From the Laboratory for Molecular Pharmacology,
Department of Pharmacology, The Panum Institute, University of
Copenhagen DK-2200 and the § 7TM Pharma A/S, DK-2100
Copenhagen, Denmark
Received for publication, January 23, 2001, and in revised form, February 14, 2001
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ABSTRACT |
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The NK1 neurokinin receptor presents two
non-ideal binding phenomena, two-component binding curves for all
agonists and significant differences between agonist affinity
determined by homologous versus heterologous competition
binding. Whole cell binding with fusion proteins constructed
between either G Many G-protein-coupled
7TM1 (transmembrane segment)
receptors have the capacity to interact with multiple G-proteins and
thus regulate more than one effector pathway. Some receptors
preferentially couple to one G-protein, but in the presence of higher,
non-physiological concentrations of agonist or in, for example
reconstitution assays, these receptors are able to couple to other
G-proteins (1, 2). Other more promiscuous receptors couple functionally
to more than one G-protein in the physiological range of agonist concentration (3-6). For example, the neurokinin NK1 receptor is
coupled to two different signaling pathways: a G Because the first fusion protein between a 7TM receptor and a G-protein
was constructed in 1994 by Marullo and co-workers (11), such constructs
have proven useful in addressing many different types of questions
related to receptor function. For example, important information has
been gained concerning ligand efficacy, intrinsic activity, kinetics of
the receptor G-protein interaction, and the importance of
post-translational acylation (12-15). The improved accessibility for
the G-protein during receptor binding was originally expected to
increase the proportion of receptors stabilized in the
G-protein-coupled high affinity state. But, as recently reviewed by
Seifert et al. (16), it is clear that a situation with 100%
active receptor conformations is not obtained in classical receptor
G-protein fusions. For many receptors it has been shown that fusion to
the appropriate G-protein does induce high constitutive signaling
activity. However, for other receptors this has not been the case, not
even with a closer interaction between the receptor and the G-protein
as has been achieved through shortening of the tail (17-21).
Nevertheless, it could be argued that although the receptor and the
G-protein are covalently coupled in these fusion proteins, the often
rather long tail of the receptor in fact still makes it possible for
the receptor to interact rather freely with other G proteins that are
highly abundant in the cell, and for which the receptor may have rather
high affinity (20).
In the present study we have constructed fusion proteins between the
NK1 receptor and G Materials--
Substance P and neurokinin A were purchased from
Peninsula (Belmont, CA).
cDNA Constructs--
The cDNA coding for the long splice
version of rat G Radioactive Ligands--
125I-Bolton-Hunter
(125I-BH)-labeled substance P was prepared using
125I-Bolton-Hunter reagent (IBM5861) from Amersham
Pharmacia Biotech (Little Chalfont, UK) and purified by reverse phase
HPLC as previously reported (22). 125I-NKA labeled at
His1 to a specific activity of 2000 Ci/mmol was purchased
from Amersham Pharmacia Biotech (IBM168).
Transfections and Tissue Culture--
COS-7 cells were grown in
Dulbecco's modified Eagle's medium 1885 supplemented with 10% fetal
calf serum, 2 mM glutamine, and 0.01 mg/ml gentamicin.
Cells were transfected using the calcium phosphate precipitation method
with chloroquine addition as previously described (23).
Competition Binding Assays--
Transfected COS-7 cells were
transferred to culture plates 1 day after transfection at a density of
1-50 × 104 cells per well for the wild-type receptor
and the NK1·Gq and NK1·Gq- Phosphatidylinositol Turnover--
One day after transfection,
COS-7 cells were incubated for 24 h with 5 µCi of
[3H]myo-inositol (Amersham Pharmacia Biotech,
PT6-271) in 1 ml of medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 0.01 mg/ml gentamicin per well. Cells
were washed twice in buffer, 20 mM HEPES, pH 7.4, supplemented with 140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM CaCl2,
10 mM glucose, 0.05% (w/v) bovine serum and were incubated
in 0.5 ml of buffer supplemented with 10 mM LiCl at
37 °C for 30 min. After stimulation with various concentrations of
peptide for 45 min at 37 °C, cells were extracted with 10% ice-cold
perchloric acid followed by incubation on ice for 30 min. The resulting
supernatants were neutralized with KOH in HEPES buffer and the
generated [3H]inositol phosphate was purified on Bio-Rad
AG 1-X8 anion-exchange resin as described (24). Determinations were
made in duplicates. The number of cells used for these experiments were
identical to those used for binding experiments, i.e.
adjusted to obtain between 5 and 8% binding of the relevant peptide agonist.
cAMP Production--
One day after transfection, COS-7 cells
(0.5-10 × 105 cells per well) were incubated for
24 h with 2 µCi of [3H]adenine (Amersham Pharmacia
Biotech, TRK 311) in 1 ml of Dulbecco's 1885 medium supplemented with
10% fetal calf serum, 2 mM glutamine, and 0.01 mg/ml
gentamicin. Cells were washed twice and incubated for 15 min at
37 °C in 1 ml of freshly prepared binding buffer supplemented with 1 mM isobutylmethylxanthine (Sigma, I5879), 40 µg/ml
bacitracin, and with various concentrations of peptides or with 50 µM
forskolin. After incubation, cells were placed on ice, medium was
removed, and cells were lysed with 1 ml of 5% (w/v) trichloroacetic
acid, supplemented with 0.1 mM cAMP and 0.1 mM
ATP for 30 min. The lysis mixtures were loaded onto a Dowex 50W-X4
(Bio-Rad, 142-1351) column (Bio-Rad, poly-prep columns, 731-1550),
which was washed with 2 ml of water and placed on top of alumina
columns (Sigma, A9003) and washed again with 10 ml of water. The
columns were eluted with 6 ml of 0.1 M imidazole (Sigma,
I0125) into 15 ml of scintillation fluid (Highsafe III). Columns were
re-used up to 10 times. Dowex columns were regenerated by adding 10 ml
of 2 N HCl followed by 10 ml of water; the alumina columns
by adding 2 ml of 1 M imidazole, 10 ml of 0.1 M
imidazole and finally 5 ml of water. Determinations were made in
triplicates. The number of cells used for these experiments were
identical to those used for binding experiments, i.e.
adjusted to obtain between 5 and 8% binding of the relevant peptide agonist.
Calculations--
IC50 and EC50 values
were determined by nonlinear regression using the Prism 3.0 software
(GraphPad Software, San Diego). Values of the dissociation and
inhibition constants (Kd and Ki)
were estimated from competition binding experiments using the equations
Kd = IC50 Signal Transduction in the G
Although a more efficient decoupling of the NK1 receptor from
G Signaling in the G Competition Binding Using 125I-Substance P in Fusion
Proteins versus Wild-type Receptor--
The competition binding curve
for substance P in the wild-type NK1 receptor, transiently expressed in
COS-7 cells and using 125I-substance P as a tracer was, as
previously described, best fitted to a two component curve, identifying
both a very high affinity state (Kd = 0.044 nM; 57%) as well as a high affinity state (0.60 nM; 43%) (Fig.
4A) (9). Similarly, the
displacement curve for the other endogenous NK1 ligand, neurokinin A
(NKA) against 125I- substance P, was in the wild-type NK1
receptor also biphasic with an ~50-fold difference between a
high affinity site (Kd = 1.3 nM; 35%)
and a low affinity state (Kd = 78 nM; 65%) (Fig. 4B).
In the simple NK1·Gq fusion construct, where the G-protein was fused
to the C-terminal end of the receptor, the binding curve for substance
P was indistinguishable from that observed in the wild-type receptor
both with respect to affinity and Hill coefficient. However, in the
NK1·Gq-
Very similarly, the two-component competition curve for NKA against
125I-substance P found in the wild-type NK1 receptor was
resolved into the two corresponding monocomponent competition curves by the NK1·Gq-
This surprising observation, namely that the complex between the
receptor and G Heterologous Agonist Competition Binding in Fusion Proteins and in
the F111S Mutant--
Several groups (25) have previously described
that in the wild-type NK1 receptor, the affinity of so-called
septide-like tachykinin peptides, here represented by NKA, is
determined to be 50-100-fold lower in heterologous competition against
125I-substance P than in homologous competition against
themselves (Fig. 6). Also, the
displacement curve for substance P determined in heterologous
competition against 125I-NKA corresponds to the
monocomponent, very high affinity state of the two high affinity states
determined for substance P in homologous binding against itself,
125I-substance P (Fig. 6A). As shown in Fig. 6,
this phenomenon was eliminated when a one-to-one receptor to G-protein
interaction was secured. Thus, in both the NK1·Gq- In the present study, fusion of the NK1 receptor to either of the
two G proteins, through which it normally couples, has resolved both
the phenomenon related to the two-component agonist binding property
and the ambiguous problem of differences between affinities for
agonists measured in homologous versus heterologous
competition. In both cases, i.e. fusion to either
G Importance of Length of Spacer Peptide for Fusion Protein
Phenotype--
It is often expected, that by simply fusing a G-protein
to the C-terminal tail of a receptor, a situation with a close
one-to-one receptor to G-protein interaction will be obtained. However,
it has in fact in several receptor systems been reported that the coupling efficiency is far from 100% in ordinary fusion constructs (11, 16, 20, 26) In our system, we even observed a 50% coupling
efficiency through the "endogenous" G
Truncation of the C-terminal tail, i.e. the "spacer
peptide" between the receptor and the G protein, could theoretically
impair the ability of the G-protein to interact correctly with the
receptor by inducing conformational constraint; however, it has
previously been demonstrated that truncation of the receptor tail of
the Two Different Active Conformations of the NK1 Receptor--
The
different pharmacological profiles of the fusion proteins of the
present study indicate that the NK1 receptor can occur in at least two
distinct active conformations. It is well known that 7TM receptors
often can couple to more than one cellular signal transduction pathway.
However, it has been unclear whether this was accomplished through one
and the same active receptor conformation being able to interact with
different G proteins or the occurrence of more than one active receptor
conformation. There is a clearly distinct monocomponent binding curves
observed both for substance P and for NKA in the two tail-truncated
NK1·G-protein fusion constructs and very importantly these two curves
closely correspond to each of the components that the complex binding curves for the agonist peptides in the wild-type NK1 receptor can be
resolved into (Fig. 4). This indicates that the receptor normally
occurs in two different active conformations in complex with two
different G proteins. This is also strongly supported by the
observation in the F111S mutant form of the NK1 receptor, which is
totally uncoupled from G
An intriguing possibility would be to be able to address each of such
active receptor conformations selectively with pharmacological tools
and drugs. In this connection it should be noted that many NK1
non-peptide antagonists act as competitive antagonists when blocking
the effect of substance P but that the same compounds in the same
receptor preparation behave as insurmountable antagonists against
septide-like agonists such as NKA (27, 28).
Discrepancy between Affinity and Potency for Agonists through
NK1·G
The fact that we observe an almost wild-type G Lack of Rapid Interchange of Molecular Phenotypes of the NK1
Receptor in the Cell Membrane--
It is clear from the present study
that the affinity for NKA on the NK1 receptor is very different when
the receptor is found in complex with G
In some receptors, for example in the chemokine system, where
many different agonist tracers are available, up to 10,000-fold difference can be observed among the affinities for agonists determined in competition assays. Thus, in the CXCR2 receptor NAP-2 competes against 125I-ENA78 with very high, subnanomolar affinity,
but it competes against 125I-IL-8 with very low micromolar
affinity (31). Similarly, in the ORF-74 receptor, IL-8 competes with
nanomolar affinity against itself, 125I-IL-8, but only with
micromolar affinity against the preferred agonist,
125I-GRO
Several of the classical theoretical receptor models have left open the
possibility for an additional "dimension," i.e. for the
receptor to interact with more than one G-protein as for example emphasized by Kenakin (39-41). Recently, Leff et al. (42)
presented a simplistic three-state variant of the two-state receptor
model, where the receptor could occur in complex with two different G proteins in the same cell (42). Interestingly, based on analysis of
data from a number of receptor systems, they suggested that the two
active states of the receptor (as opposed to the third inactive state)
could occur either in an "intact" or "isolated" form in which
there was no experimentally obvious interchange between the two active
states. The latter is obviously what we observe here for the NK1 receptor.
s or G
q and the NK1 receptor with a truncated tail, which secured non-promiscuous G-protein interaction, demonstrated monocomponent agonist binding closely corresponding to either of the two affinity states found in the
wild-type receptor. High affinity binding of both substance P and
neurokinin A was observed in the tail-truncated G
s
fusion construct, whereas the lower affinity component was displayed by
the tail-truncated G
q fusion. The elusive difference
between the affinity determined in heterologous versus
homologous binding assays for substance P and especially for neurokinin
A was eliminated in the G-protein fusions. An NK1 receptor mutant with
a single substitution at the extracellular end of TM-III-(F111S), which totally uncoupled the receptor from G
s signaling, showed
binding properties that were monocomponent and otherwise very similar to those observed in the tail-truncated G
q fusion
construct. Thus, the heterogenous pharmacological phenotype displayed
by the NK1 receptor is a reflection of the occurrence of two active conformations or molecular phenotypes representing complexes with the
G
s and G
q species, respectively.
We propose that these molecular forms do not interchange
readily, conceivably because of the occurrence of microdomains or
"signal-transductosomes" within the cell membrane.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q
pathway, which activates phospholipase C
, thereby initiating
inositol phosphate formation and a G
s pathway, which
induces cAMP formation. Both effector systems are activated by
nanomolar physiological concentrations of substance P (6). Other
endogenous neurokinin peptides such as NKA and NKB as well as
artificial peptide ligands such as septide can act as high affinity
agonists on the NK1 receptor (7). Interestingly, in whole cell binding
experiments, the NK1 receptor displays certain characteristics that
indicate that the receptor in the cell membrane occurs in more than one
high affinity active molecular form. First, competition binding
experiments using the agonist substance P as a tracer reveal
two-component binding curves for all agonists, indicating the
occurrence of two different high affinity binding sites, as opposed to
a third low affinity state corresponding to the G-protein-uncoupled
receptor as demonstrated by use of antagonist tracers or GTP analogs
(8, 9) Second, in competition binding experiments, NKA, NKB, and other
so-called septide-like agonists compete for binding against radioactively labeled substance P with surprisingly low apparent affinity. This is in contrast to the nanomolar affinity for these agonists as revealed in either homologous binding experiments or in
signal transduction assays. In other words, there are major differences
in the affinity for different agonists determined in homologous binding
experiments as opposed to heterologous binding experiments against
another agonist (7). The two proposed bindings modes of the NK1
receptor have recently been characterized by the use of a number of
synthetic analogs of substance P containing a conformationally
constrained methionine analog (10). However, the molecular basis for
the two high affinity agonist binding modes remains to be characterized.
q or G
s, respectively;
and we have truncated the C-terminal tail of the receptor in an attempt
to enable the receptor to preferentially interact with the G-protein it
has been covalently coupled to, i.e. to try to exclude
interactions with other endogenous G-proteins (Fig. 1). These
tail-truncated fusion proteins give clear molecular phenotypes with
respect to coupling and especially the agonist binding profile
corresponding closely to each of the two high affinity states of the
receptor. The fact that the two high affinity binding states for
peptide agonists correspond to two different active conformations in
complex with respectively G
s and G
q was
supported by an observed total decoupling from G
s in a
mutant NK1 receptor that closely mimicked the tail-truncated G
q
fusion protein.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s was kindly provided by Dr. Din, and
the cDNA for human G
q was a gift from Dr. Bruce Conklin. The
fusion proteins with the wild-type human NK1 receptor were constructed
by oligonucleotide-directed mutagenesis, with oligonucleotides
consisting of 10-15 base pair overlap both from the N terminus of the
G-protein and from the most C-terminal part of the receptor and the 72 amino acid-truncated C-terminal receptor in the case of the
NK1·Gq and the NK1·Gq-
tail and
NK1·Gs-
tail, respectively. The cDNAs were cloned
into the eukaryotic expression vector pTEJ8. All constructs were
verified by restriction endonuclease mapping and DNA sequencing
(ALFexpress DNA sequencer, Amersham Pharmacia Biotech).
tail
constructs aiming at 5-8% binding of the various radioactive ligands.
However, in the case of the NK1·Gq-
tail and the [F111S]NK1
constructs and using 125I-NKA as radioligand, 1 × 10 6 cells/well had to be employed to detect and
characterize the binding. Two days after transfection, competition
binding experiments were performed for 3 h at 4 °C using 25 pM of the radioiodinated peptides. Binding assays were
performed in 0.5 ml of a 50 mM Tris-HCl buffer, pH 7.4, supplemented with 150 mM NaCl, 5 mM
MnCl2, 0.1% (w/v) bovine serum albumin, 40 g/ml
bacitracin. Nonspecific binding was determined as the binding in the
presence of 1 M of the corresponding unlabeled ligand.
Cells were washed twice in 0.5 ml of ice-cold buffer, and 0.5-1 ml of
lysis buffer (8 M urea, 2% Nonidet P-40 in 3 M
acetic acid) was added, and the bound radioactivity was counted.
Determinations were made in duplicate. Initial experiments showed that
steady-state binding was reached with the various radioactive ligands
under these conditions.
L and
Ki = IC50/(1 + L/Kd), where L is the
concentration of radioactive ligand.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q Fusion Proteins and
the [F111S]NK1 Mutant--
To try to confine the NK1 receptor to
interact exclusively with one species of G-proteins, in this case
G
q and not G
s, two fusion proteins were
made between the receptor and G
q. In
NK1·Gq, the G
q protein was fused to the
far C-terminal end of the NK1 receptor, whereas in NK1·Gq-
tail,
the 72 most C-terminal residues of the C-terminal tail of the NK1
receptors were deleted, resulting in a fusion protein where
G
q was placed only 14 residues after the presumed
palmitoylation site (Fig. 1). The
NK1·Gq construct behaved rather similarly to the
wild-type NK1 receptor with respect to signaling through the
phospholipase C pathway, both in regards to Emax and
EC50 for the substance P-stimulated inositol phosphate turnover as determined in transiently transfected COS-7 cells (Fig.
2A). However, although fusion
of G
q to the far end of the C-terminal tail of the NK1
receptor impaired signaling through the adenylylcyclase pathway, this
G
s-mediated signaling was far from eliminated. In fact,
the Emax of substance P-induced cAMP accumulation in the
NK1·Gq construct was still ~50% of that observed in the wild-type
NK1 receptor (Fig. 2B and Table
I). In contrast, truncation of the tail
of the NK1 receptor, i.e. shortening the spacer between the
receptor and the G
q, severely limited the ability of the
receptor to signal through the G
s-mediated adenyl cyclase pathway. Thus, in the NK1·Gq-
tail construct,
the response in cAMP accumulation was decreased 10-fold when compared
with the wild-type NK1 receptor, i.e. from an
Emax of 46 fmol/105 cells to 4.8 fmol/105 cells (Fig. 2B and Table I). This
effect was not caused by a decrease in expression level, because the
Bmax was not decreased in the NK1·Gq-
tail
fusion protein (Table I). Moreover, the efficacy of substance P to
induce inositol phosphate turnover was also close to that observed in
the wild-type receptor, i.e. an Emax = 67 fmol/105 cells as compared with 84 fmol/105
cells in the wild-type receptor. In the NK1·Gq-
tail
fusion protein, the dose-response curve for substance P induction of
inositol phosphate turnover was however more shallow with a slope close to unity as compared with the more steep curves observed in the wild-type receptor and in the non-truncated NK1·Gq construct (Fig. 2).
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Fig. 1.
Serpetine model of the NK1 receptor with the
covalently attached G-protein. The full-length receptor has an
85-amino acid long C-terminal tail, as measured from the presumed
palmitoylation site. In the constructs termed NK1·Gq the
G-protein has been attached to the far C-terminal end, whereas in the
construct NK1·Gq or Gs- tail, 72 amino
acids have been truncated from the tail leaving only a 13-amino acid
spacer after the palmitoylation site. The black circle
indicates the single amino acid substitution Phe111 to Ser,
(F111S) in TM-III, which decouples the receptor from G
s.
In the generic nomenclature and numbering system for 7TM receptors (43,
44), Phe111 is located at position III:07, i.e.
one residue prior to the famous amine-interacting AspIII:08 of
monoamine receptors.
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Fig. 2.
Inositol phosphate and cyclic AMP turnover in
response to substance P in NK1 G q
protein fusion constructs. Inositol phosphate turnover
(A and C) and cAMP accumulation (B and
D) in response to substance P in the wild-type NK1 receptor
(
), the NK1·Gq fusion construct (
), the
NK1·Gq-
tail construct (
) (see Fig. 1 and text for
details), and the [F111S]NK1 (
) mutant transiently expressed in
COS-7 cells. Data are mean ± S.E. from 3-8 experiments performed
in duplicate and carried out in parallel against the wild-type receptor
for each of the constructs.
Competition binding experiments
q signal transduction pathway
(inositol phosphate turnover) and the presumed G
s pathway
(cAMP accumulation) performed in NK1 receptor as well as the G-protein
fusions and the [F111S]NK1 mutant in transiently transfected COS-7
cells as described in detail in the text.
s signaling was achieved by truncating the C-terminal
tail of the receptor in the NK1·Gq-
tail fusion
protein, still ~10% of the response remained. However, during our
general mutational analysis of the NK1 receptor, we discovered a point
mutation in TM-III, Phe111 to Ser, which totally decoupled
the receptor from G
s signaling. This position, which in
most 7TM receptors is occupied by a residue with a large aromatic or
aliphatic side chain, is located at the extracellular end of TM-III at
generic position III:07 facing toward II and VII (Fig. 1). If anything,
substance P induced a small decrease in cAMP production in the
[F111S]hNK1 mutant (Fig. 2D). With respect of
G
q signaling, the dose-response curve for substance
P-induced inositol phosphate turnover observed in the [F111S]hNK1
mutant was similar in shape to that found in the
NK1·Gq-
tail fusion protein, i.e. with a
Hill coefficient of approximately unity (Fig. 2C).
s NK1 Fusion Protein--
A
fusion protein similar to the NK1·Gq-
tail was
constructed, but G
q was replaced by G
s.
Although both the potency of substance P and the shape of the
dose-response curve for induction of cAMP production were very similar
to what was observed in the wild-type receptor, the Emax
was reduced to ~60% in this NK1·Gs-
tail construct (Fig. 3B). Surprisingly, the
close association of the NK1 receptor with G
s had very
little effect on the substance P-induced inositol phosphate
accumulation, albeit the slope of the dose-response curve was closer to
unity in the NK1·Gs-
tail than in the wild-type receptor. This was also observed in the NK1·Gq-
tail
(Fig. 3A).
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Fig. 3.
Inositol phosphate and cyclic AMP turnover in
response to substance P in NK1 G s
protein fusion constructs. Inositol phosphate turnover
(A) and cyclic AMP (B) in response to substance P
in the wild-type NK1 receptor (
) and in the
NK1·Gs-
tail (
) (see Fig. 1 and the text for
details) transiently expressed in COS-7 cells. Data are mean ± S.E. from at least 3 separate experiments made in duplicate and carried
out in parallel for each of the panels.
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Fig. 4.
Competition binding experiment for agonists
in the wild-type NK1 receptor and in the two tail-truncated
receptor-G fusion proteins. Displacement
of 125I-substance P by substance P (A) and
neurokinin A (B) in the wild-type NK1 receptor (
), the
NK1·Gs-
tail (
) construct, and the
NK1·Gq-
tail (
). Whole cell binding experiments were
performed in transiently transfected COS-7 cells. Data are mean ± S.E. from 3 to 8 separate experiments made in duplicate.
Kd values for substance P against
125I-substance P is indicated in Table I.
Ki for NKA against 125I-substance P is
for wild type (13 ± 3 nM), for
NK1·Gq-
tail (56 ± 4 nM) and for
NK1·Gs-
tail (1.4 ± 0.2 nM).
tail construct, a monocomponent displacement curve (Hill coefficient = 1.0) was observed for substance P with an affinity (Kd = 0.44 nM) corresponding
closely to the high affinity part of the two-component curve found in
the wild-type receptor (Fig. 4A). A monocomponent
displacement curve was also observed in the
NK1·Gs-
tail construct for substance P (Hill
coefficient = 1.1), and in this case the affinity for substance P
(Kd = 0.02 nM) corresponded to the
very-high affinity state observed for substance P in the wild-type
receptor. Thus, each of the two high affinity states for substance P
found in the wild-type NK1 receptor corresponds closely to the
monocomponent states observed for the peptide in each of the
tail-truncated fusion proteins with either G
s or
G
q, respectively.
tail and the NK1·Gs-
tail
receptor fusion proteins (Fig. 4B and Table I). Also in the
case of NKA it was the NK1·Gs-
tail construct that gave
the high affinity state, whereas the NK1·Gq-
tail construct displayed the more low affinity state (Fig.
4B).
s results in the highest affinity states
both for substance P and for NKA, was further substantiated by binding experiments using the [F111S]NK1 receptor mutant. In this mutant, which, as demonstrated above, is totally uncoupled from
G
s (Fig. 2D), substance P showed an affinity
of 0.8 nM corresponding to the lower of the two high
affinity states (Fig. 5A). NKA
showed an affinity of 93 nM also corresponding to the lower
of the two affinity states for that peptide on the wild-type NK1
receptor (Fig. 5B). In both cases the competition curves
observed in the [F111S]NK1 mutant were similar to those observed in
the NK1·Gq-
tail fusion protein, albeit slightly
shifted to the right. The F111S mutation did not affect the antagonist
binding profile, which implies that the receptor as such is not
structurally destroyed nor misfolded in this mutant form (Table I and
unpublished data).
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Fig. 5.
Competition binding experiment for agonists
in wild-type NK1 receptor and in the
G s-uncoupled [F111S]NK1
mutant. Displacement of 125I-labeled substance P with
substance P (A) and neurokinin A (B) for
wild-type NK1 receptor (
) and [F111S]NK1 (
). The displacement
curves for the NK1·Gq-
tail construct is shown as a
dashed line for comparison. Whole cell binding experiments
were performed in transiently transfected COS-7 cells. Data are
mean ± S.E. from 3 to 8 separate experiments made in duplicate.
Kd values for substance P against
125I-substance P is indicated in Table I.
Ki for NKA against 125I-substance P is
for wild type (13 ± 3 nM), and for [F111S]NK1
(93 ± 13 nM).
tail
(panels C and D), in the [F111S]hNK1 receptor
mutant (panels E and F), as well as in the
NK1·Gs-
tail (panels G and H),
the heterologous and the homologous competition binding curves were
almost superimposable for both substance P and for NKA. As described
above, using only 125I-substance P (Figs. 4 and 5), the
NK1·Gq-
tail and the [F111S]hNK1 receptors displayed
the lower of the two affinity sites for both substance P and NKA,
whereas the NK1·Gs-
tail construct displayed the high
affinity state, again for both substance P and NKA (Fig. 4). However,
the picture is not totally perfect, because the high affinity state for
NKA determined in the homologous binding experiments in the wild-type
NK1 receptor is still ~10-fold higher than the affinity determined in
both homologous and heterologous binding experiments in the
NK1·Gs-
tail construct, Kd = 0.33 nM versus 3.2 nM (homologous) and
1.4 nM (heterologous) (Fig. 6, panels B and
H). Thus, although the phenomenon of different affinities
determined in heterologous versus homologous binding assays
has been eliminated in for example the G
s fusion protein with the truncated tail, the actual affinity for NKA is lower than in
the wild-type.
View larger version (37K):
[in a new window]
Fig. 6.
Comparison of heterologous and homologous
competition binding experiment for substance P (SP)
and NKA using either 125I-substance P or
125I-NKA in the wild-type NK1 (A and
B), the
NK1·Gq- tail construct
(C and D),
NK1·Gs-
tail construct
(G and H), and the
G
s-uncoupled [F111S]NK1 mutant
(E and F). Whole cell binding
experiments were performed in transiently transfected COS-7 cells. Data
are mean ± S.E. from 3 to 8 separate experiments made in
duplicate. Kd values for substance P against
125I-substance P is indicated in Table I.
Ki for NKA against 125I-substance P is
mentioned in the legend to Figs. 4 and 5. Kd values
for NKA against 125I-NKA is for wild type (0.33 ± 0.03 nM), for NK1·Gq-
tail (13 ± 1 nM), for [F111S]NK1 (131 ± 24 nM), and
for NK1·Gs-
tail (3.2 ± 0.2 nM).
Ki for substance P against 125I-NKA is
for wild type (0.04 ± 0.01 nM), for
NK1·Gq-
tail (0.34 ± 0.07 nM), for
[F111S]NK1 (0.7 ± 0.1 nM) and for
NK1·Gs-
tail (0.07 ± 0.02 nM).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q or G
s, monocomponent binding of
agonists was accomplished, and the affinity for agonists was found to
be identical in homologous and heterologous competition. These results
indicate that the mixed pharmacological phenotype observed with the NK1
receptor in fact is a reflection of the occurrence of at least two
different molecular phenotypes, which we interpret as two different
active receptor conformations, in complex with the respective G
proteins. Interestingly, especially the fact that the affinity for an
agonist determined in homologous versus heterologous
competition binding is normally far from identical (see below), this
indicates that the molecular phenotypes in some cases are not readily
interchangeable in the plasma membrane of the whole cell.
s pathway when G
q was simply fused to the far C-terminal end of the
tail of the NK1 receptor. Importantly, truncation of the long
C-terminal tail of the NK1 receptor secured a more faithful coupling
between the receptor and the tethered G-protein, as demonstrated in the decrease from 50% to ~10% promiscuous coupling through the
G
s pathway. Moreover, in these tail-truncated constructs
monocomponent binding curves were found for the agonists, which was not
the case when, for example G
q was fused to the far
C-terminal end of the receptor.
2-adrenergic receptor is well tolerated in G-protein
fusions (21). The backbone of the remaining C terminus in the presently employed
-tail constructs can in fact still in an extended form stretch ~86 Å away from the receptor, which should ensure a
reasonable degree of conformational freedom. Nevertheless, in the
-tail constructs, we do observe a tendency toward a lower coupling
efficacy as compared with the wild-type receptor, both in cAMP and in
inositol phosphate turnover. This could possibly be ascribed to an
impaired interaction between either the receptor and the G-protein or
between the G-protein and the effector molecule (adenylylcyclase and
phospholipase C
) (21).
s and where monocomponent
binding curves were obtained that closely correspond to those observed with the tail-truncated G
q fusion construct. Thus, it
appears that the mixed pharmacological phenotype normally found in the NK1 receptor can be experimentally resolved into each of its components through appropriate fusion with each of two different G proteins. It
remains to be seen to what degree this is a general phenomenon and
whether the frequently observed two- or multicomponent agonist binding
curves, especially found among peptide receptor agonists, in fact is a
reflection of the occurrence of different active conformations of these
receptors in complex with different G proteins. It should be noted that
in many cases, the classical low affinity binding of the agonist to the
G-protein-uncoupled form of the receptor may interfere with this
picture. However, at least in the case of the NK1 receptor, we are
dealing with two high affinity states of the receptor being clearly
different from the binding observed to the uncouple receptor (8).
s Complexes--
It was surprising to find that
the very high affinity state of the NK1 receptor was the one stabilized
by G
s because it is generally believed that it is the
G
q signaling pathway that is the physiologically most
relevant for the NK1 receptor. Moreover, in the wild-type NK1 receptor
the potency of substance P for activating the G
q
signaling pathway is generally around one order of magnitude higher
than the potency for activating the G
s pathway (6). Interestingly, although the affinity for substance P in the
NK1·Gs-
tail construct is 0.02 nM
(monocomponent binding) (Fig. 4A), the EC50 for
substance P in stimulating cAMP accumulation through this construct is
100-fold lower, i.e. 1.5 nM, which is similar to what is observed in the wild-type receptor (Fig. 3B). We
cannot explain this discrepancy between G
s-mediated very
high affinity binding and significantly lower G
s
signaling potency but would point out that the discrepancy apparently
is also found in the wild-type receptor.
q
signaling even in the tail-truncated G
s fusion construct
could theoretically be attributed to a high degree of promiscuous
coupling to endogenous G
q even in this supposedly tight
receptor-G
s protein complex. However, this is not a
likely explanation because we find a clear monocomponent
"non-G
q-like" binding curve for both substance P and
NKA in this construct. Nevertheless, it is possible that the
NK1·Gs-
tail fusion construct could in fact be just
~10% promiscuous in its coupling, which the corresponding
Gq-
tail construct clearly is (Fig. 2B). Such
a low degree of molecular heterogeneity in the binding complexes may
not be appreciated in the binding curves. Thus, it is possible that
high efficacy signaling through the G
q pathway does not
require as high a degree of receptor occupancy or a high number of
receptor-G
q complexes. Another explanation could be, that a
significant degree of the observed stimulation of inositol phosphate
turnover is mediated through the
-subunits of the heterotrimeric
G proteins (29).
s (high affinity)
as opposed to when it is found in complex with G
q (much
lower affinity). In binding assays with 125I-NKA, where
only trace amounts of the radioactively labeled peptide is used, this
will preferentially bind to and display the high affinity
G
s-coupled NK1
receptor.2 The fact that
substance P has very high affinity for the NK1 receptor in complex with
G
s (Fig. 4A) thus explains why substance P
also in the wild-type receptor competes against 125I-NKA
binding with very high affinity and in a monocomponent fashion (Fig.
6A). On the other hand when using 125I-substance
P as radioligand, this tracer will bind and display the NK1 receptor
both in complex with G
s and with G
q
because the affinity for substance P is relatively high in both of
these G-protein complexes in comparison to the concentration of
125I-substance P used in the binding assays. Accordingly,
in the wild-type receptor both of the affinity states for both
substance P and for NKA are displayed when using
125I-substance P resulting in the two component binding
curve with Hill coefficients below unity for each of these peptides
(Fig. 4). Especially for NKA, the results show that there is a
considerable difference between the affinity determined in homologous
competition against 125I-NKA and the apparent affinity
determined in heterologous competition against
125I-substance P (Fig. 6B and Refs. 9,
27). Similarly certain differences in agonist potency and efficacy have
been obtained with the
2 adrenergic receptor depending
on which G-protein it was attached to (30).
(32). Such large differences suggest that the
molecular phenotypes, conformations of the receptor that preferentially bind one or the other agonist are not in a readily interchangeable equilibrium in the cell membrane. In the case of the NK1 receptor, our
data indicate that the observed distinct pharmacological phenotypes of
the receptor in fact correspond to complexes with different G proteins.
In the literature there is ample evidence, for example from
fluorescence labeling experiments that the distribution of G proteins
are not random in the cell membrane and that especially the
- but
also the
-subunits appear to be relatively immobilized (33, 34).
Moreover, it is becoming more and more evident that the receptors
themselves interact with various types of scaffolding proteins, which
secure both close proximity to other physiologically relevant receptors
and/or other proteins involved in the signal transduction mechanism
(35, 36). Thus, a picture is emerging where receptors can be envisioned
to be found in the membrane in different types of
"signal-transducisomes," i.e. in a more or less close
association with proteins involved in different types of signal
transduction pathways including G proteins, effector proteins, and
other associated proteins (37, 38). If the different receptor
conformations, such as those observed in the present study, occur in
the cell membrane in different types of "signal-transducisomes," then it is not difficult to imagine that the interchange between these
molecular phenotypes would not necessarily occur readily. Nevertheless,
the present study does provide evidence that the complex
pharmacological phenotype of the NK1 receptor can be resolved into two
distinct molecular phenotypes corresponding to two different receptor
conformations in complexes with either G
s or
G
q.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Susanne Hummelgaard and Mette Simons for expert technical assistance.
![]() |
FOOTNOTES |
---|
* This study was supported by grants from the Danish Medical Research Council and from the Lundbeck Research Foundation.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.
¶ Supported by a grant from the P. Carl Pedersen Foundation.
To whom correspondence should be addressed: Laboratory for
Molecular Pharmacology, Dept. of Pharmacology, The Panum Inst., Bldg.
18.6, Blegdamsvej 3, DK-2200, Copenhagen, Denmark. Tel.: 45 3532 7603;
Fax: 45 3535 2995; E-mail: schwartz@molpharm.dk.
Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M100621200
2
It should be noted that 100-fold or more cells
are needed to detect and characterize 125I-NKA binding to
the Gs-uncoupled constructs,
NK1·Gq-
tail and [F111S]NK1 as compared with the
wild-type and G
s-coupled constructs (see "Experimental
Procedures").
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: 7TM, seven transmembrane; NKA, neurokinin A; NK1, neurokinin.
![]() |
REFERENCES |
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