From the Laboratoire de Chimie Biologique et de la Nutrition, Faculté de Médecine, Université Libre de Bruxelles, 808 route de Lennik, Building G/E, CP 611, B-1070 Brussels, Belgium
Received for publication, August 23, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We mutated the vasoactive intestinal peptide
(VIP) Asp3 residue and two VPAC1
receptor second transmembrane helix basic residues (Arg188
and Lys195). VIP had a lower affinity for R188Q,
R188L, K195Q, and K195I VPAC1 receptors than for
VPAC1 receptors. [Asn3] VIP and
[Gln3] VIP had lower affinities than VIP for
VPAC1 receptors but higher affinities for the mutant
receptors; the two basic amino acids facilitated the introduction of
the negatively charged aspartate inside the transmembrane domain. The
resulting interaction was necessary for receptor activation.
1/[Asn3] VIP and [Gln3] VIP were partial
agonists at VPAC1 receptors; 2/VIP did not fully activate
the K195Q, K195I, R188Q, and R188L VPAC1 receptors; a VIP
analogue ([Arg16] VIP) was more efficient than VIP at the
four mutated receptors; and [Asn3] VIP and
[Gln3] VIP were more efficient than VIP at the R188Q and
R188L VPAC1 receptors; 3/the [Asp3] negative
charge did not contribute to the recognition of the VIP1
antagonist,
[AcHis1,D-Phe2,Lys15,Arg16,Leu27]
VIP (1-7)/growth hormone releasing factor (8-27). This is the first demonstration that, to activate the VPAC1 receptor,
the Asp3 side chain of VIP must penetrate
within the transmembrane domain, in close proximity to two highly
conserved basic amino acids from transmembrane 2.
The neuropeptides vasoactive intestinal polypeptide
(VIP)1 and pituitary
adenylate cyclase-activating polypeptide (PACAP) contribute to the
regulation of intestinal secretion and motility, of the vascular tone,
of the exocrine and endocrine secretions, of immunological responses,
and to the development of the central nervous system (1-3). The
effects of VIP are mediated through interaction with two receptor
subclasses named the VPAC1 and VPAC2 receptors;
the effects of PACAP are also mediated through interactions with the same receptors, as well as through a selective receptor named PAC1 (3, 4).
VPAC1, VPAC2, and PAC1 receptors
are encoded by different genes and expressed in different cell
populations in both the central nervous system and peripheral tissues
(3, 5, 6). They are preferentially coupled to G The positioning of the ligand on the receptors is still poorly
understood. Investigations of chimeric receptors and mutants have
indicated that the large amino-terminal domain (10-13) structured by
disulfide bridges (14-16) makes a key contribution to ligand recognition that several other highly conserved residues play a role in
the general structure (17, 18) and that creating constitutively active
receptors through mutations in the intracellular part of the receptor
is possible (19).
The amino-terminal part of the ligand is necessary for high affinity
binding and for second messenger activation; its deletion in VIP,
PACAP, and secretin reduced both the affinity and the intrinsic
activity of the peptide (20). The identification of the receptor
residues interacting with the amino terminus of the ligand is a
prerequisite to model the active form of the receptor and conceive new
ligands, preferably non-peptidic, that could be of therapeutic
interest. We focused in this work on the human VPAC1
receptors and investigated the contribution of two basic residues
located in the second transmembrane helix to ligand recognition and
adenylate cyclase activation.
We obtained evidence that both basic residues are important for
recognition of the Asp3 of VIP and stabilization of the
active VIP-receptor complex conformation. Our results also suggested
that a second binding mode, that does not involve recognition of the
VIP Asp3 residue and does not induce receptor activation,
is also possible.
Construction of the Four Mutated Receptors, R188L, R188Q, K195Q,
and K195I, and their Stable Expression in Chinese Hamster Ovary (CHO)
Cells--
The human VPAC1 receptor cDNA was cloned by
PCR according to the previously reported sequence (21), using specific
primers. Generation of the four mutated receptors was achieved using
the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla CA)
essentially according to the manufacturer's instructions. Briefly, the
human VPAC1 receptor-coding region, inserted into the
mammalian expression vector pcDNA3.1 (Invitrogen Corp.), was submitted to 22 cycles of PCR (95 °C for 30 s, 54 °C for 1 min, and 68 °C for 14 min) in a 50-µl reaction volume. The forward and reverse primers were complementary and contained the desired nucleotide changes, flanked on either side by 15 perfectly matched nucleotides (only the forward primers are shown):
Arg188 to Leu,
CATATCCTTCATCCTGCTGGCTGCCGCTGTCTTC; Arg188 to
Gln, CATATCCTTCATCCTGCAGGCTGCCGCTGTCTTC; Lys195
to Ile, GCCGCTGTCTTCATCATAGACTTGGCCCTCTTC;
Lys195 to Gln,
GCCGCTGTCTTCATCCAAGACTTGGCCCTCTTC.
Following PCR, 10 µl were analyzed by agarose gel electrophoresis,
and the remaining 40 µl were digested for at least 2 h by 1 µl
of DpnI restriction enzyme (Stratagene, La Jolla, CA) to
remove the parental methylated DNA. The digested PCR products were
transformed into TOP10 One Shot competent Escherichia
coli bacterial cells (Invitrogen Corp.). Of several colonies
verified by agarose gel electrophoresis of miniprep plasmid DNA (22), three were retained and further purified on Qiaquick PCR purification spin columns, and the mutations were checked for by DNA sequencing on
an ABI automated sequencing apparatus, using the BigDye Terminator sequencing prism kit from ABI (Perkin-Elmer). Plasmid DNA from one
clone for each mutation, containing the correct nucleotide substitutions, was prepared using a midiprep endotoxin-free kit (Stratagene, La Jolla, CA), the complete nucleotide sequence of the
receptor coding region was verified by DNA sequencing, and 20 µg were
electroporated (Electroporator II; Invitrogen Corp.) into wild type
CHO-K1 cells. Selection was carried out in culture medium (50%
HamF12, 50% Dulbecco's modified Eagle's medium, 10% fetal calf
serum, 1% penicillin (10 milliunits/ml), 1% streptomycin (10 µg/ml), 1% L-glutamine (200 mM; Life
Technologies LTD, Paisley, United Kingdom), supplemented with 600 µg
of geneticin (Gly418)/ml culture medium. After 10 to 15 days of selection, isolated colonies were transferred to 24-well
microtiter plates and grown until confluence, trypsinized, and further
expanded in 6-well microtiter plates, from which cells were scraped and
membranes prepared for screening by an adenylate cyclase activity assay in the presence of 10 µM VIP.
Membrane Preparation--
Membranes were prepared from scraped
cells lysed in 1 mM NaHCO3 by immediate
freezing in liquid nitrogen. After thawing, the lysate was first
centrifuged at 4 °C for 10 min at 400 × g, and the
supernatant was further centrifuged at 20,000 × g for
10 min. The resulting pellet, resuspended in 1 mM
NaHCO3, was used immediately as a crude membrane fraction.
Radioiodination of Three Different Tracers and Binding
Studies--
Binding studies were performed as described (23) using
125I-VIP, 125I-[Gln3] VIP, or
125I-VIP1 antagonist. The three tracers were
radiolabeled similarly and had comparable specific radioactivity (6,
23). In all cases, the nonspecific binding was defined as residual
binding in the presence of the corresponding unlabeled peptide (1 µM). Binding was performed at 20 °C in a total volume
of 120 µl containing 20 mM Tris-maleate, 2 mM
MgCl2, 0.1 mg/ml bacitracin, 1% bovine serum albumin (pH
7.4) buffer. 3 to 30 µg of protein were used per assay. Bound and
free radioactivity were separated by filtration through glass-fiber
GF/C filters presoaked for 24 h in 0.01% polyethyleneimine and
rinsed three times with a 20 mM (pH 7.4) sodium phosphate buffer containing 1% bovine serum albumin.
Adenylate Cyclase Activation--
Adenylate cyclase activity was
determined by the procedure of Salomon et al. (24), as
described previously. Membrane proteins (3-15 µg) were incubated in
a total volume of 60 µl containing 0.5 mM
[ Peptide Synthesis--
VIP(1-28)-amide (VIP),
[Asn3] VIP(1-28)-amide ([Asn3] VIP),
[Glu3] VIP(1-28)-amide ([Glu3] VIP),
[Gln3] VIP(1-28)-amide ([Gln3] VIP),
[Arg16] VIP(1-28)-amide ([Arg16] VIP),
[Lys15,Arg16,Leu27]
VIP(1-7)/GRF(8-27)-amide (VIP1 agonist),
[AcHis1,DPhe2,Lys15,Arg16,Leu27]
VIP(1-7)/GRF(8-27)-amide (PG 97, 269, or VIP1
antagonist),
[Asn3,Lys15,Arg16,Leu27]
VIP(1-7)/GRF(8-27)-amide ([Asn3] VIP1
agonist) and
[AcHis1,DPhe2,Asn3,Lys15, Arg16,Leu27]
VIP (1-7)/GRF(8-27)-amide ([Asn3] VIP1
antagonist) were synthesized in our laboratory by the Fmoc (9-fluorenylmethoxy carbonyl) strategy on an Applied Biosystems Apparatus 431A (Foster City, CA). The peptide purity (>97%) was assessed by capillary electrophoresis, and the conformity was verified
by electrospray mass spectrometry.
Data Analysis--
All competition curves and dose-effect curves
were analyzed by a non-linear regression program (Graph Pad Prism, San
Diego, CA).
Interaction of VIP Analogues with the Human Wild Type Recombinant
VPAC1 Receptor--
The VPAC1 IC50
values, measured in 125I-VIP competition curves on
membranes of CHO cells expressing
900 ± 50 fmol of receptor/mg of protein, are summarized
in Table I. The agonists
EC50 values, evaluated from complete dose-effect curves
(Fig. 1), are summarized in Table
II. Calculation of the maximal
stimulation of adenylate cyclase activity indicated that VIP and
[Arg16] VIP (25) reached the same maximum effect, higher
than that obtained in the presence of [Glu3] VIP,
[Asn3] VIP, [Gln3] VIP (this work), or
VIP1 agonist (26) (Table II). The VIP1 antagonist, PG 97 269 (23), did not stimulate adenylate cyclase at any
concentration but inhibited competitively the effect of VIP with a
Ki value of 2-3 nM.
Analysis of the Mutated R188Q, R188L, K195Q, and K195I
VPAC1 Receptors--
The clones transfected with the DNA
coding for the mutated receptors were selected on the basis of the
ability of 10 µM VIP membranes to activate adenylate
cyclase. The selected clones were then tested for 125I-VIP
binding. Binding was non-significant in all the clones tested. As
expected from the binding data, the EC50 values for VIP
were 30 to 100-fold higher than at VPAC1 receptors (Fig.
1). There was no noticeable difference between the R188L and R188Q
receptors on one hand and the K195I and K195Q on the other hand.
We then tested the ability of VIP analogues to stimulate adenylate
cyclase. [Arg16] VIP, [Asn3] VIP, and
[Gln3] VIP were more potent and more efficient than VIP
at the R188L- and R188Q-VPAC1 receptors, and
[Glu3] VIP and the VIP1 agonist behaved as
partial agonists on both mutant receptors (Fig. 1 and results not
shown). The results are compared in Table II with the data obtained at
wild type receptors with the same peptides.
[Arg16] VIP was also more potent and more efficient than
VIP on the K195Q- (Fig. 1) and K195I-VPAC1 receptors (data
not shown). [Glu3] VIP, [Gln3] VIP, and
[Asn3] VIP behaved as partial agonists on this construct,
and the VIP1 agonists' ability to activate adenylate
cyclase through K195Q- and K195I-VPAC1 receptor mutants was
barely detectable (Table II).
The VIP1 antagonists' affinity for the mutated receptors
was evaluated by comparing VIP, [Arg16] VIP, or
VIP1 agonist dose-effect curves in the absence and presence of 0.1, 0.3, or 1.0 µM antagonist (Fig.
2 and results not shown). The VIP
dose-effect curves were shifted dose-dependently to higher concentrations in the presence of antagonist, as expected for Ki values
Taken together, these results suggested that the affinity of
[Gln3] VIP and of the VIP1 antagonist for the
mutant receptors might be sufficient to allow binding studies using
these radiolabeled peptides. This was indeed the case. The binding
experiments were performed at 20 °C as tracer binding was unstable
at 37 °C (probably because of a receptor instability, as already
noted for chimeric receptors (10, 25)). The binding of both tracers was
rapid (equilibrium was achieved within 20 min) and reversible (data not
shown). The peptides' IC50 values (reported in Table I)
did not depend on the tracer used. Representative competition curves are shown in Fig.
3.
The R188Q and K195Q receptor concentrations were comparable (within
2-fold) to the VPAC1 receptor concentration.
We were surprised that, even though the presence of a negative
charge in position 3 was clearly deleterious for recognition of the
R188Q, R188L, K195Q, and K195I mutant VPAC1 receptors by agonists, the VIP1 antagonist (that also possesses an
Asp3 residue) retained a high affinity for the mutated
receptors. We therefore synthesized and tested two additional peptides
with an Asn3 residue, the Asn3 VIP1
agonist and Asn3 VIP1 antagonist. As shown in
Tables I and II, replacing the Asp3 by an Asn3
residue reduced the affinity and efficacy of the VIP1
agonist at wild type receptors but increased its affinity and efficacy on the mutated R188Q receptor. In contrast, replacing Asp3
by an Asn3 residue in the VIP1 antagonist did
not affect its affinity for the receptors studied in this work; the
modified peptide always behaved as a high affinity antagonist (Fig.
4).
VIP receptors belong to a G protein-coupled receptor family that
does not share any sequence homology with rhodopsin or with the
In view of the significant sequence homologies between secretin, VIP,
PACAP, GRF, glucagon, glucagon-like peptide (7-36), and gastric
inhibitory peptide on the one hand and between their receptors on the
other hand, we hypothesized that these different peptides recognize
"equivalent" binding sites in their respective receptors. The
observation that all the aforementioned peptides except glucagon
possess an acidic residue (Asp or Glu) in position 3, and that all the
receptors (except glucagon's) possess aligned basic Arg and Lys
residues at the extracellular end of TM2, led us to suspect that these
residues might interact in the peptide-receptor complex.
Our preliminary results confirmed that, as in secretin receptors (17,
38), the VPAC1 receptor Arg188 and
Lys195 were important for VIP recognition; we were unable
to obtain significant 125I-VIP binding at the mutated
receptors, and very high VIP concentrations were necessary to activate
the adenylate cyclase. The following results suggested in addition that
VIP was unable to stabilize the mutated receptors' active
conformations sufficiently to ensure full receptor activation:
[Asn3] VIP, [Gln3] VIP, and
[Arg16] VIP were more efficient than VIP at the R188Q and
R188L VPAC1 receptors, and [Arg16] VIP was
more efficient than VIP at the K195Q and K195I VPAC1 receptors.
When VIP and its receptor are free, the VIP Asp3 and the
receptor Arg188 and Lys195 side chains probably
form dipole-ion interactions with surrounding water molecules. These
favorable interactions are disrupted upon ligand binding; they must be
compensated by ligand-receptor interactions to allow high affinity
binding. The "uncharged VIP analogues" [Asn3] VIP and
[Gln3] VIP had a higher affinity than VIP for the
"uncharged receptor mutants" (R188Q, R188L, K195Q, and K195I
VPAC1 receptors), suggesting that the VIP Asp3
and the receptor Arg188 and Lys195 side chains
were in close proximity in the agonist-receptor complex. The affinity
loss that we observed upon replacement of the VIP Asp3,
VPAC1 receptor Arg188, and VPAC1
receptor Lys195 (30- to 100-fold) was however comparatively
small and did not support the hypothesis that the Asp negative charge
is close enough to the receptor Arg and Lys positive charges to form
ionic bonds (40). It is more likely that Asp3,
Arg188, and Lys195 formed strong hydrogen bonds
(i.e. dipole-dipole or ion-dipole interactions); the two
receptor basic residues probably participated in the formation of an
electrophilic pocket that recognized the negatively charged VIP
Asp3 side chain.
[Asn3] VIP and [Gln3] VIP behaved as
partial agonists at wild type receptors but were more efficient than
VIP at the R188Q and R188L mutant receptors. The incomplete activation
of the mutant receptors by VIP might be caused by difficulties in
burying the anionic Asp3 side chain deep enough in the
(uncharged) mutant receptors' binding site. This hypothesis is
indirectly supported by the observation that replacing the VIP
Asp3 or the VPAC1 receptor Arg188
or Lys195 with uncharged amino acids affected the
recognition of the VIP1 agonist (an efficient partial
agonist) somewhat less than the recognition of VIP and did not affect
binding of the VIP1 antagonist, a compound that does not
induce receptor activation. Due perhaps to steric hindrance between the
receptor and the large D-Phe2 side chain, the
VIP1 antagonist Asp3 was apparently unable to
enter the agonist binding pocket and to trigger receptor activation.
The parathyroid hormone (PTH) receptor belongs to the same receptor
family as the VPAC1 receptor. A molecular model of the PTH-receptor interaction has been developed, based on experimental data
from cross-linking studies, spectroscopic investigations of the hormone
and receptor fragments, and theoretical structure predictions (39).
According to this model, PTH recognizes extracellular receptor domains
(the amino-terminal domain and extracellular loops) but penetrates very
little if at all inside the compact transmembrane helices bundle. It is
tempting to suggest that, like PTH, VIP initially recognizes an
extracellular binding site. In a second step, driven and stabilized
i.e. by the
Asp3-Lys195/Arg188 interactions, a
transmembrane binding pocket opens and recognizes the agonists'
amino-terminal amino acids, and the receptor activates intracellular G
proteins. "Too large" amino-terminal VIP amino acids
(D-Phe2 instead of Ser2 and
pCl-Phe6 instead of Phe6) might prevent the
recognition of this activated receptor conformation by steric
hindrance; [D-Phe2] and
[pCl-Phe6] VIP or VIP/GRF analogues usually behave as VIP
antagonists (27, 28).
The location of the VIP and secretin "Asp3 binding
site" to transmembrane helix 2 was somewhat unexpected; indeed, in
the rhodopsin-like To conclude, our present results suggested that the VIP
Asp3 side chain fitted inside the transmembrane helix
bundle, in close proximity to TM2 Lys195 and
Arg188. This interaction was essential for receptor
activation. The VIP1 antagonist Asp3 residue
did not recognize the same binding pocket perhaps because of
unfavorable coulombic interactions between the
D-Phe2 side chain and the receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s
proteins that stimulate adenylate cyclase activity. The
PAC1 and VPAC1 receptors may stimulate, in
addition, inositol trisphosphate synthesis and calcium mobilization (7,
8). This effect is however detected only at high VPAC1 receptor expression levels (8). VIP and PACAP receptors are members of
a large family of G protein-coupled receptors, often referred as the
GPCR-B family (4, 9), that includes the secretin, glucagon,
glucagon-like peptide-1, calcitonin, parathyroid hormone, and growth
hormone releasing factor (GRF) receptors. The VIP, PACAP, secretin, and
GRF receptors constitute a subfamily based on the homology of the
ligands and of the receptors. Each receptor recognizes its own cognate
ligand with a high affinity but recognizes at least one other parent
peptide with a comparable or a lower affinity (4). Because of the
sequence homology of the ligands and the receptors, the information
obtained on one receptor-ligand pair can be anticipated to be relevant
also in the other systems.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
32P]ATP, 10 µM GTP, 5 mM
MgCl2, 0.5 mM EGTA, 1 mM cAMP, 1 mM theophylline, 10 mM phospho(enol)pyruvate,
30 µg/ml pyruvate kinase, and 30 mM Tris-HCl at a final
pH value of 7.8. The reaction was initiated by membranes addition and
was terminated after a 15-min incubation at 37 °C by adding 0.5 ml
of a 0.5% sodium dodecyl sulfate solution containing 0.5 mM ATP, 0.5 mM cAMP, and 20,000 cpm
[3H]cAMP. cAMP was separated from ATP by two successive
chromatographies on Dowex 50W × 8 and neutral alumina.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Binding properties of VPAC1, R188Q, and
K195I-VPAC1 receptors
log
Ki) values was always below 0.15 log units.
IC50 values, measured in 125I-VIP,
125I-[Gln3]VIP, or 125I-VIP1
antagonist competition curves on membranes of CHO cells expressing
VPAC1, R188Q-, or K195Q-VPAC1 receptors, respectively.
View larger version (14K):
[in a new window]
Fig. 1.
Adenylate cyclase stimulation through
VPAC1 receptors (top panel), K195Q-
(center panel), and R188Q VPAC1 receptors
(bottom panel). VIP (closed circles),
[Arg16] VIP (open triangles), and
[Gln3] VIP (open squares) dose-effect curves
were obtained at CHO cell membranes expressing the wild type or mutated
VPAC1 receptors. The figure is representative of at least
three experiments performed in duplicate.
Adenylate cyclase stimulation through VPAC1 R188Q, and
K195I-VPAC1
log
EC50) values was always below 0.15 log units, and the standard
deviation of the agonists' efficiency (Emax, in % of VIPs) was lower than ± 10%. n.d., not determined.
2-3 nM at the five
receptors (Fig. 2 and results not shown).
View larger version (18K):
[in a new window]
Fig. 2.
Competitive inhibition of adenylate cyclase
stimulation by the VIP1 antagonist, PG 97 269. VIP
dose-effect curves in the absence (open circles) and
presence (closed circles) of 100 nM
VIP1 antagonist were obtained at CHO cell membranes
expressing the R188L VPAC1 receptors (top
panel), and [Arg16] VIP dose-effect curves in the
absence (open circles) and presence (closed
circles) of 100 nM VIP1 antagonist were
obtained at CHO cell membranes expressing the K195I VPAC1
receptors (bottom panel). The figure is representative of at
least three experiments performed in duplicate.
View larger version (14K):
[in a new window]
Fig. 3.
Binding to VPAC1, K195Q-, and
R188Q-VPAC1 receptors. VIP (closed
circles), [Arg16] VIP (open triangles),
and [Gln3] VIP (open squares) competition
curves were obtained at wild type (top panel), K195Q-
(center panel), or R188Q VPAC1 (bottom
panel) receptors, using 125I-VIP,
125I-VIP1 antagonist, or
125I-[Gln3] VIP as tracer, respectively. The
figure represents the average of three experiments performed in
duplicate.
View larger version (18K):
[in a new window]
Fig. 4.
Importance of the [Asp3] for
VIP1 agonist and antagonist binding. Competition
curves using the VIP1 agonist (closed squares,
left panels), [Asn3] VIP1 agonist
(open squares, left panels), VIP1
antagonist (closed circles, right panels), and
[Asn3] VIP1 antagonist (open
circles, right panels) at wild type (top
panels), K195Q- (center panels), and R188Q
VPAC1 receptors (bottom panels), using
125I-VIP, 125I-VIP1 antagonist, or
125I-[Gln3] VIP as tracer, respectively. The
figure represents the average of three experiments performed in
duplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenergic receptor family. Previous work with chimeric receptors
(see Refs. 10-13 and 29-34) and cross-linking studies (35-37) led to
a general VIP positioning model; the amino-terminal and
carboxyl-terminal sequences of VIP and of its analogues seemed to
interact with the "7TM" (transmembrane) receptor domain, whereas recognition of the central VIP sequence (from positions
6 to
22?)
depends on the amino-terminal extracellular receptor domain.
-adrenergic G protein-coupled receptor family,
the ligand binding pocket is lined by TMs 3 to 7 and does not involve
TMs 1 and 2. It is important to note in this respect that most of the
"signature" amino acids that define the
-adrenergic G
protein-coupled receptor family, including the proline residues that
participate in the formation of the agonist binding pocket, are absent
from the secretin receptor family (including VPAC1
receptors). Our results suggest that (in contrast with the G protein
binding site that appears to involve the same intracellular loops in
both receptor families) the agonist binding site was located in very
different transmembrane regions in the secretin- and
-adrenergic-receptor families. Further studies will be needed to
extend this observation and allow the construction of an activated
agonist-receptor complex model.
![]() |
FOOTNOTES |
---|
* Supported by Fonds de la Recherche Scientifique Médicale Grant 3.4507.98, by an "Action de Recherche Concertée" from the Communauté Française de Belgique, by an "Interuniversity Poles of Attraction Program-Belgian State, Prime Minister's Office, Federal Office for Scientific, Technical and Cultural Affairs" and by a grant from the European Community (PACAP and VIP Euronetwork project).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.
Recipient of a post-doctoral fellowship from the F.R.S.M. (Belgium).
§ Recipient of a Marie Curie training grant from the European Commission. Present address: Dpt. Bioquimica y Biologia Molecular, Facultad de Medicine, Universitad de Alcalà, Ctra Madrid Barcelona, Km: 33,600, 28871 Madrid, Espana.
¶ To whom correspondence should be addressed. Tel.: 32 2 555 62 11; Fax: 32 2 555 62 30; E-mail: mawaelbr@ulb.ac.be.
Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M007696200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: VIP, vasoactive intestinal peptide; TM, transmembrane helix; PACAP, pituitary adenylate cyclase-activating polypeptide; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; GRF, growth hormone releasing factor; PTH, parathyroid hormone.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Christophe, J. (1993) Biochim. Biophys. Acta 1154, 183-199[Medline] [Order article via Infotrieve] |
2. | Rawlings, S. R., and Hezareh, M. (1996) Endocr. Rev. 17, 4-29[Medline] [Order article via Infotrieve] |
3. |
Vaudry, D.,
Gonzalez, B. J.,
Basille, M.,
Yon, L.,
Fournier, A.,
and Vaudry, H.
(2000)
Pharmacol. Rev.
52,
269-324 |
4. |
Harmar, A. J.,
Arimura, A.,
Gozes, I.,
Journot, L.,
Laburthe, M.,
Pisegna, J. R.,
Rawlings, S. R.,
Robberecht, P.,
Said, S. I.,
Sreedharan, S. P.,
Wank, S. A.,
and Waschek, J. A.
(1998)
Pharmacol. Rev.
50,
265-270 |
5. | Usdin, T. B., Bonner, T. I., and Mezey, E. (1994) Endocrinology 135, 2662-2680[Abstract] |
6. |
Vertongen, P.,
Schiffmann, S. N.,
Gourlet, P.,
and Robberecht, P.
(1998)
Ann. N. Y. Acad. Sci.
865,
412-415 |
7. | Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P. H., and Journot, L. (1993) Nature 365, 170-175[CrossRef][Medline] [Order article via Infotrieve] |
8. | Van Rampelbergh, J., Poloczek, P., Francoys, I., Delporte, C., Winand, J., Robberecht, P., and Waelbroeck, M. (1997) Biochim. Biophys. Acta 1357, 249-255[Medline] [Order article via Infotrieve] |
9. | Rawlings, S. R. (1994) Mol. Cell. Endocrinol. 101, C5-C9[CrossRef][Medline] [Order article via Infotrieve] |
10. | Vilardaga, J. P., De Neef, P., Di Paolo, E., Bollen, A., Waelbroeck, M., and Robberecht, P. (1995) Biochem. Biophys. Res. Commun. 211, 885-891[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Hashimoto, H.,
Ogawa, N.,
Hagihara, N.,
Yamamoto, K.,
Imanishi, K.,
Nogi, H.,
Nishino, A.,
Fujita, T.,
Matsuda, T.,
Nagata, S.,
and Baba, A.
(1997)
Mol. Pharmacol.
52,
128-135 |
12. |
Holtmann, M. H.,
Ganguli, S.,
Hadac, E. M.,
Dolu, V.,
and Miller, L. J.
(1996)
J. Biol. Chem.
271,
14944-14949 |
13. |
Holtmann, M. H.,
Hadac, E. M.,
and Miller, L. J.
(1995)
J. Biol. Chem.
270,
14394-14398 |
14. | Knudsen, S. M., Tams, J. W., Wulff, B. S., and Fahrenkrug, J. (1997) FEBS Lett. 412, 141-143[CrossRef][Medline] [Order article via Infotrieve] |
15. | Vilardaga, J. P., Di Paolo, E., Bialek, C., De Neef, P., Waelbroeck, M., Bollen, A., and Robberecht, P. (1997) Eur. J. Biochem. 246, 173-180[Abstract] |
16. | Grauschopf, U., Lilie, H., Honold, K., Wozny, M., Reusch, D., Esswein, A., Schafer, W., Rucknagel, K. P., and Rudolph, R. (2000) Biochemistry 39, 8878-8887[CrossRef][Medline] [Order article via Infotrieve] |
17. | Di Paolo, E., Vilardaga, J. P., Petry, H., Moguilevsky, N., Bollen, A., Robberecht, P., and Waelbroeck, M. (1999) Peptides 20, 1187-1193[CrossRef][Medline] [Order article via Infotrieve] |
18. | Du, K., Nicole, P., Couvineau, A., and Laburthe, M. (1997) Biochem. Biophys. Res. Commun. 230, 289-292[CrossRef][Medline] [Order article via Infotrieve] |
19. | Gaudin, P., Couvineau, A., Rouyer-Fessard, C., Maoret, J. J., and Laburthe, M. (1999) Biochem. Biophys. Res. Commun. 254, 15-20[CrossRef][Medline] [Order article via Infotrieve] |
20. | Ciccarelli, E., Vilardaga, J. P., De Neef, P., Di Paolo, E., Waelbroeck, M., Bollen, A., and Robberecht, P. (1994) Regul. Pept. 54, 397-407[CrossRef][Medline] [Order article via Infotrieve] |
21. | Couvineau, A., Rouyer-Fessard, C., Darmoul, D., Maoret, J. J., Carrero, I., Ogier-Denis, E., and Laburthe, M. (1994) Biochem. Biophys. Res. Commun. 200, 769-776[CrossRef][Medline] [Order article via Infotrieve] |
22. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York |
23. | Gourlet, P., De Neef, P., Cnudde, J., Waelbroeck, M., and Robberecht, P. (1997) Peptides 18, 1555-1560[CrossRef][Medline] [Order article via Infotrieve] |
24. | Salomon, Y., Londos, C., and Rodbell, M. (1974) Anal. Biochem. 58, 541-548[Medline] [Order article via Infotrieve] |
25. | Gourlet, P., Vandermeers, A., Vandermeers-Piret, M. C., De Neef, P., Waelbroeck, M., and Robberecht, P. (1996) Biochim. Biophys. Acta 1314, 267-273[Medline] [Order article via Infotrieve] |
26. | Gourlet, P., Vandermeers, A., Vertongen, P., Rathe, J., De Neef, P., Cnudde, J., Waelbroeck, M., and Robberecht, P. (1997) Peptides 18, 1539-1545[CrossRef][Medline] [Order article via Infotrieve] |
27. | Rolz, C., Pellegrini, M., and Mierke, D. F. (1999) Biochemistry 38, 6397-6405[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Rekasi, Z.,
Varga, J. L.,
Schally, A. V.,
Halmos, G.,
Groot, K.,
and Czompoly, T.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
1218-1223 |
29. |
Van Rampelbergh, J.,
Juarranz, M. G.,
Perret, J.,
Bondue, A.,
Solano, R. M.,
Delporte, C.,
De Neef, P.,
Robberecht, P.,
and Waelbroeck, M.
(2000)
Br. J. Pharmacol.
130,
819-826 |
30. |
Juarranz, M. G.,
Van Rampelbergh, J.,
Gourlet, P.,
De Neef, P.,
Cnudde, J.,
Robberecht, P.,
and Waelbroeck, M.
(1999)
Eur. J. Biochem.
265,
449-456 |
31. |
Juarranz, M. G.,
Van Rampelbergh, J.,
Gourlet, P.,
De Neef, P.,
Cnudde, J.,
Robberecht, P.,
and Waelbroeck, M.
(1999)
Mol. Pharmacol.
56,
1280-1287 |
32. | Gourlet, P., Vilardaga, J. P., De Neef, P., Waelbroeck, M., Vandermeers, A., and Robberecht, P. (1996) Peptides 17, 825-829[CrossRef][Medline] [Order article via Infotrieve] |
33. | Gourlet, P., Vilardaga, J. P., De Neef, P., Vandermeers, A., Waelbroeck, M., Bollen, A., and Robberecht, P. (1996) Eur. J. Biochem. 239, 349-355[Abstract] |
34. | Gourlet, P., Vandermeers, A., Vandermeers-Piret, M. C., Rathe, J., De Neef, P., and Robberecht, P. (1996) Regul. Pept. 62, 125-130[CrossRef][Medline] [Order article via Infotrieve] |
35. | Dong, M., Asmann, Y. W., Zang, M., Pinon, D. I., and Miller, L. J. (2000) J. Biol. Chem. |
36. |
Dong, M.,
Wang, Y.,
Hadac, E. M.,
Pinon, D. I.,
Holicky, E.,
and Miller, L. J.
(1999)
J. Biol. Chem.
274,
19161-19167 |
37. |
Dong, M.,
Wang, Y.,
Pinon, D. I.,
Hadac, E. M.,
and Miller, L. J.
(1999)
J. Biol. Chem.
274,
903-909 |
38. | Vilardaga, J. P., Di Paolo, E., De Neef, P., Waelbroeck, M., Bollen, A., and Robberecht, P. (1996) Biochem. Biophys. Res. Commun. 218, 842-846[CrossRef][Medline] [Order article via Infotrieve] |
39. | Pandol, S. J., Dharmsathaphorn, K., Schoeffield, M. S., Vale, W., and Rivier, J. (1986) Am. J. Physiol. 250, G553-G557[Medline] [Order article via Infotrieve] |
40. | Andrews, P. (1986) Trends Pharmacol. Sci. 7, 148-151 |