ends at the end of the first exon. Potential Spl binding sites are underlined. A potential AP-2 binding site is underlined by a thick line (nucleotides -136 to -129). The Ynitiatoi’ sequence surroundiug the transcription start site is underlined with a double hue. A potential thyroid response element is underlined with a thick dashed line (266 to 277). The open reading frame in the S-uutrauslated region is underlined with a dashed line. 1116
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BIOCHEMICAL
AND
extended 128 bases upstream from this initiation confirmed the location of the transcription that were complementary Y-untranslated
BIOPHYSICAL
RESEARCH
site, produced a single protected band that
start site at nucleotide 1. Antisense RNA probes
to nucleotides
204 to 1056 (covering the remainder
region) were fully protected, confirming the single transcription
(data not shown). Thus, the R737P3 clone contains 480 nucleotides and a 1038-bp S-untranslated 5’-flanking
COMMUNICATIONS
region.
No TATA
or CCAAT
of the
initiation
of 5’-flanking
site region
elements are found in the
region, despite the fact that there is a single specific transcription
initiation
site.
However, the region between nucleotides -6 to + 11 is very similar to the recently described “initiator” element
sequence that directs specific transcription
initiation
in the absence of a TATA
(15). Both the S-flanking
and S-untranslated
regions are highly GC-rich, with 75% and
68% GC content each. There are numerous potential The type I IGFR promoter
Spl binding sites (16) in both regions.
is therefore similar to the promoters of many housekeeping
cellular growth control genes that lack TATA
elements and are GC-rich,
insulin receptor (17), EGF receptor (18), and NGF receptor (19) promoters. sites have been shown to interact with the “initiator” transcription
including
sequence to direct high levels of initiation
element (20). A potential binding site for the transcription
AI’-2 is found at nucleotides
the
Spl binding
from a single start site (IS), and may be necessary for transcription
in genes lacking a TATA
and
-136 to -129 in the 5’-flanking
factor
region. The AP-2 element can
mediate both cyclic AMP and phorbol ester control of transcription
(21,22), and its presence
in the type I IGFR gene may explain previous observations that cyclic Ah4P (6) and phorbol esters (9) stimulate type I IGFR expression, and this may be the site through which folliclestimulating
hormone regulates IGFR
the 5’-untranslated response element
expression (6). The region from bases 266 to 277 in
region includes one-half of the palindromic and is similar
sequence of the thyroid
to thyroid response elements found in the rat growth
hormone gene (23) and the human TSH a gene (24). The presence of this potential response elements supports the observation that thyroid hormone expression (S), and suggests a mechanism whereby this regulation
thyroid
regulates type I IGFR can occur at the level of
transcription. The type I IGFR gene is unusual in that the 5’-untranslated Interestingly,
many of the rare genes that have long S-untranslated
involved in the regulation
of cellular proliferation,
be examined,
regions seem to be
including growth factors and many of the
cellular oncogenes (25). The role of these long 5’-untranslated translation
region is extremely long.
regions is just beginning to
but there is growing evidence that they are involved in the regulation
of
(26-28). Kozak (27) and others (28-30) have shown that secondary structures in 1117
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HUEII-I Rat Human Rat Human
AND
BIOPHYSICAL
RESEARCH
ttttcaagaaccggggaaacgcgctttccagccgcgctgttgttgttttc :: : : ::: tt--------ccggg--.--------------------------------
-432 -405
aatgaacctctcccagccccgcactccccgcccacccctcccctctcctg : : :: :: :::::::::::: ------------------ccgcactccccgcccaccgcgcccg------cccacccctcccctctcctgcccacccctcccctgcctagcctttccctg :: ::: ::: ::::
-382 : ::: -379 -332 ::::
Rat
--cagcccgccc-----.-gccc----------tgcc--tg==--g~=--------
Human
gctacccacccctgccccgccgagaccggaccggcggcgggggcattgtt :: ::::: : : :: ::: :: : :::::: gc------ccccttggccaccgagtcccg-ccggcgcccggggcattgtt
Rat Human Rat Human Rat Human Rat Human Rat Human Rat Human Rat Human Rat
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-358 -282
: :: : : ::::::: -316
ttt-ggagtcgggcgggaggggagggcgcgtgcgggt--ggccggcgcag : : : : : :::: ::::: : : : : :: :: ::: : :: ttttggagtcctgcgggtggggagggtgcggacagggccggccggcgcag
-235 ::::::::::: -266
tgcgg-tgggggcgggagcgggtgggacgcgcgcgtgtctctgtgtgcgc ::::: ::::::::: :::: :::: : : : : :::::: aagcg--tggg-cgcgcgcgtggctcagtgtgcgc tgcgggtgegagCgg gcgggaggcggtggggcgggagat--gggggcggcgcctcgcagtctcg..,.... . . . : ::: : :: :::: :::::::: gcgggggccggcgcgggtggaggcgc~tgcctcctgggcccgg
:::
-186 :::
:::::::: -219 -139
:::::
: : :: -169
--gt--------------------cgccccacgcccgggctccg----::::::::::: :::::: ctccccacgcccgcgctccgtccgcacgtccctgcgatcccgaactccgg
-11-J -119
-tttttgcgcgcgccg-gcctgggccgggccctcggcggcgcgccgctgctcg :: ::: : : :: : : ::::: : : :::::::::::::: ctcttggcgactgccgagtc-gggcccggccctcggcgcgccgggactcg gcggtggccgctcg-agtgtgcgagcgggcgcgtgtgcgcgggccagggc : : : : : : : : :: : :::::::: : : : : : : : : ::::::::: gctgtagccgcttggagtgtgcgcgcgggcacgtgtgcgcggccccgaga
-69 :::: -70 -20 ::
: : -20
gccgcgcgcgc-gagcccccAGTGTGTGGC :: ::: ::: :::::::::::::::::: gcg-CgcgcgtagaecccccAGTGTGTGGC
10 10
+ Transcription start site
Figure 3. Comparison of the S-flanking regions of the rat and human type I IGFR genes. Nudeotide 1 corresponds to the transcription initiation site (arrow). Homology is shown as dots between identical nucleotides, the dashes represent gaps inserted to a.IIow optimal alignment. There is 75% overall homology. Conserved potential Spl binding sites are underlined, the potential conserved AP-2 site is underlined with a thick line (nucleotides 136 to -129 in the human gene), and the “initiator” sequence is underlined with a double line.
the S-untranslated inhibit
translation.
region with stability in the range of AG = -60 k&/mole Computer
analysis of the S’-untranslated
can severely
region of the human type I
IGFR gene (FOLD, (31)) reveals the potential for significant secondary structures including three hairpin
throughout
structures with free energies greater than AG = -60 kcal/mole the S-untranslated
region.
Another 1118
located
unusual feature that is found in the
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S-untranslated
BIOCHEMICAL
AND
region of the type I IGFR
frame upstream of the actual translation at nucleotide translation
741 with an ATG initiator
mRNAs,
RESEARCH
COMMUNICATIONS
gene is the presence of a short open reading
start site.
This 84-bp open reading frame begins
codon that conforms to the requirements
(26) and terminates
of a strong
prior to the major coding sequence.
occurs in a small subset of cellular mRNAs oncogene
BIOPHYSICAL
as well as mRNAs
that again includes a remarkable for other genes requiring
carefully
This motif number of regulated
expression (32). The presence of a short upstream open reading frame may regulate gene expression by reducing translation observed for the rat ornithine
of the downstream open reading frame (26), as has been
decarboxylase gene (28).
The 5’-region of the human type I IGFR gene is very similar to that of the rat IGFR gene (10). There is 75% homology in the 5’-flanking in the 5’-untranslated conservation
regions.
to this high overall homology, there is marked
of sequence at many of the potential
Many of the potential 5’-untranslated potential
In addition
Spl binding
regions.
regions (Fig. 3), and 85% homology regulatory
sites are conserved in both the S-flanking
The potential
AP-2 binding site in the 5’-flanking
thyroid response element in the 5’untranslated
is almost
complete
transcription
initiation
homology
over a large segment
site, including
In summary, characterization human type I IGFR
sites found in these genes. region and the
region are also conserved. There of the region
the putative “initiator” of the nucleotide
and the
surrounding
the
sequence.
sequence of the 5’-region
of the
gene has provided further evidence that this gene is highly regulated.
Prior in vitro and in viva studies have shown that type I IGFR
expression is stimulated
by
a number of factors (6-9) and is regulated during development
in a tissue-specific manner
(5). The present work identifies a number of potential sites and mechanisms through which this regulation
may be achieved. Many of these regulatory elements are conserved between
the human and the rat genes, further supporting expression the type I IGFR gene is important
Acknowledements: We thank Haim Sierra for her technical assistance. Fellowship (DWC), the American (Cl’& Jr.), the Genentech Clinical (SJC).
the hypothesis that regulation
in the control of hormonally
of the
mediated growth.
Werner for his helpful discussions and Maria de la Luz This work was supported by the Harriet Lane Pediatric Diabetes Association Washington, D.C. area affiliate Scholar Award (SJC), and NIDDK grant R29 DK38542
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