From the Department of Biochemistry and Nutrition, Medical School, Université Libre de Bruxelles, Building G/E, CP 611, 808 Route de Lennik, B-1070 Brussels, Belgium
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ABSTRACT |
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We cloned the 5' upstream region of the rat
glucagon receptor gene, demonstrating that the 5' noncoding domain of
the glucagon receptor mRNA contained two untranslated exons of 131 and 166 nucleotides (nt), respectively, separated by two introns of 0.6 and 3.2 kilobase pairs. We also observed an alternative splicing involving the 166-base pair exon. Cloning of up to 2 kilobase pairs of
the newly identified genomic domain and transfection of various
constructs driving a reporter gene, in pancreatic islet cell line
INS-1, uncovered a strong glucose regulation of the promoter activity
of plasmids containing up to nucleotide The primary physiological role of glucagon, together with insulin,
is maintenance of normal glycemia. The liver has a central role in
handling absorbed nutrients and in the regulation of hepatic glycogen
disposability; this requires high density of glucagon receptors in the
liver (for review see Refs. 1 and 2). The glucagon receptor mRNA
has also been detected at variable levels in other tissues such as
heart, kidney, adrenal gland, and adipose tissue (3-5), as well as in
pancreatic islets, especially in B cells (6, 7). The expression of the
glucagon receptor mRNA is stimulated by glucose and inhibited by
cyclic AMP, both in liver (8) and in cultured endocrine cells (9).
Consequently, the promoter of the glucagon receptor gene could contain
regulatory elements for these factors. It has been recently suggested
by Burcelin et al. (10) that the glucose regulation of the
glucagon receptor mRNA level differs from the glucose regulation of
other genes such as Glut2, and might be mediated by triose metabolites, suggesting the existence of new enhancer sequences. To address this
question, a prerequisite is the precise knowledge of the complete 5'
mRNA sequence. For the glucagon receptor mRNA, there was an
ambiguity; the glucagon receptor cDNA has been first cloned (from
rat) simultaneously by us (11, 12) and by Jelinek et al.
(13). Our sequence (GenBank accession number L04796) was almost
identical to that of Jelinek (GenBank accession number M96674) except
for the 5' end; the first 25 nt1 in our sequence
(corresponding to positions General Methods--
Rat liver and heart fragments were
homogenized in 4 M guanidine isothiocyanate, and total RNA
was purified on CsCl gradient (15); poly(A) RNA was purified on
oligo(dT)-cellulose (Invitrogen).
Reverse transcription was performed using Superscript IITM (Life
Technologies, Inc.). Anchored PCR of the 5' end was performed after
single-strand ligation of phosphorylated oligonucleotide to cDNA
using the 5' RACE kit (CLONTECH). Subcloning of PCR
product was carried out by TA ligation to the pCRII plasmid (Invitrogen).
Sequencing of plasmids purified on QiagenTM columns was performed with
the Sequenase or Thermosequenase kit (U.S. Biochemical Corp; Amersham).
Polymerase chain reaction was performed as indicated in the legend of
figures. Analysis of amplified DNA fragments on agarose gel,
transferred to nitrocellulose filters and hybridized to
5'-32P-labeled oligodeoxynucleotide was, performed as
detailed by Sambrook et al. (15).
Genomic Cloning and Construction of Reporter Gene
Plasmids--
Rat genomic DNA, partially digested with
Sau3AI (15), was cloned in XhoI-digested and
partially filled
In a second screening we used 32P-labeled oligonucleotides
J.for and S.for (described below). One positive
An oligonucleotide probe based on the 5'end of this fragment was used
as a probe to subclone a 2-kb PstI/SacI fragment
in pBluescript SK+ resulting in clone "P/S." The two subcloned
fragments contain a 130-bp overlap with a unique XbaI site.
We ligated a HindIII/XbaI fragment of clone P/S
with a XbaI/XbaI fragment of the clone B/B in the
pBlCat6 vector digested with HindIII/XbaI. The
sequence of this constructed plasmid (called clone P/ctr/B) was
deposited in GenBank under accession number U63022.
In the first transfection experiments, we used clones B/B and P/ctr/B.
These plasmids gave only a low reporter gene activity in transfected
cells (see below); these plasmids bear approximately 1 kb of sequence
situated downstream from the mRNA start point. This intervening
sequence may prevent the efficient transcription of the reporter gene.
We therefore prepared shortened plasmids by deletion of the sequence
located immediately after the end of the first untranslated exon, using
a unique ApaI site.
The clone B/B was digested by ApaI and XhoI,
blunted and ligated to give clone "pCB." The clone P/ctr/B was
digested by ApaI and NotI, blunted and ligated to
give the clone "pCC". The latter clone contained a XhoI
site located 1 kb upstream from its 3'end (ex-ApaI site),
and a second XhoI site in the polylinker, so that XhoI digestion excised a 1-kb insert, which was subcloned in
XhoI site of pBlCat6 vector in both its normal and reverse
orientation (clones pCX and pCXi).
The clone pCC was digested by BamHI, and a 0.5-kb fragment
was removed; the plasmid was then recirculated by ligation. The first
BamHI site is in position Mutations--
Mutations were introduced using the QuickChange
site-directed mutagenesis kit from Stratagene according to
manufacturer's instruction. The principal is based on Pfu
amplification of the whole plasmid and digestion of nonmutated DNA
using DpnI digestion. DpnI does not digest
unmethylated, Pfu-amplified DNA.
We used as forward primer GCACGTGTGACAGTTGCAATCTTTC for the
preparation of pTkCd-m1 clone, and
CCACACACGGTGGTCGTGTGAGTGCTGCAATCTTTC as forward
primer for the preparation of pTkCd-m2 clone. Clone pTkCd-m1 carries a
single nucleotide change. Clone pTkCd-m2 contains a double mutation of
the two first nucleotides of each palindromic E-box (change of four nucleotides).
We used CTTTGCTCCCACACACGGTGCAATCTTTCACCCAGGAGCC as
forward primer to produce the pCX Transfection Studies--
Insulin-secreting cell line INS-1 was
kindly provided by Prof. Wollheim (University Medical Center,
Geneva, Switzerland). These cells were cultured in modified RPMI
1640 as described previously (16). Cell culture material and media were
from Life Technologies, Inc. Transfections were usually performed on
cells 4-5 days after seeding in six-well plates, in order to achieve
roughly 60% cell confluence. The day before transfection, the usual
cell medium was replaced with by RPMI 1640 medium with a low glucose
concentration (5 mM instead of 11 mM). The
cationic liposome Dosper (Boehringer Mannheim) was used according to
the manufacturer's instructions. In standard transfections, we used
1.5 µg of plasmid DNA and 10 µl of Dosper reagent/35-mm diameter
well. After 5 h, the transfection medium was replaced by RPMI 1640 medium, with variable glucose concentrations.
In several experiments, the transfection yield was estimated by
cotransfection with 0.5 µg of pcDNA3.1/his/lacZ (Invitrogen). Galactosidase activity was quantified on 2% of the cell extract using
o-nitrophenyl-
Chloramphenicol acetyltransferase (CAT) activity of transfected cells
was assayed by acylation of [14C]chloramphenicol (ICN)
using n-butyryl-CoA (Promega) as indicated in the Promega
protocol. An aliquot of the cell extract from individual wells was used
for CAT assays and TLC analysis. Autoradiographic densities (measure of
"volume" of absorption: integrated optical densities of the spot
surface) were estimated using "Viber-Lourmat" image analysis
apparatus. In control experiments we found a linear correlation between
the amount of CAT in the assay and the "volume" of absorption
measured in the range of 0-125 milliunits. Activity of our samples
from transfected cells never exceeded 100 milliunits of CAT.
Amplification of cDNA Using Primers Based on the Conflictual
Sequences--
Analysis of the 5' end sequence of the glucagon
receptor mRNA was obtained by amplification of rat liver and heart
cDNA using two forward primers J.for and S.for, based on the
conflictual sequences reported in the literature. A forward primer
based on an unambiguous sequence in the 5' end was used as a control.
Fig. 1A shows the amplified
products electrophoretic pattern. The amplification specificity was
confirmed by Southern blotting, using internal oligodeoxynucleotides as probes (data not shown).
The major products were subcloned and sequenced. Obtained sequences,
confirmed later by sequencing products of anchored PCR and by
sequencing cloned genomic DNA (see below), demonstrated that glucagon
receptor mRNAs contain two untranslated exons, and that the second
one could be spliced out. We called the first 5' untranslated exon
(described by Jelinek et al.; Ref. 13) UJ, and the second
alternatively spliced exon (described by Svoboda et al.;
Ref. 11) US. These results are illustrated in Fig. 1B.
Anchored PCR of cDNA 5' Ends (5' RACE)--
To identify the
mRNA transcription start site, and estimate the relative abundance
of these polymorphous glucagon receptor mRNA populations, we
performed 5' end-anchored PCR.
We ligated liver and heart single-stranded cDNA to a phosphorylated
anchor primer, and then used three different reverse primers to amplify
this anchored cDNA. The procedure is detailed in the legend of Fig.
2. Hybridization using the internal
labeled oligonucleotide probe (R-20) allowed us to visualize a major
band with a smear for the liver and two separate bands for the heart
(Fig. 2). The same pattern was observed in the three tested conditions.
Shorter exposure clearly indicated that most of the DNA amplified from liver had the same mobility as the smaller fragment amplified from the
heart. The smaller band corresponded to approximately 350 bp with R147,
and about 160 bp with R-20 reverse primer. The size of the larger
fragment amplified from the heart was in good agreement with the
results expected in the presence of an additional 166-bp unspliced 5'
exon. These results indicated that the average size of the 5'
untranslated end of the glucagon receptor mRNA was approximately
180 bp or 180 + 166 bp.
We subcloned the products of the second step of the anchored PCR into
the pCRII vector, and hybridized recombinant colonies with 5'-labeled
J.for and S.for oligonucleotides. 90% of clones originating from the
liver hybridized with "J.for" only, and did not contain the US exon
(confirmed by sequencing). In contrast, among the 55 colonies
originating from the heart, 21 hybridized with the "J.for" probe
only, 31 (representing the longer form comprising the 166-bp US exon)
hybridized with both "J.for" and "S.for" probes, and 3 colonies
hybridized with "S.for" only and contained truncated US exon
sequences. This result suggested that all transcripts originated
from a single promoter located upstream UJ exon, rather that from two
separate promoters found upstream from each untranslated exon. The
reason for a tissue-specific use of the second US exon remains unclear.
To identify the transcription start point, we subcloned the products of
the second anchored PCR step in the pCRII vector, and sequenced the 5'
end of 30 clones originating from both liver and heart. All the inserts
yielded the same sequence, but starting at various points ranging from
73 to 131 nt upstream from the 3' end of exon UJ (see Fig.
3). These sizes were in good agreement with the average size of the 5' RACE amplified fragments shown in Fig.
2.
A supplement verification of the 5' mRNA length was performed by
two additional independent anchored PCR. Primers were located on exon
UJ, in order to favor the longest 5' transcripts. In a population of 20 clones, we did not find a sequence exceeding the point indicated in
Fig. 3B by "start exon UJ," strongly suggesting that we
had indeed identified the actual mRNA 5'end.
Screening of Rat Genomic Library and Genomic Organization--
We
constructed and screened a bacteriophage
In the second screening, we obtained DNA fragments located up to 5 kb
upstream from the initiation codon (GenBank accession number U63022).
The genomic organization of the whole glucagon receptor gene deduced
from these data is illustrated in Fig. 3A. The comparison of
the rat glucagon receptor genomic sequence with published cDNA
sequences confirmed the existence of two exons in the 5' untranslated
domain. Even our longest 5'-cDNA clones sequences were identical to
the genomic DNA sequence. These clones indicate the putative
transcription start site (Fig. 3B). The experimentally
determined size of 5' domain of cDNA (Fig. 2) is in good agreement
with this position of the start point.
The sequence neighboring the putative start site was in relative
agreement with the consensus sequence of promoters lacking TATA boxes.
The genomic sequence upstream from this putative start site was highly
(G + C)-rich and could correspond to the basal gene promoter. Moreover,
we found at position Reporter Gene Study of the Promoter--
We subcloned the putative
promoter domain of the glucagon receptor gene in the pBlCat6 plasmid as
illustrated in Fig. 4A. For
the transfection studies, we deleted all sequences downstream from the
first exon (UJ). Transfections were performed in INS-1 cells: a cell
line established (16) and kindly provided by Prof. Wollheim. These
cells contain the glucagon receptor mRNA and express functional
glucagon receptors (18). Glucose regulated expression of the glucagon
receptor in these cells was demonstrated by the adenylyl cyclase
stimulation: the activity was higher (225 pmol of cAMP/min/mg of
protein; 4.2-fold stimulation) using membranes from cells grown in the
presence of 20 mM glucose, then with the membranes from
cells grown in the presence of 5 mM glucose (100 pmol of
cAMP/min/mg of protein; 2-fold stimulation). An INS-1 subclone that
expresses low levels of the glucagon receptor and a RIN cell line that
does not express the glucagon receptor were used as controls of
transfection specificity (data not shown).
We studied the glucagon receptor promoter activity and its regulation
by glucose in the INS-1 cells expressing greatly glucagon receptors. We
constructed CAT plasmid variants containing various putative promoter
sequences. The DNA fragments tested are described in Fig. 4. We
observed that the transfection with the short pCB (
In contrast, cells transfected by the longer plasmids pCX (
The shortened plasmid pCP (
We also tested the promoter activity of plasmids bearing inserts in the
reversed orientation (Fig. 6). Plasmid
pCXi (+156 to
We removed the basal promoter (insert of the clone pCB) by
BamHI digestion of the whole promoter domain (plasmid pCC).
We subcloned this genomic fragment (
We realized a glucose activation dose response curve. Average results
are shown in Fig. 8. Most of activity
variation was observed between 5 and 10 mM glucose, with
about half-maximal activity observed at an average of 7.5 mM glucose. These glucose concentrations are within
physiological glycemia range. In addition, the glucose stimulation of
the gene transcription suggests a threshold-like behavior. Indeed,
analysis of individual results indicates that the majority of
experimental points offer a low activity up to 5 mM
glucose, and high activity as of 10 mM glucose. Three
dose-response experiments were performed with pCX plasmid (pBlCat6
derivative) Fig. 8, and one, with similar relative results, with pTkCd
plasmid (pBlCat5 derivative including HSV tk promoter).
We used the Quick Change site-directed mutagenesis kit for the
preparation of several mutated clones. First, we deleted the G-box from
the pCX clone, yielding the pCX We have cloned the 5' upstream domain of the glucagon receptor
gene, we demonstrated that it contains a functional glucose-regulated promoter, and we located its glucose regulatory element. This is the
first time that a G protein-coupled receptor gene promoter regulated by
glucose has been cloned.
Discussion of Gene Organization--
The glucagon receptor belongs
to the type B G protein-coupled receptor family (GPCRs; see GPCR data
base (Ref. 19)). Several genes of this receptor family have been
described, and share a similar coding domain, i.e.
exon/intron organization pattern (14, 20-27). This pattern is distinct
from the exon/intron organization of the other GPCR families.
Two types of 5' untranslated domains, with or without introns, have
been described in this B type receptor family. In other words, the gene
promoter could be located either immediately upstream of the ATG
initiation site, or at a more distal position, such as the promoter of
the porcine calcitonin receptor gene located 20 kb from the ATG
initiation site and separated from this site by two 5' untranslated
exons and two introns (21). The rat PTH receptor possesses two
promoters, located upstream from the first or the third untranslated
exon respectively, with different tissue specificities (22, 23). An
alternative splicing of at least four untranslated exons was described
for the human PACAP receptor (24).
In contrast, the gene promoter is close to the ATG initiation site in
both the human and the rat VPAC1 receptor genes (formerly VIP1 receptor) (25, 26), as well as in the rat GLP-1
receptor gene (27).
Conflicting data have been published for the 5' end of glucagon
receptor gene: Buggy et al. reported the sequence of a
putative human glucagon receptor promoter, separated from the
initiation codon by an undescribed 5-kb intron (28). In contrast,
Burcelin et al. (17) reported the sequence of a putative
mouse glucagon receptor promoter adjacent to the initiation codon.
However, no experimental data demonstrating the promoter activity were
provided. Our results suggest that sequences upstream from the ATG of
the mouse glucagon receptor belong to an intron (85% homology with our
rat intronic sequence). A comparison of our experimentally active rat
promoter with the sequence of the putative human glucagon receptor
promoter indicates that the best significant homology (65% over 120 nt) is located in the vicinity of the mRNA start point. The size of
the human 5' untranslated domain, estimated by primer extension assay,
was larger (475 nt) than in rat (376 nt). The complete 5' sequence of
human gene has not yet been reported (28), but comparison of the rat
and the human glucagon receptor cDNAs shows a high (82%) homology
in the coding domain (29, 30).
Herein we provide evidence for a polymorphism of the 5' untranslated
domain of the rat glucagon receptor mRNA. Indeed, two introns of
0.6 and 3.2 kb place the transcription start site (of exon UJ) at more
than 4 kb from the ATG initiation codon (Fig. 3B). In the 5'
untranslated domain, a 166-bp exon (US) was present between the two
introns. This exon seems to be spliced in the mature mRNA from
liver and less frequently in the heart, but this requires further investigation.
The mRNA 5' end polymorphism could be explained a priori
either by the existence of two alternative promoters or by an
alternative splicing. Two alternative promoters have indeed been
described for the PTH receptor (23) as well as for several genes
involved in the glucose metabolism (31, 32). Our experimental results show that the majority of the mRNA molecules containing the US exon
also contains the UJ exon, suggesting a unique transcriptional initiation domain. Nevertheless, we cannot rule out the existence of an
alternative promoter, active in other tissues expressing the glucagon
receptor, or active only during specific life periods.
The size of the products synthesized by anchored PCR at the 5' end of
the rat cDNA (Fig. 2) yielded an average size of 180-nt untranslated mRNA (without exon US) and of 370 nt (with exon US) (Fig. 2). This size is comparable to that obtained by sequencing, supporting that most transcription start at either 210 or 376 nt
(without or with exon US) upstream from the ATG. However, other (downstream) putative start positions are also observed. This situation
is similar for the PTH receptor (22) or GLP-1 receptor (27) genes, in
which multiple start points are described. A cDNA with multiple
start points is generally observed for genes without TATA boxes but
with GC-rich domain in the basal promoter, which is the case for all
genes described to date for this receptor family.
However, we cannot rule out a gene organization similar to the PTH
receptor gene; this would imply existence of a third and very short
third 5' untranslated exon together with the existence of two basal
promoters, located, respectively, upstream from the first and second
exon (23). Indeed, the sequence TCTGGAAAGTTTGCAGG, located 9 nucleotides downstream of the 5'-most mRNA experimental start point
of, is, according to the GeneID program (Ref. 33, geneind{at}darwin.bu.edu), a potential acceptor splicing site. Thus, few nucleotides upstream may represent the third 5'exon, present on a
still unsequenced genomic fragment. In this hypothesis, the genomic
sequence upstream from the start point would have a basal promoter
activity (becoming a second basal promoter as for the PTH receptor
gene; Ref. 23), and the G-box would be located on the first intron.
Such a location of a glucose regulatory element in the first intron has
been described for the fatty acid synthase gene (see below).
Discussion of Glucose Regulation--
Several gene promoters are
up-regulated by increasing of extracellular glucose concentration.
Glucose activation of two genes expressed in the liver, pyruvate kinase
L (PKL) and Spot14 (a lipogenesis-associated protein) have been
extensively studied. It appears that glucose stimulates the
transcription of these two genes via similar motifs present in the
promoter. These carbohydrate response elements were called GIRE (34) or
ChoRE (35) for the PKL or Spot14 genes, respectively. The core motif in
these elements is the so called E-box, constituted by the palindromic sequence CACGTG, the consensus recognition sequence of transcription factors belonging to the c-Myc family. Glucose regulation of PKL gene
uses two imperfect E-boxes, separated by 5 nt, forming a perfect
palindrome coined L4 box: CACGGGn5CCCGTG (36). A
similar feature is required for glucose activation of the Spot14 gene: CACGTGn5CCCGTG (37). The spacing distance of 5 nt between these two E-boxes was shown to be essential for its activity
(37). It is noteworthy that the L4 box found in the pyruvate kinase L
gene is active in INS-1 cell line, whereas neither the proinsulin I nor
the glucokinase mRNA levels are increased under the same conditions
(38).
These glucose regulatory elements have different locations in the
different promoters: located only at
We observed that the sequence motif
tgCACGTGtgaCAGCTGca in
the promoter domain of the glucagon receptor located at
The E-boxes found in our G-box motif are more palindromic than in the
L4 box and in the L4-like glucose regulatory elements described
previously. The palindromic nature of the E-boxes seems to be important
for the glucose regulation; mutations that increase the palindromic
nature of the second E-box of the L4 box in the Spot14 promoter
strongly increase its glucose-stimulated enhancer activity (37).
As this enhancer is palindromic, it is not surprising that the glucose
stimulation was also observed in the reverse orientation (Fig. 6).
Absolute activity of the reversed plasmids is lower, which is expected,
as in this orientation the normal basal promoter is also in the reverse
orientation. The transcription start sites were not investigated for
reverse orientation plasmids. Consequently, we did not determine which
domain acts as a basal promoter in these plasmids. However, a GC-rich
domain may act as a promoter in the reverse orientation (40), and a
similar glucose regulatory element may act as enhancer downstream from
the transcription start site (39).
Recently, USF-2 (upstream stimulatory factor 2) was shown to be
directly involved in the glucose-induced stimulation of PKL and Spot14
gene transcription (41, 42). USF-2 is a member of the c-Myc family of
transcription factors characterized by helix-loop-helix/leucine zipper
domains. However, another transcription factor may act on the glucagon
receptor G-box including a 3-nt spacing; indeed, the 5 nt inserted
between the two E-boxes are essential for the previously described L4
box activation (37).
It was clearly demonstrated that, in a first time, glucose has to
be phosphorylated to glucose 6-phosphate before the stimulation of the described genes occurs (38, 43). According to Doiron et
al. (44), an intermediate of the nonoxidative branch of pentose phosphate pathway, the xylulose 5-phosphate, mediates the glucose signal to the transcriptional machinery. An involvement of a
5'-AMP-activated protein kinase has recently been described (45).
According to Burcelin et al. (10), the glucose stimulation
of the hepatic glucagon receptor mRNA level, involves triose phosphate intermediates of the glucose metabolism. This again suggests
that the L4-box and the G-box do not interact with the same factors.
The aldolase A, pyruvate kinase M2, and probably Glut2 genes are
regulated by glucose via dephosphorylation of Sp1 (46). Burcelin
et al. (10) recently demonstrated that, in mouse, the glucose-induced up-regulation of the Glut2 and glucagon receptor mRNA levels involve different mechanisms, supporting our data.
The present results have established for the first time the
localization and the sequence of a highly active, glucose-regulated glucagon receptor promoter, involving a novel L4-like box. A better understanding of the regulation will require the study of the protein
elements involved in the glucose regulation of the glucagon receptor,
as well the study of the metabolites of glucose that are involved
within the regulatory pathway.
868, or more, upstream from
the transcriptional start point. This promoter activity displayed
threshold-like behavior, with low activity of the promoter below 5 mM glucose, and maximal activation as of 10 mM glucose. This glucose regulation was mapped to a highly palindromic 19-nucleotide region between nt
545 and
527. Indeed, deletion or mutation of this sequence abolished the glucose regulation. This domain contained two palindromic "E-boxes" CACGTG and CAGCTG separated by 3 nt, a feature similar to the "L4 box" found in the
pyruvate kinase L gene promoter. This is the first description of a G
protein-coupled receptor gene promoter regulated by glucose.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
105 to
80 from the ATG codon) differed
from the sequence published by Jelinek (13). The coding domain of the
rat glucagon receptor gene is highly fragmented; it contains 12 introns
with uneven splicing maturation (14) that may produce cloning artifacts
resulting in the observed differences described for the 5' sequences.
Therefore, we first identified the correct organization of the 5' end
of the rat glucagon receptor mRNA. Thereafter, we cloned a fragment of genomic DNA located upstream from the transcription start point, and
identified this fragment as a glucose activable promoter region using
reporter gene studies. Finally, we localized a L4-like box sequence as
the central motif of the glucose-induced gene stimulation.
MATERIALS AND METHODS
-FixII bacteriophage DNA (Stratagene), and packaged
as recommended by the manufacturer. The resulting primary genomic
library was screened at 40,000 plaque-forming units/145-mm plate. We
initially screened the
-FixII library with 32P-labeled
oligonucleotides complementary to nt +147 to +128 and to nt + 772 to
+739 of the glucagon receptor CDS. One positive clone (P 10-C) was
isolated. First, a 3.5-kb XbaI/PstI hybridizing fragment was subcloned into pUC18. Second, a 1-kb
XbaI/XbaI fragment (located upstream from of the
XbaI/PstI fragment) was subcloned in
pZErOTM-1 (Invitrogen). The XbaI/PstI
fragment contained a 2.5-kb sequence corresponding to the 5' end of the
coding domain of the glucagon receptor gene (Ref. 14; GenBank accession
number L31574), and, in addition, a 1-kb sequence upstream from the
previously described initiation codon. The 5' end extended sequence is
listed in GenBank under accession number U63021.
-FixII clone (JS 3-A) was isolated, and a 1.3-kb BamHI/BamHI
hybridizing fragment was subcloned first in pZErOTM-1
(Invitrogen) and then in the BamHI site of pBlCat6 vector
(clone B/B).
287, and the second
BamHI site is in the polylinker of pBlCat6, so
BamHI digestion removed from the plasmid pCC, fragment an
equivalent to the pCB plasmid insert. The remaining promoter domain
(
2 kb to
287) was then excised by
HindIII/BamHI and subcloned in the
HindIII/BamHI sites of the pBlCat5 plasmid,
i.e. upstream from the HSV thymidine kinase basal promoter.
The resulting plasmid was called pTkCd.
clone. This clone is characterized by a 15-nt deletion located between
543 to
529, i.e.
deletion of the fragment containing the two E-boxes of the palindromic G-box (L4-like box). Reverse primers were complementary to the above
described forward primers. Mutated clones were sequenced as described above.
-D-galactopyranoside
(Invitrogen) according to the manufacturer's recommended procedure.
Measurement of galactosidase activity was highly reproducible, with
intra-assay variation of less than 25%.
RESULTS
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Fig. 1.
Determination of the structure of the 5' ends
of glucagon receptor cDNA. Panel
A, PCR of 5' ends of glucagon receptor cDNA. Heart or
liver cDNA fragments were amplified using two alternative forward
primers fitting conflicting glucagon receptor 5' end sequences; S.for
was based on sequence nt 101 to
82 of our sequence (L04796), and
J.for was based on sequence
105 to
86 of Jelinek's sequence
(M96674). We used a forward primer G.for based on nt
15 to +3
(located downstream from the position where the glucagon receptor
sequences diverged) as a control. The same reverse primer R147
(corresponding to nt +128 to +147 of glucagon receptor CDS) was used
with the three forward primers. This reverse primer is located
downstream of the 100-bp intron present in the genomic DNA at position
nt +63. After 30 cycles of PCR (94, 60, and 72 °C, 1 min. each) the
reaction products were analyzed on 1.2% agarose gel. The intensity of
cDNA fragments amplification was lower in heart as compared with
liver, as expected since the glucagon receptor mRNA content is 7 times lower in heart than in the liver (3). The specificity of the
amplification was confirmed by Southern blotting, using internal
oligodeoxynucleotides as probe (data not shown).Panel B,
schema of structure of the 5' end of the glucagon receptor mRNA
deduced from the PCR results. The S.for primer, based on our 5'end
sequence, amplified a single 250-bp fragment as expected (246 bp) for
the mRNA corresponding to the sequence L04796 (3). Likewise the
G.for primers also yield only the expected 189-bp DNA fragment. Minor
fragments, 100 bp longer, originated from immature mRNA containing
the 100-bp intron I (14). Primer J.for, based on the 5' end published
by Jelinek, amplified two fragments of 250 and 400 bp in both heart and
liver. The larger fragment predominated in the heart. The 250-bp
fragment corresponded to the published sequence, M96674 (252 bp). The
400-bp fragment possessed an additional 166-bp exon anchored at
position nt
79. The 3' end of this additional exon corresponded to
our sequence L04796.
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Fig. 2.
Southern blot of anchored PCR of the 5' end
the of glucagon receptor cDNA (5' RACE) from liver and heart.
The liver and heart mRNA were reverse transcribed using the R426
reverse primer (complementary to nt +426 to 408) of the glucagon
receptor CDS. The reaction product was purified and ligated to a
phosphorylated anchor primer as recommended by the manufacturer
(CLONTECH 5' RACE kit). In the first amplification
step, the ligated cDNA was amplified using the PCR anchor primer
(CLONTECH) together with R356 (based on nt +356 to
+339, samples 1-4) or R147 (based on nt +147 to +128, samples 5 and 6)
reverse primers. The product of the first PCR was diluted 1000 times
and reamplified using the same PCR anchor primer, and reverse primers
R147 (lanes 1 and 2) or R-20 (based on
nt 20 to
39, lanes 3-6). The product of the
second PCR was separated on 1.2% agarose gel, transferred to
nitrocellulose membrane, hybridized using R-20-labeled oligonucleotide
as a probe, and autoradiographed.
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Fig. 3.
Structure of the 5' end domain of the rat
glucagon receptor gene. Panel A, schematic
description of the whole rat glucagon receptor gene. Exons,
closed bars; introns, single
line; promoter, open box. The splicing
pattern of the 5' untranslated domain is indicated by the
dashed lines. The top line
presents a partial glucagon receptor gene restriction map:
A, ApaI; B, BamHI;
P, PstI; S, SacI;
Xb, XbaI; Xh, XhoI
restriction sites. Panel B, sequence of the 5' end of the
rat glucagon receptor gene: promoter domain and exon UJ (clone pCX).
Genomic DNA sequences were obtained by sequencing the subcloned
-FixII genomic clones as indicated under "Materials and
Methods." Uppercase letters, promoter domain;
lowercase letters, exon UJ; italics,
first intron; arrow, the position of 5'-most transcription
start site; bold, the 2 E-boxes, forming the L4-like box
(underlined). The BamHI, PmlI, and
DraIII restriction enzyme-cut sites, which allowed the
construction of the plasmids used for the transfection studies, are
indicated over the sequence.
-FixII rat genomic DNA
library. The first genomic clone obtained contained the complete
glucagon receptor coding domain, and 1.5-kb sequence upstream from the
ATG initiation codon. The genomic DNA sequence upstream from position
79 (GenBank accession number U63021) diverged from both published
cDNA 5' end sequences and represented thus obviously an intron.
This sequence showed 83% identity (over 992 nt upstream from the ATG)
with the reported putative mouse glucagon receptor gene promoter
(GenBank accession number L38612) (17).
527 to
545 upstream from the
transcription start point, highly palindromic sequence of 19 nucleotides containing a canonical E-box "CACGTG", followed by
three nucleotides "tga" and another palindromic E-box "CAGCTG" (Fig. 3B). This feature is very similar to the "L4 box"
found in pyruvate kinase L or Spot14 promoter, in which it is thought to be the glucose response element (GIRE or ChoRE; see
"Discussion"). We call this nucleotide stretch the "G-box".
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Fig. 4.
Construction of plasmids used for
transfection studies. The ligation of a
HindIII/XbaI fragment of clone P/S and a
XbaI/XbaI fragment of clone B/B with the pBlCat6
vector digested by HindIII/XbaI yielded
clone P/ctr/B. P/ctr/B was digested by ApaI and
NotI, then blunted and ligated to obtain clone pCC.
This clone contained a XhoI site 1 kb upstream from
its 3' end (i.e. ApaI site). A second XhoI site
is located in the polylinker, so that XhoI digestion
excised a 1-kb insert that was subcloned in the XhoI site of
the pBlCat6 vector to obtain clone pCX. DraIII cuts at
position 550 and
267 nt. DraIII-digested plasmid was
blunted with T4 DNA polymerase and ligated to obtain clone pCD.
236 to +156)
plasmid gave the same, relatively large CAT activity in cells grown in
medium containing either 5 or 20 mM glucose (Fig.
5).
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Fig. 5.
CAT activity of INS-1 cell extracts
transfected with plasmids containing the glucagon receptor
promoter. The left panel summarizes the
pBlCat6 plasmids used, with the various rat glucagon receptor gene 5'
sequences: clone pCC ( 2 kb to +156); pCX (
869 to +156) and the
short clone pCB (
236 to +156) were constructed as indicated in Fig.
4. The pCP clone (
541 to +156) was pCX shortened with
EcoRV (cutting in polylinker) and PmlI (cutting in the first
E-box, see Fig. 3B). The pCD plasmid (
869 to
550,
267
to +156) was prepared by internal deletion (between
267 and
550 nt)
of pCX with the DraIII enzyme. The right
panel shows the results of transfection experiments. The CAT
activity of transfected cells was assayed by acylation of
[14C]chloramphenicol by n-butyryl-CoA, after
cell culture in media containing 5 mM (hatched
bars) or 20 mM (open bars)
glucose. The spot densities were calculated in each experiment as
percentage of activity produced by pCX plasmid. The mean of three to
five separate experiments performed in duplicates ± S.E. is
represented.
869 to
+156) and pCC (
2 kb to +156) expressed a low CAT activity in the
presence of glucose 5 mM and a spectacularly higher CAT activity in the presence of 20 mM glucose (Fig. 5).
Transfection realized with plasmid pCX (
869 to +156) and plasmid pCC
(
2 kb to +156), gave similar results, suggesting that clone pCX
(
869 to +156) contained the DNA fragment responsible for the glucose responsiveness. This enhancer should be located upstream from the
plasmid pCB insert sequence (i.e. between position
236 to
869 upstream from the transcription start site). Results obtained with the short pCB plasmid shown that these regulatory elements inhibited gene transcription at low, 5 mM glucose and
activated the transcription at high, 20 mM glucose concentrations.
541 to +156) gave a lower activity, with
reduced glucose stimulation. The deleted plasmid pCD (pCX deleted
between
267 and
550 nt) had a lower, nonregulated activity (Fig.
5). These results suggest that the glucose regulatory element is
located close to the PmlI site and DraIII
restriction sites, including nucleotides
541 to
550. This
corresponds indeed to the position of the 19-nt palindromic sequence
"G-box" indicated above (Fig. 3B).
868) possessed a low activity that remained glucose
regulated. Plasmid pCBi (+156 to
236) possessed a low activity that
was no regulated by glucose (Fig. 6). As the putative glucose enhancer sequence is highly palindromic, it is not surprising that glucose stimulation was also observed in reverse orientation of plasmid pCXii. Absolute activity value of reversed plasmids was
lower, which was expected, as in this orientation the normal basal
promoter was also in the reverse orientation. This was confirmed by the low activity of the reversed plasmid pCBi containing only the putative
basal promoter (i.e. GC-rich domain) (Fig. 6).
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Fig. 6.
CAT activity of INS-1 cell extract
transfected with plasmids containing the glucagon receptor promoter in
both orientations. Left panel, schematic
representation of the pBlCat6 plasmids with inserts in the normal
orientation (plasmids pCX ( 869 to +156) and pCB (
236 to +156)), and
in the reverse orientation (plasmid pCXi (+156 to
868)
and pCBi (+156 to
236)). Right
panel, results of transfection experiments performed as in
Fig. 5 using a culture medium containing 5 mM glucose
(hatched bars) or 20 mM glucose
(open bars). Results represented the mean of
three separate paired experiments performed in duplicates ± S.E.
2 kb to
287) upstream from the
HSV thymidine kinase basal promoter in the pBlCat5 plasmid. Cells
transfected with this plasmid, called pTkCd, which contains the
reporter gene driven by the recombinant glucagon receptor enhancer and
the tk basal promoter, displayed much higher activities that pCX
plasmid. The pTkCd plasmid retained the same level of sensitivity that
the pCX plasmid to glucose activation (Fig.
7). Therefore, this plasmid will be used
as a model for further studies of the glucose enhancer.
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Fig. 7.
Increasing CAT activity in INS-1 cells
transfected with plasmids containing the glucagon receptor promoter
subcloned upstream of a heterologous tk promoter. Cells were
transfected by control pBlCat6 plasmid, reference pCX ( 869 to +156)
plasmid, or pTkCd plasmid. In pTkCd plasmid, the promoter domain
containing the glucose regulatory element (
2 kb to
287) was
subcloned upstream from the HSV tk promoter of the pBlCat5 plasmid.
Transfected cells were grown in either 5 mM glucose or 20 mM glucose and CAT activity of cells extracts was assayed
as described under "Material and Methods." Figure shows an
autoradiography of thin layer chromatography of a representative
experiment made in duplicates (duplicates were migrated beside each
other).
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Fig. 8.
Dose-response curve of extracellular glucose
concentration on the CAT activity in INS-1 cells transfected by
plasmids containing the glucagon receptor promoter. INS-1 cells
transfected by pCX ( 869 to +156) plasmid as described under
"Materials and Methods" were grown for 48 h in the medium
containing indicated concentration of glucose. The CAT activity of cell
extracts were assayed as described under "Material and Methods."
The mean ± S.E. of three independent experiments, performed at
least in duplicate, is represented.
clone that lacks a 15-nucleotide
domain that includes the two E-boxes. This deletion produced a total
suppression of the glucose stimulation of the CAT activity in the
transfected cells (Fig. 9A).
In addition, two mutations were introduced in the G-box present in the
pTkCd plasmid. In the first clone pTkCd-m1, we mutated the second E-box CAGCTG to CAGTTG. In the
second mutated clone pTkCd-m2, we changed CA of the two E-boxes to TG.
Transfections of INS-1 cells with the mutated plasmids showed that,
whereas the C
T point mutation of pTkCd-m1 did not impair glucose
stimulation of the reporter gene expression, the CA
TG mutations of
pTkCd-m2 almost totally suppressed glucose activation of the reporter
gene. These results demonstrate the key role of the G-box in the
glucose activation of the rat glucagon receptor gene
expression.
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Fig. 9.
Effect of deletion (panel A)
or mutations (panel B) of G-box on the CAT activity in
transfected INS-1 cells. Panel A, Ins-1
cells were transfected by pCX ( 869 to +156) plasmid, or the G-box
deleted plasmid pCX
prepared by QuickChange site-directed
mutagenesis kit (Stratagene). Transfected cells were grown in either 5 mM glucose or 20 mM glucose, and the CAT
activity of cells extracts was assayed as described under "Material
and Methods." The mean ± S.E. of three (for pCX
) or five
(for pCX) independent experiments, performed in duplicate, is
represented. Panel B, INS-1 cells were
transfected by pTkCd plasmid (pBlCat5, tk basal promoter with fragment
2 kb to
287) and two mutants of the G-box prepared by QuickChange
site-directed mutagenesis kit (Stratagene) as described under
"Materials and Methods." Mutations are indicated in bold
and underlined. Transfected cells were grown in either 5 mM glucose or 20 mM glucose, and the CAT
activity of cell extracts was assayed as described under "Material
and Methods." The mean of two independent experiments, performed in
duplicate, is represented.
DISCUSSION
168 to
144 nt from the start
point of the pyruvate kinase L gene, whereas in the Spot14 gene it is
found at nt
1448 to
1431. The gene of fatty acid synthase, another
glucose-regulated gene, also possesses a sequence feature closely
related to L4 box. However, this glucose regulatory element is located
in the first intron (+283 to +303) (39).
545 to
527
is very similar to the motif of the regulatory elements described above. This highly palindromic (underlined), 19-nucleotide sequence contained two perfect, 6-nucleotide palindromes (uppercase). The first
one represents a canonical E-box sequence. Results observed with
different constructs obtained by with restriction digestion (Fig. 5)
suggested that the glucose regulation is centered on this domain. We
confirmed the essential role of this motif, the coined G-box, by the
mutational studies (Fig. 9), where the deletion or mutation of this
motif suppressed glucose stimulation.
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ACKNOWLEDGEMENTS |
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We are grateful to Prof. Wollheim (University Medical Center, Geneva, Switzerland) for the grant of the INS-1 cell line. We thank Drs. P. Robberecht and M. Waelbroeck for the manuscript revision, M. Stiévenart for the graphical representation of the results, and N. De Gendt-Peuchot for secretarial assistance.
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FOOTNOTES |
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* This work was supported in part by a grant from the Fonds de la Recherche Scientifique Médicale 3.4507.98, a Bekales grant, and a grant from the Association Belge du Diabète (Brussels, Belgium).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U63021 and U63022.
Recipient of a doctoral fellowship from the Interuniversity Poles
of Attraction Program, Belgian State Prime Minister's Office, Federal
Office for Scientific, Technical and Cultural Affairs.
§ Recipient of a doctoral fellowship from Fonds Pour la Formation a la Recherche Dans L'Industrie et Dans L'Agriculture (FRIA)-Belgium.
¶ To whom correspondence should be addressed. Tel.: 32-2-555-62-25; Fax: 32-2-555-62-30; E-mail: msvobod{at}ulb.ac.be.
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ABBREVIATIONS |
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The abbreviations used are: nt, nucleotide(s); PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; CDS, coding DNA sequence; GPCR, G protein-coupled receptor; GLP-1, glucagon-like peptide 1; PACAP, pituitary adenylyl cyclase activating peptide; PKL, pyruvate kinase L; VIP, vasoactive intestinal peptide; bp, base pair(s); kb, kilobase pair(s); RACE, rapid amplification of cDNA ends; tk, thymidine kinase; HSV, herpes simplex virus; PTH, parathyroid hormone.
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REFERENCES |
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