From the
Secretin and vasoactive intestinal polypeptide (VIP) receptors
are closely related G protein-coupled receptors in a recently described
family possessing a large amino-terminal ectodomain. We postulated that
this domain might be critical for agonist recognition and therefore
constructed a series of six chimeric receptors, exchanging the amino
terminus, the first extracellular loop, or both in secretin and VIP
receptors. Constructs were expressed in COS cells and characterized by
cAMP generation and binding of both secretin and VIP radioligands. Wild
type receptors demonstrated high affinity binding of respective ligands
(IC
Secretin and vasoactive intestinal polypeptide (VIP)
The
ligand-binding domains of several G protein-coupled receptors have been
well explored(5, 6) . Investigations have largely
focused on members of the
To date, however,
few details exist regarding molecular mechanisms of ligand binding and
receptor activation in the calcitonin-parathyroid hormone receptor
family(8, 9) . In this work, we focus on the receptors
for secretin and VIP that are 44% identical in sequence (10, 11, 12) and signal similarly(13) .
In spite of their structural and functional similarities, however, they
display distinct specificity for agonist ligands. This makes them ideal
candidates for analysis using chimeric receptor proteins.
Using this
approach, we have demonstrated that 1) the extracellular amino terminus
of the VIP receptor is the critical domain responsible for the
selectivity of activation by VIP; 2) the same domain of the secretin
receptor is also important for the selectivity of high affinity binding
and activation by secretin but is not sufficient by itself to provide
this function; 3) the first extracellular loop of the secretin receptor
is also a key domain for selective agonist binding and activation; and
4) in this series of constructs high affinity secretin binding
correlated well with secretin-like biological activity, whereas VIP
binding could be dissociated from its biological responses.
We cloned the secretin receptor cDNA from a rat pancreatic
cDNA library (11) and acquired the rat pancreatic VIP receptor
cDNA from Professor Nagata (Osaka Bioscience Institute, Osaka, Japan).
Synthetic rat secretin and VIP were from Peninsula Laboratories.
[Tyr
The chimeric receptors utilized in this study include
portions of wild type rat secretin and VIP receptors ().
Chimeras were constructed by excising sequences of the wild type
receptor cDNAs and replacing them with the corresponding sequences of
the other receptor. Naturally occurring restriction sites were used
when possible; otherwise PCR mutagenesis was performed.
All PCR reactions were
performed with Taq DNA polymerase in a thermocycler running 35
cycles: 94 °C for 1 min, 55 °C for 2 min, and 72 °C for 3
min. Products were separated on 1% agarose gels and purified on Qiaex
resin. Transformations of constructs in pcDNAI/Neo and pcDM8 were
performed in MC1061-P3 cells, whereas those in pBK-CMV were performed
in XLI-Blue MRF`. Correct sequences of all constructs were confirmed by
DNA sequencing using the dideoxynucleotide chain termination
method(15) .
Constructs were expressed in COS-7 cells maintained in
culture in Dulbecco's modified Eagle's medium with 5% fetal
clone 2. Cells were transfected with 2-4 µg of DNA using a
modification of the DEAE-dextran method, which included
Me
Intracellular cAMP levels were assayed with a
[
The
generation of standard curves and the measurement of cAMP levels in
supernatants of cell lysates were performed as per the
manufacturer's instructions. Radioactivity was quantified by
scintillation counting in a Beckman LS6000. All assays were performed
in duplicate and repeated at least 3 times.
Binding studies were performed with enriched plasma membranes
prepared from COS cells 72 h after transfection, as we previously
described(11) . The secretin analogue,
[
Receptor-bearing
membrane preparations (1-10 µg) were incubated with a
constant amount of radioiodinated peptide (3-5 pM VIP or
secretin analogue(11) ) and increasing concentrations of
non-radiolabeled peptide (0-1 µM) for 1 h at room
temperature in Krebs-Ringer-HEPES medium. Bound and free radioligand
were separated using a Skatron cell harvester with glass fiber filter
mats that had been soaked in 0.3% polybrene with bound radioactivity
quantified in a
All observations were repeated at least three times in
independent experiments and are expressed as means ± S.E.
Differences were determined by using the Mann-Whitney non-parametric
test for unpaired values with p < 0.05 considered to be
significant.
The converse chimera, the VIP
receptor with the amino-terminal domain of the secretin receptor
(Se
In searching for additional receptor domains
necessary for secretin responsiveness, a chimera was generated that
possessed the secretin receptor sequence for the extracellular amino
terminus as well as the first extracellular loop, with the rest of the
receptor corresponding to the VIP receptor sequence. The additional
replacement of the first extracellular loop by the analogous secretin
receptor sequence provided full responsiveness to secretin (EC
The first extracellular loop of the secretin receptor did not
by itself provide a substantial change in the selectivity of the VIP
receptor (Se
The secretin receptor with the first extracellular
loop replaced with the corresponding sequence of the VIP receptor
responded maximally only to secretin (within the range of
concentrations used). The EC
Binding data for all constructs are summarized in Fig. 4. All
receptors that had demonstrated biological responses to nanomolar
concentrations of secretin bound secretin with high affinity (IC
General rules have not yet evolved for mechanisms of binding
and activation of receptors in the recently described
calcitonin-parathyroid hormone family that have as natural agonists
peptide ligands with moderately large pharmacophoric domains. In this
work, we have utilized a chimeric receptor approach to focus primarily
on the potential role of the amino terminus in agonist binding and
biological responsiveness. We have chosen the secretin and VIP
receptors, which are structurally quite similar yet have distinct
agonist specificities. This complements the deletion mutagenesis
approach recently utilized for the parathyroid hormone
receptor(8, 9) .
The amino terminus is a particularly
interesting domain in this family, being quite large (approximately 150
residues) and cysteine-rich. This family of receptors has 8 conserved
Cys residues in predicted ectodomains, with 6 of these in the amino
terminus. Further evidence for the importance of this domain is a
naturally occurring mutation in the growth hormone-releasing hormone
receptor. A mis-sense mutation changing Asp-60 (conserved throughout
the family) to Gly is reported to disrupt receptor function in the
Dwarf Little Mouse(18) .
By replacing the amino terminus of
the secretin receptor with the corresponding sequence of the VIP
receptor (Ve
The pattern of secretin-specific determinants on
the receptor are different than the VIP-specific determinants. The
reciprocal chimeric construct in which the amino terminus of the
secretin receptor (Se
The
first extracellular loop of the secretin receptor (Se
The Ve
Organizing
the chimeras according to their responsiveness to secretin and VIP
facilitates envisioning the distribution of determinants critical for
receptor activation by the respective ligands (Fig. 2). Three
groups can be distinguished based on secretin responsiveness: the wild
type secretin receptor and the Se
Wild type secretin and VIP receptors exhibit sensitive responses to
their natural ligands, with selectivities of at least 2 orders of
magnitude. For both receptors, the presence of the amino terminus is
necessary for the activation of the receptors by low concentrations of
native agonists. Although this domain of the VIP receptor can alone
account for a selectivity of 2 orders of magnitude, in the secretin
receptor additional determinants are necessary to activate the receptor
with physiological concentrations and high selectivity. Further
addition of the first extracellular loop of the secretin receptor can
provide full responsiveness to secretin.
The ligand binding studies
nicely complement the biological activity studies. These showed high
affinity binding of secretin to the wild type secretin receptor and the
Se
In contrast, VIP
bound with high affinity to both wild type VIP and secretin receptors
and to almost all chimeric constructs. However, this binding only
resulted in a biological response at VIP-like receptors. At the
secretin receptor, the cAMP response generated by VIP correlated with
its low affinity binding to the site that bound the secretin
radioligand. This is a novel observation that was previously not
possible with receptors on native cells in which more than one receptor
might have been present. It was previously felt that there were
distinct ``secretin-preferring'' receptors and
``VIP-preferring'' receptors(19) . Whereas distinct
wild type secretin and VIP receptors do exist and may coexist on a
single native cell, binding data for the recombinant wild type rat
secretin receptor in a system in which we can be certain that a VIP
receptor does not exist demonstrate both types of binding. Biological
activities, however, clearly distinguish between these. It will be of
interest to determine if high affinity binding of VIP is a property of
all secretin receptors or if this is peculiar to the rat receptor.
In this study we have demonstrated that the amino terminus of
secretin and VIP receptors with their characteristic structural
features play a critical role for the specificity of binding affinity
and receptor activation. For the secretin receptor, other extracellular
sites are critical as well. Our results provide a basis for future
studies defining the specific determinants for affinity and specificity
of these receptors. Although chimeric constructs substituting smaller
portions of these receptors may localize determinants more precisely,
roles of distinct amino acid residues will also need to be addressed.
The numbers in
parentheses represent the amino acid codons in the wild type receptor
proteins.
We acknowledge the excellent technical assistance of
E. Holicky, D. Pinon, and I. Ferber and the excellent secretarial
assistance of S. Erickson.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
values (in nM): at the secretin receptor: 2.2
for secretin, >1000 for VIP; at the VIP receptor: 2.2 for VIP,
>1000 for secretin) and appropriately sensitive and selective
biological responses (EC
values (in nM): at the
secretin receptor: 1.5 for secretin, 127 for VIP; at the VIP receptor:
1.0 for VIP, 273 for secretin). Replacement of the secretin receptor
amino terminus with that of the VIP receptor resulted in biological
responsiveness typical of the VIP receptor (EC
=
120 nM for secretin, 1.7 nM for VIP). The converse
was not true, with this domain of the secretin receptor not able to
provide the same response when incorporated into the VIP receptor
(EC
= 50 nM for VIP, 30 nM for
secretin). The addition of both the first loop and the amino terminus
of the secretin receptor was effective in yielding a secretin
receptor-like response (EC
= 2.0 nM for
secretin, 47 nM for VIP). All chimeric constructs expressing
selectivity for secretin-stimulated activity bound this hormone with
high affinity (IC
= 0.2-2.2 nM);
however, there was divergence between VIP binding and biological
activity. Thus, the amino terminus of secretin and VIP receptors plays
a key role in agonist recognition and responsiveness, with the first
loop playing a critical complementary role for the secretin receptor.
(
)receptors belong to a recently described family of G
protein-coupled receptors (1, 2, 3) that are
only 12% homologous with other members of this superfamily and lack the
signature sequences that are conserved in the
-adrenergic receptor
family. Notable features of this group include a long extracellular
amino terminus and a series of 8 conserved cysteine residues in the
predicted ectodomain. Of interest, ligands for these receptors tend to
be moderately large peptides having diffuse pharmacophoric
domains(4) . A central hypothesis for the current work is that
these large and complex domains interact with each other.
-adrenergic receptor family, which bind
very small molecules in the outer third of the plasma membrane between
transmembrane helices(5) . Receptors in this family that bind
small peptide ligands have also been examined recently, suggesting a
theme that proximal loop regions are important binding
determinants(7) . Binding themes have also been established for
the metabotropic glutamate receptors, the glycoprotein hormone
receptors, and the thrombin receptor(6) .
,pNO
-Phe
]rat
secretin 27 was synthesized as we described(11) . All other
reagents were analytical grade.
Receptor Constructs
Ve
This chimera was
generated using a unique restriction site for BstXI present in
analogous positions in VIP receptor (SLASLS
LVA) and secretin
receptor (SLAML
LVA) cDNAs. The HindIII-BstXI
fragment of the VIP receptor cDNA was ligated into the secretin
receptor cDNA.
Se
The VIP receptor
sequence between codons 117 and 429 was amplified by PCR introducing a
new restriction site for BsrGI at the 5` end and a stop codon
and HindIII site at the 3` end. The PCR product was then
ligated into the secretin receptor cDNA.
V
Ve
A
74-base pair oligonucleotide was designed as a reverse primer carrying
the sequence of the first extracellular loop of the wild type VIP
receptor and including an RcaI site downstream of the
mutagenized region. PCR was performed using the previously generated
Vee
S
S chimera as a template. The PCR product was ligated into
the HindIII and RcaI sites of the wild type secretin
receptor construct.
Ve
This chimeric
construct was produced in the same way as VeS
e
S.
The wild type secretin receptor construct was used as a template
instead of Ve
S for PCR mutagenesis.
Se
Overlap
extension PCR (14) was used to construct this chimera, replacing
VIP receptor codons 168-183 with secretin receptor codons
175-191.
e
V
Se
The part of
SeV
e
V downstream of the BstXI site was
excised and ligated into the BstXI and NotI sites of
the wild type VIP receptor construct.
Receptor Expression
SO shock and treatment with 0.1 mM chloroquine
diphosphate (16). Transfected cells were harvested after 72 h.
Biological Activity Studies
H]cAMP assay kit from Diagnostic Products
Corporation (Los Angeles, CA). COS cells were harvested mechanically 72
h after transfection. These were washed with phosphate-buffered saline,
resuspended in Krebs-Ringer-HEPES medium incorporating 25 mM HEPES, pH 7.4, 104 mM NaCl, 5 mM KCl, 1.2
mM MgSO
, 2 mM CaCl
, 1 mM KH
PO
, 0.2% bovine serum albumin, 0.01%
soybean trypsin inhibitor, and 1 mM 3-isobutyl-1-methylxanthine. Hormonal stimulation was performed
for 10 min at 37 °C, and the reaction was stopped with ice-cold 6%
perchloric acid. The pH was adjusted to 6 with KHCO
, and
lysates were cleared by centrifugation at 2000 rpm for 10 min.
Ligand Binding Studies
I-Tyr
,pNO
-Phe
]rat
secretin 27, was synthesized and radioiodinated as we
described(11) . VIP was radioiodinated oxidatively and purified
by high pressure liquid chromatography(17) .
-counter. Non-specific binding, assessed in the
presence of excess unlabeled analogous peptide (1 µM secretin or VIP), represented less than 2.9 ± 0.4%, with
total binding representing 10 ± 1% of radioligand in the
incubation.
Statistical Analysis
Biological Activity
Both wild type receptors
showed a selectivity for their native agonist ligands of 2 or more
orders of magnitude (Fig. 1). The secretin receptor was activated
by secretin with an EC of 1.5 nM and by VIP with
an EC
of 127 nM. The VIP receptor was activated
by VIP with an EC
of 1.0 nM and by secretin with
an EC
of 273 nM. Biological activity data for
chimeric constructs are also shown in Fig. 1. These data are
further summarized in Fig. 2, in which the responsiveness to each
hormone was used to organize the data.
Figure 1:
Biological responses of wild type and
chimeric receptors. Shown are cAMP responses to secretin () and
VIP (
) in the wild type and chimeric secretin and VIP receptors.
Values are expressed as the means ± S.E. of at least three
independent experiments with data normalized relative to the maximal
response to one of the natural agonists. In all constructs, this
represented an increase of 2.4 ± 0.1 times the basal
levels.
Figure 2:
Cyclic AMP responses listed in the order
of responsiveness to each hormone. Shown are the means ± S.E. of
the concentrations of secretin and VIP that stimulated half-maximal
biological responses in at least three independent experiments. A, biological activity ordered according to the responsiveness
of chimeras to secretin; B, biological activity ordered
according to the responsiveness of chimeras to
VIP.
Replacing the amino terminus
of the secretin receptor with the same domain of the VIP receptor
(VeS) resulted in a construct that changed its specificity
from that of the secretin receptor to that of the VIP receptor. This
construct was activated by secretin with an EC
of 120
nM and by VIP with an EC
of 1.7 nM,
representing a 100-fold decrease in responsiveness to secretin and a
similar increase in responsiveness to VIP. This responsiveness to VIP
was not statistically different from the responsiveness of the wild
type VIP receptor (p = 0.38), and it had a selectivity
of 2 orders of magnitude between VIP and secretin responsiveness like
the wild type receptor. This result strongly suggested that the
extracellular amino terminus plays a critical role for receptor
activation by its natural ligand.
V), did not, however, exhibit analogous behavior.
Although its maximal increase in cAMP was comparable with that of other
constructs, its responsiveness to VIP was reduced 50-fold (EC
= 50 nM) while gaining only partial
responsiveness to secretin (EC
= 30 nM) (p < 0.0001). This construct responded to VIP similarly to
the wild type secretin receptor (p = 0.12), further
supporting the importance of the amino terminus of the VIP receptor for
VIP responsiveness.
= 2 nM) (p = 0.37).
Interestingly, this chimera was also activated by VIP, with an
EC
of 47 nM, demonstrating similar responsiveness
to VIP to that of the wild type secretin receptor (p =
0.15).
V). Like the chimera with only the amino
terminus replaced (Se
V), the secretin responsiveness was
slightly better (EC
= 47 nM) than that of
the wild type VIP receptor (p = 0.058). Because this
construct still possessed the amino terminus of the wild type VIP
receptor, it was very responsive to VIP (EC
= 0.5
nM).
for secretin was 4
nM, whereas the EC
for VIP was greater than 500
nM.
Ligand Binding
Receptor binding affinity was
determined by homologous competition whenever possible. For low
affinity ligands, this was not possible and was confirmed by using such
a ligand to compete for binding of a higher affinity radioligand. Fig. 3illustrates the competition binding data for the parent
wild type receptor. At the wild type secretin receptor, competition for
secretin radioligand binding with secretin (IC =
2.2 nM) and VIP (IC
> 1 µM)
suggested relative binding affinities that correlated with cAMP
responses to these hormones. VIP radioligand binding to the wild type
VIP receptor yielded analogous data with VIP having an IC
of 2.2 nM and secretin having an IC
greater
than 1 µM.
Figure 3:
Binding
characteristics of wild type receptors. Shown are results from
competition binding experiments utilizing enriched plasma membranes
from COS cells transfected with wild type receptor constructs and the
noted radioligands. Values are expressed as the means ± S.E. of
at least three independent experiments with data representing saturable
binding relative to the control condition in the absence of
competitor.
Although direct binding of the secretin
radioligand to the wild type VIP receptor confirmed its apparent low
affinity, the binding of the VIP radioligand to the wild type secretin
receptor yielded unexpected results. Homologous competition for that
radioligand demonstrated high affinity binding with an IC of 0.6 nM (Fig. 3); however, this binding did not
correlate with cAMP generation. VIP-stimulated cAMP generation
correlated better with the low affinity binding that was observed when
VIP competed for secretin radioligand binding. This suggests that there
is a separate and distinct non-biologically relevant high affinity
VIP-binding site on the rat secretin receptor. Studies are underway to
determine if this is an intrinsic property of the secretin receptor
from other species or if this is a unique property of the rat receptor.
= 0.2-2.2 nM), as well as binding VIP with
high affinity (IC
= 0.6-1.4 nM). All
constructs that demonstrated selectivity for biological responses that
was similar to that of the wild type VIP receptor displayed high
affinity binding of VIP (IC
= 1.0-9.3
nM) and low affinity or absent binding of secretin. The
Se
V construct that showed no selectivity for biological
responses to either hormone did not bind either secretin or VIP
radioligand with adequate affinity to interpret binding data.
Figure 4:
Summary of competition binding data. Shown
are the means ± S.E. of the concentrations of secretin and VIP
that inhibited half of the saturable ligand binding of the noted
radioligand in at least three independent experiments ordered according
to the responsiveness to secretin.
Competition binding curves for key chimeric constructs are
illustrated in Fig. 5. The VeS construct bound VIP
with high affinity. The Se
e
V construct, like
the wild type secretin receptor, bound both secretin and VIP with high
affinity.
Figure 5:
Binding characteristics of chimeric
constructs. Shown are the results from competition binding experiments
utilizing enriched plasma membranes from the COS cells transfected with
chimeric constructs. All curves represent homologous
competition of the same radioligand and cold peptide noted. Values are
expressed as the means ± S.E. of at least three independent
experiments with data representing saturable binding relative to the
control condition in the absence of
competitor.
S), a chimera was generated that behaved like
the VIP receptor. It responded to VIP with a similar EC
to
that of the wild type VIP receptor (EC
= 1.7
nMversus EC
= 1.0 nM) (p = 0.38). Additional receptor domains will likely
also contribute to binding and activation of this receptor. It is
possible that such domains are sufficiently provided by homologous or
identical residues within the secretin receptor sequence in this
chimeric construct.
V) replaced this domain in the VIP
receptor displayed only a slight improvement in its secretin
responsiveness (p = 0.041). The shift in EC
from 273 nM secretin for the wild type VIP receptor to
30 nM secretin for this chimeric construct is consistent with
the amino terminus contributing to receptor activation but clearly does
not account for full responsiveness (p < 0.0001).
V)
also contributed a small amount to the secretin responsiveness of the
VIP receptor. Adding both the amino terminus and the first
extracellular loop of the secretin receptor together
(Se
e
V) resulted in a marked effect in the
secretin responsiveness of the VIP receptor, being similar to that of
the wild type secretin receptor (p = 0.37). Of
interest, the VIP responsiveness of this construct decreased from that
of the VIP receptor to that of the secretin receptor, consistent with
the critical role of the amino terminus in VIP effects. As expected,
such an effect was not seen when only the first extracellular loop was
replaced in the VIP receptor.
e
S
chimera representing the secretin receptor with the extracellular amino
terminus and the first extracellular loop of the VIP receptor further
confirmed this evolving theme. The amino terminus is critical for
receptor activation by VIP, and the first extracellular loop did not
contribute further to distinguishing VIP from secretin.
e
V and
Ve
S chimeras responded well to secretin; the
Se
V and Se
V chimeras showed intermediate
responses to secretin; and Ve
S,
Ve
e
S, and the wild type VIP receptor showed
poor responses to secretin. Based on the responsiveness to VIP, again
three groups could be distinguished. The wild type VIP receptor and the
Se
V, Ve
S, and Ve
e
S
chimeras responded well to VIP; Se
V and
Se
e
V showed intermediate responses; and
Ve
S and the wild type secretin receptor responded poorly.
e
V and Ve
S chimeras, correlating
with the sensitive biological responses to secretin. All other
constructs and the wild type VIP receptor bound secretin poorly. The
correlation between high affinity secretin binding and biological
responses might suggest that the determinants critical for receptor
binding also mediate activation. However, a more detailed survey will
likely reveal distinct residues that will distinguish between
contributions to ligand binding and receptor activation.
Secretin-binding determinants are clearly present in the extracellular
amino terminus and in the first extracellular loop.
Table: Secretin-VIP receptor chimeras
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.