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NEUROPHARMACOLOGY
Juvantia Pharma Ltd., Turku, Finland (M.E., S.W., J.-M.S.); Department of Biology, Åbo Akademi University, Turku, Finland (A.B., P.P.); and Institute of Biomedicine/Anatomy, University of Helsinki, Helsinki, Finland (P.P.)
Received November 25, 2002; accepted February 14, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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;
Vilim et al., 1999),
prolactin-releasing peptide (PrRP) (Hinuma
et al., 1998), RFamide-related peptides (RFRPs)
(Hinuma et al., 2000), and
KiSS-1 (Ohtaki et al.,
2001).
NPFF, the first RFamide peptide identified in mammals, acts as a modulator
of morphine analgesia, tolerance, and dependence, and influences many
functions including pain mechanisms
(Panula et al., 1996).
Recently, two human receptors for NPFF have been identified: hNPFF1 and hNPFF2
(Bonini et al., 2000;
Elshourbagy et al., 2000;
Hinuma et al., 2000). These
receptor subtypes have different tissue localizations in human and rat
(Bonini et al., 2000). The
recently cloned NPFF2 receptor (named HLWAR77 by
Elshourbagy et al., 2000) has
been described as Gi/o-coupled when stably expressed in HEK 293
cells (Elshourbagy et al.,
2000) or CHO cells (Kotani et
al., 2001). Among the receptors with the highest amino acid
sequence homology to NPFF1 and NPFF2 are members of the orexin, human
neuropeptide Y (NPY), and cholecystokinin family, which have been implicated
in feeding (Bonini et al.,
2000). BIBP3226, an anorexigenic Y1 receptor ligand, has been
shown to bind to the NPFF1 receptor, thus further suggesting a potential role
of the NPFF1 receptor in regulation of feeding
(Bonini et al., 2000).
PrRP, the second RFamide peptide, was identified as the endogenous ligand
for the orphan G protein-coupled receptor (GPCR), hGR3
(Hinuma et al., 1998). The full
physiological role of PrRP remains to be elucidated, but it has been
envisioned to play a broader role in brain function than originally suggested,
and the ability to stimulate prolactin release may not represent its primary
biological function (Samson et al.,
2000). The hGR3 receptor (named GPR10 by
Marchese et al., 1995; and in
this paper referred to as the hPrRP receptor) and its rat counterpart, UHR-1,
have been shown to couple to at least Gq and Gi but not
to Gs in CHO cells (Hinuma et al.,
1998,
1999).
Mammalian genes encoding a third class of RFamide peptides, namely hRFRP1
and hRFRP3 and their receptor OT7T022, have recently been reported
(Hinuma et al., 2000). OT7T022
is activated by NPFF and corresponds to the NPFF1 receptor characterized by
Bonini et al. (2000). RFRPs
(more specifically hRFRP1) have been shown to regulate prolactin secretion
(Hinuma et al., 2000), but
additional physiological functions cannot be ruled out.
KiSS-1 is a metastasis suppressor gene that encodes the fourth class of
RFamide peptides (Ohtaki et al.,
2001). The gene product of KiSS-1, also known as
"metastin," was found to be the endogenous ligand of an orphan
GPCR, hOT7T175 (Ohtaki et al.,
2001).
The cellular mechanisms by which NPFF, PrRP, and RFRP exert their functions
in vivo are poorly understood. To clarify the cellular and pharmacological
actions activated by these RFamide peptides, well characterized, functional in
vitro assays on recombinant cell lines and/or cell lines expressing these
receptors endogenously need to be developed. In the present report, we have
used [35S]guanosine-5'-O-(3-thio)triphosphate
([35S]GTP
S) binding induced by RFamide peptides on the
hNPFF2 and hPrRP receptors stably transfected in CHO cells (CHO-hNPFF2 and
CHO-hPrRP-R, respectively) to determine agonist efficacies and rank orders of
potency. The agonist potencies of RFamide peptides were compared with their
affinities obtained in competition binding assays with cell membrane
preparations. Receptor autoradiography was conducted to visualize and
quantitate the NPFF binding sites in rat cervical spinal cord sections to
investigate the binding properties of RFamide peptides at the NPFF receptor in
a native environment.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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S was purchased from PerkinElmer Life Sciences
(Boston, MA). The specific activity of [35S]GTPS was 1250
Ci/mmol.
125I-(1DMe
[PDB]
)Y8Fa Binding Assays on Rat Spinal Cord
Membranes. Rat spinal cord membrane preparations and binding assays were
performed using essentially the method for 125I-YLFQPQRF-amide
(125I-Y8Fa) binding as described by Allard et al.
(1989) and modified by Payza
and Yang (1993). Briefly,
membranes (100–160 µg/sample) were incubated with 0.02 to 0.07 nM
125I-(1DMe
[PDB]
)Y8Fa, and various concentrations of ligands in 50 mM
Tris-HCl, pH 7.5 at RT, 60 mM NaCl, 1 mM MgCl2, 3 µg/ml
aprotinin, 7.5 g/l BSA, and 30 µM bestatin. Total binding (TB) and
nonspecific binding (NSB) were determined in the absence and presence,
respectively, of 1 µM (1DMe
[PDB]
)Y8Fa. After 45 min at RT, incubations were
terminated by rapid filtration (Tomtec Harvester96; Tomtec Inc., Hamden, CT)
through GF/B glass fiber filter mats (presoaked at 4°C in 200 ml of 50 mM
Tris-HCl, 60 mM NaCl, 1 mM MgCl2, 1 g/l BSA, 5 g/l
polyethyleneimine, pH 7.4 at 4°C for 45 min). Filters were washed four
times with 5 ml of ice-cold wash buffer (50 mM Tris-HCl, 60 mM NaCl, 1 mM
MgCl2, pH 7.4 at 4°C). Specific binding (SB) was calculated as
TB - NSB. The SB in the presence of various concentrations of test compounds
was expressed as percentage of control SB, which was obtained as the TB in the
absence of any competing compound minus NSB. Analysis of competition binding
experiments was carried out by nonlinear least squares curve fitting with Hill
slopes (nH) being set to unity
(Devillers et al., 1994).
Affinity constants (Ki) were calculated from the
IC50 values according to the Cheng-Prusoff equation
(Cheng and Prusoff, 1973). The
KD for this purpose was 0.19 nM, as determined in a pilot
study.
125I-(1DMe [PDB] )Y8Fa Binding Assay on CHO-hNPFF2 Membranes. Recombinant CHO cells expressing the hNPFF2 receptor (Euroscreen S.A., Brussels, Belgium) were grown in Ham's F12 medium (Nutrient Mixture Ham's F12; Invitrogen, Glasgow, UK) supplemented with 10% fetal bovine calf serum and 400 µg/ml G418. Cells were harvested in phosphate-buffered saline and frozen at -70°C. To prepare membranes, thawed cell pellets were homogenized on ice in a Potter-Elvehjem homogenizer in 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5 at RT supplemented with 320 mM sucrose. The nuclear pellet obtained by centrifugation of the homogenate at 1,000g for 15 min at 4°C was discarded. From the supernatant, a membrane fraction was collected by centrifugation at 48,000gmax for 30 min at 4°C. The 48,000g pellet was re-suspended in 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5 at RT without sucrose and centrifuged again at 48,000gmax for 30 min. Binding of 125I-(1DMe [PDB] )Y8Fa to membranes (2.5 µg/sample) of hNPFF2 receptor expressing CHO cells and analysis of the binding experiments were carried out as described above for rat spinal cord membranes. The KD used in the analysis of the competition binding experiments was 0.10 nM, as determined in a pilot study.
125I-PrRP Binding Assay on CHO-hPrRP-R Membranes. Recombinant CHO cells expressing the human PrRP (hPrRP) receptor hGR3/GPR10 (Euroscreen S.A.) were grown, and membranes from these cells were prepared by the same method as described above for hNPFF2 expressing CHO cells.
125I-PrRP20 competition binding experiments were performed by incubating membranes (0.75 µg/sample) with 0.02 to 0.05 nM iodinated peptide and various concentrations of ligands in 25 mM Hepes, pH 7.4 at RT, 1 mM CaCl2, 5 mM MgCl2, 3 µg/ml aprotinin, 30 µM bestatin, and 7.5 mg/ml BSA. NSB was determined in the presence of 1 µM hPrRP20. Incubations were terminated after 45 min at RT as described above for rat spinal cord membranes except that 25 mM Hepes, 1 mM CaCl2, 5 mM MgCl2, and 0.5 M NaCl, pH 7.4, at 4°C was used as the washing buffer. The analysis of the binding data was also carried out as described above using a KD value of 0.3 nM, as determined in saturation binding experiments.
[35S]GTPS Binding Assay. The agonist
activity of various ligands was determined by their ability to stimulate the
receptor-mediated binding of [35S]GTPS to G proteins in
membranes of CHO-hNPFF2 or CHO-hPrRP-R cells. Membranes (2 and 10 µg/sample
of CHO-hNPFF2 and CHO-hPrRP-R cells, respectively) were incubated in 50 mM
Tris-HCl, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 1 µM GDP,
20 mM NaCl (CHO-hNPFF2 assay), or 100 mM NaCl (CHO-hPrRP-R assay), pH 7.4 at
RT with 6 to 12 concentrations of test ligands and a tracer concentration of
[35S]GTPS (0.07–0.16 nM). After a 60-min incubation at
RT (30-min preincubation without label followed by a 30-min stimulation after
addition of label in CHO-hNPFF2 assay or 60 min stimulation in CHO-hPrRP-R
assay without preincubation), the reaction was terminated by rapid vacuum
filtration through glass fiber filters. Filters were washed four times with 5
ml of ice-cold wash buffer (20 mM Tris-HCl, 5 mM MgCl2, 1 mM EDTA,
pH 7.4 at RT), dried, and counted for radioactivity in a scintillation
counter. Experimental results were calculated with nonlinear least squares
curve fitting. Agonist effects were normalized against the stimulation
obtained with reference compounds (1DMe
[PDB]
)Y8Fa, in the case of the hNPFF2
receptor, and hPrRP20, in the case of the hPrRP receptor. The response of the
reference agonists was set as 100%. Experiments were repeated at least three
times, unless indicated otherwise.
Quantitative Receptor Autoradiography. Autoradiography was performed
on rat spinal cord sections with 125I-(1DMe
[PDB]
)Y8Fa and was carried
out essentially as described earlier for 125I-Y8Fa
(Allard et al., 1992). Coronal
20-µm cryosections at the cervical level of the rat spinal cord were
collected onto poly-L-lysine-coated slides (Menzel-Gläser;
Merck, Darmstadt, Germany) and stored dry at -70°C. The mounted sections
were rehydrated in 50 mM Tris-HCl (pH 7.5), 140 mM NaCl and 0.5% BSA for 20
min at RT. The slides were washed twice with ice-cold 50 mM Tris-HCl (pH 7.5)
for 2 min before the incubation with 0.05 nM 125I-(1DMe
[PDB]
)Y8Fa in 50
mM Tris-HCl (pH 7.5), 120 mM NaCl, 0.5% BSA, 0.1 mM bestatin, 1 mM EDTA, and 3
µM aprotinin. Displacement of 125I-(1DMe
[PDB]
)Y8Fa was analyzed by
including unlabeled competing agents at the following concentrations: rNPSF
(500 nM, 5 nM), NPFF (20 nM, 0.2 nM), (1DMe
[PDB]
)Y8Fa (20 nM, 0.2 nM), bPrRP20 (200
nM, 2 nM), hRFRP-1 (1 µM, 10 nM), hRFRP(6G)-3 (1 µM, 10 nM), BIBP3226 (1
µM, 10 nM), and substance P (1 µM). The concentrations of the ligands
[except hRFRP(6G)-3 and BIBP3226, which were tested at 10 nM and 1 µM] were
chosen to represent approximately the Ki and the 100-fold
Ki value (Table
1). After 60 min of incubation at RT, the slides were washed three
times for 2 min in ice-cold Tris-HCl (pH 7.5) and were finally dipped in
ice-cold distilled water, air-dried, and exposed on film (Biomax MR; Eastman
Kodak, Rochester, NY). Autoradiographic films were analyzed using the MCID
image analysis system (Imaging Research, St. Catherines, ON, Canada). The
optical density of the area of the upper dorsal horn (laminae I–II) was
measured and the integrated optical density values were determined based on a
calibration curve derived from 14C standards (American Radiolabeled
Chemicals, St. Louis, MO) exposed simultaneously with the samples to the
films. The means ± S.E.M. of three animals and three sections in each
area of every animal are given as the quantitative assessment of ligand
binding activity. Statistical analysis was performed by one-way analysis of
variance of grouped data (Newman-Keuls test; MicroComputer Specialists,
Wynnewood, PA).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). In
accordance with previous results, the high affinity of NPFF is lost in its
nonamidated analog, NPFF-OH (Payza et al.,
1993; Mazarguil et al.,
2001). rNPSF had a 40-fold and 7-fold lower affinity than NPFF for
the rNPFF and hNPFF2 receptors, respectively. hNPY and the C-terminal fragment
18–36 of hNPY (hNPY18-36) had an affinity of about 1 µM for the rNPFF
receptor and a slightly lower affinity for the hNPFF2 receptor. BIBP3226,
which has been designed to mimic the C terminus of the NPY molecule
(Rudolf et al., 1994) bound to
the rNPFF and hNPFF2 receptors with affinities of 390 ± 50 and 640
± 80 nM, respectively. The human endogenous RFamide peptides hPrRP20, hPrRP31, hRFRP-1, and hRFRP-3 had affinities better than 20 nM for the rNPFF receptor, whereas the affinities of these RFamide peptides for the hNPFF2 receptor were better than 60 nM. The affinity of hPrRP20 was comparable to the affinity obtained for the invertebrate neuropeptide FMRFamide. Interestingly, even a peptide containing only eight C-terminal amino acids of hPrRP31, hPrRP24–31, was able to bind to the NPFF receptors with high affinity.
Agonist Activity of RFamide Peptides at the hNPFF2 Receptor.
[35S]GTPS binding experiments on membranes of CHO-hNPFF2
cells were performed in the presence of 20 mM NaCl and 1 µM GDP. The
agonist potencies are directly proportional to the binding affinities (Tables
1 and
2: r = 0.9549,
p < 0.0001). The efficacy of NPFF [79 ± 10% compared with
(1DMe
[PDB]
)Y8Fa] was lower than that for the metabolically stable analog (1DMe
[PDB]
)Y8Fa
(100% stimulation; Table 2).
Interestingly, all other tested RFamide peptides had efficacy approximately
similar to or even greater than that of (1DMe
[PDB]
)Y8Fa. The most efficacious among
the RFamide peptides was hPrRP31, for which an agonist response 1.7-fold that
of (1DMe
[PDB]
)Y8Fa was obtained (Table
2, Fig. 1A). The
dose-dependent increase in [35S]GTPS binding caused by the
RFamide peptides was clearly mediated through hNPFF2 receptors, since
membranes from CHO cells not expressing this receptor failed to give rise to
any significant increase in [35S]GTPS binding when
challenged with micromolar concentrations of hPrRP20, (1DMe
[PDB]
)Y8Fa, hPrRP31, or
hRFRP-1 (data not shown). Concerning agonist potencies, the following rank
order was observed in the [35S]GTPS binding assay on the
hNPFF2 receptor: (1DMe
[PDB]
)Y8Fa
NPFF > FMRFamide > hRFRP-1 hPrRP20
hPrRP24–31 hPrRP31 > rNPSF > hRFRP-3. The dose-response
curves for NPFF-OH, hNPY, and hNPY18-36 did not reach plateau and did not,
therefore, allow for calculation of reliable estimates of agonist potency
(data not shown).
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Affinities and Activities of RFamide Peptides at the hPrRP Receptor.
The affinities of various ligands for the hPrRP receptor hGR3/GPR10 expressed
in CHO cells were determined in 125I-PrRP competition binding
assays. The affinities of the two naturally occurring PrRPs, the full-length
peptide hPrRP31 and the 20-amino acid-long C-terminal fragment hPrRP20, at the
hPrRP receptor hGR3/GRP10 were 0.68 nM ± 0.06 nM and 1.0 ± 0.3
nM, respectively (Table 3). The
C-terminal octapeptide hPrRP24-31 displayed considerably lower affinity, with
a Ki of 47 ± 11 nM. This result is in line with the
findings published by Roland et al.
(1999). In contrast to the
generally quite high affinities of RFamide peptides at the NPFF receptor, the
hPrRP receptor showed strong discrimination among the related mammalian
RFamide peptides; NPFF, hRFRP-1, and hRFRP-3 did not display any noticeable
interaction with the hPrRP receptor even in the micromolar range
(Table 3). However, the
C-terminal fragment of hNPY (hNPY18-36) and BIBP3226 bound to the hPrRP
receptor with micromolar affinity.
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[35S]GTPS binding was also performed with membranes of
CHO-hPrRP-R cells. Both hPrRP20 and hPrRP31 dose dependently increased
[35S]GTPS binding, and the signal over basal was about 50%
for both peptides (Fig. 2). PTX
uncouples Gi/o proteins from GPCRs and thereby disrupts the
corresponding signaling pathways. Following PTX pretreatment, over 80% of the
[35S]GTPS binding stimulated by hPrRP20 or hPrRP31 and over
50% of the basal [35S]GTPS binding was abolished
(Fig. 2). The PrRP analog
hPrRP24-31 showed reduced potency to stimulate [35S]GTPS
binding (EC50 values were 230 ± 140 and 1.7 ± 0.7 nM
for hPrRP24–31 and hPrRP20, respectively), which is consistent with an
earlier report, where the heptapeptide PrRP25-31 was described to be able to
elicit a calcium response albeit with reduced potency compared with PrRP20
(Roland et al., 1999).
However, in agreement with the binding results, we did not observe any
activation of the hPrRP receptor in response to NPFF, hRFRP-1, or hRFRP-3
(Fig. 1B).
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Quantitative Receptor Autoradiography on Rat Spinal Cord Sections.
NPFF binding sites in the superficial layers of the dorsal horn in the rat
spinal cord represent mainly NPFF2 receptors
(Bonini et al., 2000). A panel
of ligands was analyzed by quantitative receptor autoradiography to visualize
the displacement of 125I-(1DMe
[PDB]
)Y8Fa in rat spinal cord sections.
The ligands were tested at two concentrations on cryostat sections from three
different animals on the level of the cervical spinal cord (See Materials
and Methods). Substance P was included as a negative control, because it
did not inhibit specific 125I-(1DMe
[PDB]
)Y8Fa binding in the competition
binding assay on rat spinal cord membranes (data not shown). In accordance
with previous reports, we observed a high density of binding sites in the
superficial layers of the dorsal horn and around the central canal (Allard et
al., 1989,
1992). The receptor density
around the central canal was approximately 50% of that in the dorsal horn
(Fig. 3, A and B). At the
highest concentration tested, only substance P did not significantly affect
125I-(1DMe
[PDB]
)Y8Fa binding, whereas NPFF, (1DMe
[PDB]
)Y8Fa, hRFRP-1, rNPSF,
bPrRP20, BIBP3226, and hRFRP(6G)-3 competed for the same binding sites as
125I-(1DMe
[PDB]
)Y8Fa in the superficial layers of the dorsal horn and in
the central canal (Fig. 3, A and
B). Change of the C-terminal amino acid 6Q to 6G resulted in
significant reduction of the binding of hRFRP(6G)-3 in comparison to other
RFamides, demonstrating that the C-terminal structure beyond the RFamide motif
is also critical for binding. It should be noted that the NPY1
antagonist BIBP3226 at a concentration of 1 µM displaced
125I(1DMe
[PDB]
)Y8Fa binding by roughly 50% (as calculated in percentage
of total binding in the absence of competing ligand) both in the dorsal horn
and around the central canal.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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;
Roumy and Zajac, 1998;
Panula et al., 1999;
Vilim et al., 1999), NPFF also
modulates a variety of other physiological processes such as insulin release,
prolactin release, blood pressure, food intake, electrolyte balance, and
morphine abstinence syndrome (Panula et
al., 1996; Aarnisalo et al.,
1997). PrRP, which was initially found to stimulate the release of
prolactin from anterior pituitary lactotrophs
(Hinuma et al., 1998), has been
suggested to have a broader role in the central nervous system, and its
ability to stimulate prolactin release might not represent its primary
biological function (Samson et al.,
2000). RFRPs have also been reported to exhibit regulatory effects
on prolactin secretion, although by mechanisms other than PrRP
(Hinuma et al., 2000). It
therefore seems that RFamide peptides and their receptors may have evolved in
close association with regulatory mechanisms for prolactin secretion in
mammals (Hinuma et al., 2000).
Nonetheless, the possibility that RFRPs might have other, still unknown,
important functions cannot be ruled out.
In the present study, we found that the RFamide peptides hPrRP20, hPrRP31,
hRFRP-1, and hRFRP-3 bind with high affinity to the rNPFF receptor as well as
to the hNPFF2 receptor, suggesting that different RFamide peptides may exert
their in vivo functions through the NPFF receptors. In contrast, the hPrRP
receptor seems to require an additional motif beyond the C-terminal RFamide,
since none of the related mammalian RFamide peptides, NPFF, hRFRP-1, and
hRFRP-3, were able to compete with 125I-PrRP for binding to
recombinant hPrRP receptor. The affinities of hNPY and BIBP3226, a
NPY1 receptor antagonist, at hNPFF2 and rNPFF receptors were
moderate. NPY and NPFF possess similar C-terminal sequences containing an
arginine and a C-terminally amidated aromatic residue. Despite the only
moderate affinities of hNPY and BIPB3226 at hNPFF2 and rNPFF receptors, it is
thus conceivable that NPY ligands might cross-react with NPFF receptors, as
has already been suggested by Mollereau et al.
(2001). BIBP3226 has
previously been reported to display 10- to 60-fold higher affinity for the
hNPFF1 and rNPFF1 receptors as compared with the hNPFF2 and rNPFF2 receptors
(Bonini et al., 2000).
NPFF and NPY, as well as their receptors, share common structural features that could explain the interaction of some NPY ligands with NPFF receptors. 1) the C-terminal sequences of NPY (RYamide) and the RFamide peptides (RF-amide), which are essential for interaction with their receptors, are very similar, with a difference of only a single hydroxyl group; and 2) the receptor sequences are 30 to 35% identical. However, based on the results presented in this paper, PrRP ligands also have to be considered as likely candidates for such cross-reactivity with the NPFF receptor.
Compared to the NPFF receptor, the hPrRP receptor is more clearly capable of differentiating between different RF-amide peptides; none of the tested related mammalian RF-amide peptides were able to bind to the PrRP receptor with affinity comparable to that of the PrRP peptides. Likewise, BIBP3226 and the C-terminal fragment of hNPY bound to the hPrRP receptor with only very low affinity.
We do note that in our assay conditions, the affinities of NPFF and
BIBP3226 for the hNPFF2 receptors expressed in CHO cells were clearly lower
than the affinities reported by Mollereau et al.
(2001) for the same cell line
using a novel radioligand, 125I-EYF
(125I-EYWSLAAPQRF-NH2). This discrepancy might be due to
differences in the binding characteristics of 125I-EYF and
125I-(1DMe
[PDB]
)Y8Fa. However, our affinity value for NPFF at the hNPFF2
receptor is in good agreement with earlier reports in which
125I-(1DMe
[PDB]
)Y8Fa or 125I-Y8Fa was used as radioligand
(Bonini et al., 2000;
Liu et al., 2001).
Our quantitative receptor autoradiography results are in good agreement
with the binding assay on rat spinal cord membranes. All tested RFamide
peptides, as well as BIBP3226, were able to displace the binding of
125I-(1DMe
[PDB]
)Y8Fa at concentrations about 100-fold above their
estimated Ki value or at 1 µM (hRFRP(6G)-3 and
BIBP3226), both in the dorsal horn and around the central canal, where
specific binding of 125I-(1DMe
[PDB]
)Y8Fa has previously been reported
(Allard et al., 1989,
1992). The results indicate
that NPFF, rNPSF, bPrRP20, and hRFRP-1 are able to compete for the same NPFF
binding sites in rat spinal cord. Even though our autoradiographic data do not
allow us to discriminate between different NPFF receptor subtypes, earlier
reports have indicated that NPFF2 is the predominant receptor subtype in rat
spinal cord (Bonini et al.,
2000). Expression of rNPFF2 mRNA has also been reported both in
the laminae I–II in the dorsal horn and the central canal region of the
rat spinal cord (Liu et al.,
2001). We cannot, however, exclude the possibility that
additional, still uncharacterized, receptor subtypes exist in rat spinal
cord.
The functional properties of the RFamide peptides were tested for their
ability to stimulate [35S]GTPS binding. It was recently
shown that an analog of NPFF dose dependently enhanced the binding of
[35S]GTPS to membranes of CHO-hNPFF2 cells, and this
stimulation was abolished by PTX pretreatment
(Kotani et al., 2001). In our
study more than 80% of the hPrRP20 or hPrRP31-induced
[35S]GTPS binding via the hPrRP receptor was prevented by
PTX. Taken together, both the hNPFF2 and the hPrRP receptor seem to couple to
the Gi/o class of G proteins in CHO cells. It has previously been
suggested that the hPrRP receptor hGR3/GRP10 signals through the Gq
pathway in both GH3 rat pituitary tumor cells and primary cultures of rat
anterior pituitary, but the coupling clearly depends on the specific cellular
system in which the receptor is expressed
(Kimura et al., 2000;
Langmead et al., 2000).
The observation that hPrRP31 and hPrRP20 can interact with the hNPFF2
receptor was also confirmed in functional experiments. Both PrRP ligands
caused clear increases in [35S]GTPS binding over basal.
Unexpectedly, we found that hPrRP31 consistently produced significantly
greater responses than NPFF or (1DMe
[PDB]
)Y8Fa at the hNPFF2 receptor. This is an
important finding, since the NPFF2 receptor has been suggested to be involved
in mediating the analgesic effect of NPFF in the rat
(Bonini et al., 2000;
Yang et al., 2001). The higher
efficacy of a ligand other than the putative endogenous ligand is surprising,
but not unprecedented. A publication by Narita et al.
(1998) represents a convincing
example of a situation in which endogenous ligands do not produce the highest
agonist response in a given test system. They show that the opioid peptides
endomorphin-1 and -2 have lower efficacy in the [35S]GTPS
binding assay on mouse spinal cord membranes than has the synthetic
µ-opioid agonist
[D-Ala2,NHPhe4,Gly-ol]enkephalin
(Narita et al., 1998). Perret
et al. (2002) reported that
two vasoactive intestinal peptide (VIP) analogs are more efficacious than the
natural agonist, VIP, at mutated VIPAC receptors and referred to these
synthetic compounds producing a larger response than the endogenous ligand as
"superagonists". More surprising, however, and to our knowledge
not previously reported, is the observation that the endogenous agonist of one
GPCR possesses a higher efficacy on another GPCR than the purported endogenous
agonist for the second receptor itself. To exclude the possibility that the
higher response by hPrRP31 relative to that of NPFF was caused by the
activation of an additional receptor in the cellular background of CHO cells,
we also tested the effect of hPrRP31 in membranes of CHO-K1 cells that did not
express the hNPFF2 receptor. No significant agonist effect of hPrRP31 could be
detected in these experiments (data not shown). Our results therefore strongly
suggest that, at least on the hNPFF2 receptor subtype, NPFF is not the most
efficacious agonist, and hPrRP31 acts as a superagonist. In addition, this
report provides a quantitative examination of the relative efficacies of
several RFamide peptides on the hNPFF2 receptor. On the hPrRP receptor, NPFF
was unable to elicit any functional response, as expected from the observation
that NPFF interacts poorly with the hPrRP receptor.
Our observation that hPrRP31 binds to NPFF receptors with relatively high
affinity and activates at least the hNPFF2 receptor subtype with high efficacy
points to the possibility that PrRP might exert physiological effects through
NPFF receptors. A physiologically significant interaction that might regulate,
for example, autonomic functions could occur in the nucleus of the solitary
tract, where both the NPFF2 receptor (Liu
et al., 2001) and PrRP (Roland
et al., 1999; Maruyama et al.,
1999) have been shown to be present.
Very low numbers of PrRP immunoreactive fibers and nerve terminals have
been observed in the laminae I–II of the dorsal horn and around the
central canal in the rat spinal cord (P. Panula and K. Kuokkaner, unpublished
observations). Tissue distribution studies indicate that PrRP mRNA, but no
RFRP mRNA, is present in the rat spinal cord as detected by reverse
transcription-polymerase chain reaction
(Fujii et al., 1999;
Hinuma et al., 2000). However,
there is the possibility that the RFRP peptides could be transported to the
spinal cord after translation. In future studies it will be very important to
examine in detail the coexpression of the NPFF2 receptor and RFamide peptides,
although a close physical association between the ligand-storing neurons and
receptors is not necessary for interactions
Thus, it is conceivable that the RFamide peptides, NPFF, PrRP, and possibly RFRP, may compete for NPFF receptor under physiological and/or pathological conditions. However, whether the observed in vitro efficacy of hPrRP31 at the hNPFF2 receptor does indeed translate into an in vivo efficacy remains to be established.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: NPFF, neuropeptide FF; PrRP, prolactin-releasing
peptide; RFRP, RFamide-related peptide; hNPFF2, human neuropeptide FF receptor
subtype 2; CHO, Chinese hamster ovary; NPY, neuropeptide Y; GPCR, G
protein-coupled receptor; [35S]GTPS,
[35S]guanosine-5'-O-(3-thio)triphosphate;
(1DMe
[PDB]
)Y8Fa, DYL(NMe)FQPQRF-NH2; rNPSF, rat neuropeptide SF; r, rat;
b, bovine; h, human; HEK, human embryonic kidney cell; FMRFamide,
phenylalanyl-methionyl-arginyl-phenylalaninamide; 125I-Y8Fa,
125I-YLFQPQRFamide; 125I-EYF,
125I-EYWSLAAPQRF-NH2; RT, room temperature; BSA, bovine
serum albumin; TB, total binding; NSB, nonspecific binding; SB, specific
binding; PTX, pertussis toxin; VIP, vasoactive intestinal polypeptide.
Address correspondence to: Mia Engström, Lemminkäisenkatu 5, FIN-20520 Turku, Finland; e-mail: mia.engstrom{at}juvantia.com
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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) NPFF
(
), hRFRP-1 (
), and hPrRP31 (
) (A) or to membranes of
CHO-hPrRP-R cells (5–10 µg/sample) and the indicated concentration of
hPrRP24–31 (
, 
