Two Active Molecular Phenotypes of the Tachykinin NK1 Receptor Revealed by G-protein Fusions and Mutagenesis*

Birgitte HolstDagger §, Hanne HastrupDagger , Ute RaffetsederDagger , Lene MartiniDagger §, and Thue W. SchwartzDagger §||

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Galpha s or Galpha 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 Galpha s fusion construct, whereas the lower affinity component was displayed by the tail-truncated Galpha 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 Galpha s signaling, showed binding properties that were monocomponent and otherwise very similar to those observed in the tail-truncated Galpha 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 Galpha s and Galpha 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

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 Galpha q pathway, which activates phospholipase C beta , thereby initiating inositol phosphate formation and a Galpha 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.

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 Galpha q or Galpha 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 Galpha s and Galpha q was supported by an observed total decoupling from Galpha s in a mutant NK1 receptor that closely mimicked the tail-truncated Galpha q fusion protein.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Materials-- Substance P and neurokinin A were purchased from Peninsula (Belmont, CA).

cDNA Constructs-- The cDNA coding for the long splice version of rat Galpha s was kindly provided by Dr. Din, and the cDNA for human Galpha 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-Delta tail and NK1·Gs-Delta 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).

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-Delta tail constructs aiming at 5-8% binding of the various radioactive ligands. However, in the case of the NK1·Gq-Delta 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.

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 - L and Ki = IC50/(1 L/Kd), where L is the concentration of radioactive ligand.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Signal Transduction in the Galpha 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 Galpha q and not Galpha s, two fusion proteins were made between the receptor and Galpha q. In NK1·Gq, the Galpha q protein was fused to the far C-terminal end of the NK1 receptor, whereas in NK1·Gq-Delta tail, the 72 most C-terminal residues of the C-terminal tail of the NK1 receptors were deleted, resulting in a fusion protein where Galpha 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 Galpha q to the far end of the C-terminal tail of the NK1 receptor impaired signaling through the adenylylcyclase pathway, this Galpha 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 Galpha q, severely limited the ability of the receptor to signal through the Galpha s-mediated adenyl cyclase pathway. Thus, in the NK1·Gq-Delta 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-Delta 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-Delta 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-Delta 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 Galpha 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 Galpha 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-Delta tail construct (black-square) (see Fig. 1 and text for details), and the [F111S]NK1 (black-triangle) 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.

                              
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Table I
Competition binding experiments
Competition binding experiments using 125I-substance P as tracer and substance P-induced signal transduction assays for, respectively, the presumed Galpha q signal transduction pathway (inositol phosphate turnover) and the presumed Galpha 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.

Although a more efficient decoupling of the NK1 receptor from Galpha s signaling was achieved by truncating the C-terminal tail of the receptor in the NK1·Gq-Delta 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 Galpha 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 Galpha 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-Delta tail fusion protein, i.e. with a Hill coefficient of approximately unity (Fig. 2C).

Signaling in the Galpha s NK1 Fusion Protein-- A fusion protein similar to the NK1·Gq-Delta tail was constructed, but Galpha q was replaced by Galpha 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-Delta tail construct (Fig. 3B). Surprisingly, the close association of the NK1 receptor with Galpha 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-Delta tail than in the wild-type receptor. This was also observed in the NK1·Gq-Delta tail (Fig. 3A).


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Fig. 3.   Inositol phosphate and cyclic AMP turnover in response to substance P in NK1 Galpha 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-Delta tail (black-triangle) (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.

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).


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Fig. 4.   Competition binding experiment for agonists in the wild-type NK1 receptor and in the two tail-truncated receptor-Galpha fusion proteins. Displacement of 125I-substance P by substance P (A) and neurokinin A (B) in the wild-type NK1 receptor (), the NK1·Gs-Delta tail (triangle ) construct, and the NK1·Gq-Delta 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-Delta tail (56 ± 4 nM) and for NK1·Gs-Delta tail (1.4 ± 0.2 nM).

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-Delta 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-Delta 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 Galpha s or Galpha q, respectively.

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-Delta tail and the NK1·Gs-Delta tail receptor fusion proteins (Fig. 4B and Table I). Also in the case of NKA it was the NK1·Gs-Delta tail construct that gave the high affinity state, whereas the NK1·Gq-Delta tail construct displayed the more low affinity state (Fig. 4B).

This surprising observation, namely that the complex between the receptor and Galpha 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 Galpha 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-Delta 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 Galpha 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 (triangle ). The displacement curves for the NK1·Gq-Delta 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).

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-Delta tail (panels C and D), in the [F111S]hNK1 receptor mutant (panels E and F), as well as in the NK1·Gs-Delta 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-Delta tail and the [F111S]hNK1 receptors displayed the lower of the two affinity sites for both substance P and NKA, whereas the NK1·Gs-Delta 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-Delta 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 Galpha s fusion protein with the truncated tail, the actual affinity for NKA is lower than in the wild-type.


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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-Delta tail construct (C and D), NK1·Gs-Delta tail construct (G and H), and the Galpha 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-Delta tail (13 ± 1 nM), for [F111S]NK1 (131 ± 24 nM), and for NK1·Gs-Delta 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-Delta tail (0.34 ± 0.07 nM), for [F111S]NK1 (0.7 ± 0.1 nM) and for NK1·Gs-Delta tail (0.07 ± 0.02 nM).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Galpha q or Galpha 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.

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" Galpha s pathway when Galpha 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 Galpha s pathway. Moreover, in these tail-truncated constructs monocomponent binding curves were found for the agonists, which was not the case when, for example Galpha q was fused to the far C-terminal end of the receptor.

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 beta 2-adrenergic receptor is well tolerated in G-protein fusions (21). The backbone of the remaining C terminus in the presently employed Delta -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 Delta -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 beta ) (21).

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 Galpha s and where monocomponent binding curves were obtained that closely correspond to those observed with the tail-truncated Galpha 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).

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·Galpha s Complexes-- It was surprising to find that the very high affinity state of the NK1 receptor was the one stabilized by Galpha s because it is generally believed that it is the Galpha 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 Galpha q signaling pathway is generally around one order of magnitude higher than the potency for activating the Galpha s pathway (6). Interestingly, although the affinity for substance P in the NK1·Gs-Delta 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 Galpha s-mediated very high affinity binding and significantly lower Galpha s signaling potency but would point out that the discrepancy apparently is also found in the wild-type receptor.

The fact that we observe an almost wild-type Galpha q signaling even in the tail-truncated Galpha s fusion construct could theoretically be attributed to a high degree of promiscuous coupling to endogenous Galpha q even in this supposedly tight receptor-Galpha s protein complex. However, this is not a likely explanation because we find a clear monocomponent "non-Galpha q-like" binding curve for both substance P and NKA in this construct. Nevertheless, it is possible that the NK1·Gs-Delta tail fusion construct could in fact be just ~10% promiscuous in its coupling, which the corresponding Gq-Delta 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 Galpha q pathway does not require as high a degree of receptor occupancy or a high number of receptor-Galpha q complexes. Another explanation could be, that a significant degree of the observed stimulation of inositol phosphate turnover is mediated through the beta gamma -subunits of the heterotrimeric G proteins (29).

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 Galpha s (high affinity) as opposed to when it is found in complex with Galpha 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 Galpha s-coupled NK1 receptor.2 The fact that substance P has very high affinity for the NK1 receptor in complex with Galpha 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 Galpha s and with Galpha 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 beta 2 adrenergic receptor depending on which G-protein it was attached to (30).

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-GROalpha (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 beta gamma - but also the alpha -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 Galpha s or Galpha q.

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.

    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 Galpha s-uncoupled constructs, NK1·Gq-Delta tail and [F111S]NK1 as compared with the wild-type and Galpha s-coupled constructs (see "Experimental Procedures").

    ABBREVIATIONS

The abbreviations used are: 7TM, seven transmembrane; NKA, neurokinin A; NK1, neurokinin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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