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J. Biol. Chem., Vol. 277, Issue 44, 42034-42048, November 1, 2002

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Mutations in the Extracellular Amino-terminal Domain of the NK2 Neurokinin Receptor Abolish cAMP Signaling but Preserve Intracellular Calcium Responses^*

Sandra Lecat^§, Bernard Bucher^¶, Yves Mely^¶, and Jean-Luc Galzi

From  CNRS UPR9050, Récepteurs et Protéines Membranaires, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brandt, Illkirch 67400, France and ^¶ CNRS UMR7034 Pharmacologie et Physicochimie des Interactions Cellulaires et Moléculaires, Université Louis Pasteur de Strasbourg, Faculté de Pharmacie 74, Route du Rhin, Illkirch BP 24 67401, France

Received for publication, April 15, 2002, and in revised form, August 7, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

By combining real time measurements of agonist binding, by fluorescence resonance energy transfer, and of subsequent responses, we proposed previously that the neurokinin NK2 receptor preexists in equilibrium between three states: inactive, calcium-triggering, and cAMP-producing. Thr²⁴ and Phe²⁶ of the NK2 receptor extracellular domain are considered to interact with neuropeptide agonists based on the reduction of affinity when they are substituted by alanine. Using fluorescence resonance energy transfer, we now quantify the binding kinetics of two Texas Red-modified neurokinin A agonists to the fluorescent wild-type (Y-NK2wt) and the mutant (Y-NK2mut) receptor carrying Thr²⁴  Ala and Phe²⁶  Ala mutations. TR1-neurokinin A binds with a fast component and a slow component to the Y-NK2wt receptor and triggers both a calcium and a cAMP response. In contrast, on the mutant receptor, it binds in a single fast step with a lower apparent affinity and activates only the calcium response. Another agonist, TRC4-neurokinin A, binds to both wild-type and mutant receptors in a single fast step, with similar affinities and kinetics and promotes only calcium signaling. Kinetic modeling of ligand binding and receptor interconversions is carried out to analyze phenotypic changes in terms of binding alterations or changes in the transitions between conformational states. We show that the binding and response properties of the Y-NK2mut receptor are best described according to a phenotype where a reduction of the transition between the inactive and the active states occurs.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

G-protein-coupled receptors (GPCRs)¹ represent the largest family of cell surface receptors and have multiple effects on the activated cell. Despite the variety of molecular types of ligands (hormones, proteins, small peptides, ions, lipids, and sensory stimuli such as odorants, pheromones, and photons), GPCRs share homology in their core domain composed of seven transmembrane -helices. The conservation of the same overall hydrophobic structure exemplifies the essential role of this structure for the activation process.

GPCR activation promotes GDP to GTP exchange on the  subunit of the heterotrimeric G protein associated with its cytoplasmic loops, thereby initializing the intracellular cascade of signaling events. Among the theoretical models describing the activation mechanism, the two-state allosteric model of activation has been proposed, following the discovery of inverse agonists and of constitutive activity for GPCRs (1-4). In this model, a GPCR preexists in equilibrium between an active and an inactive state in the absence of ligand. As such, agonists, higher affinity for the active state having displace the equilibrium toward activation, whereas inverse agonists have higher affinities for the inactive state. Constitutive activity is explained by an intrinsic tendency for a receptor to spontaneously isomerize toward the active conformation. Although the multiple conformational states theory is an attractive model for describing GPCR activation, there is a need for more data correlating the parameters of possible intermediate conversion states with physiological responses.

Indeed, several GPCRs have been reported to mediate multiple signaling pathways through activation of different heterotrimeric G proteins: the receptors for dopamine D1 (G_s and G_o (5), G_s and G_q (6)) and D5 (G_s and G_z (7)), the receptors for the parathyroid hormone (G_s, G_q, and G_i (8)), for the corticotropin-releasing hormone (G_s, G_i, G_q, G_o, and G_z (9)), for the melanin-concentrating hormone (G_i and G_o and maybe G_q (10)), for the vasoactive intestinal peptide (G_s and G_i (11)), for prostacyclin (G_s, G_i, and G_q (12)), the adenosine A1 receptor (G_o and G_i (13)), the ₂-adrenergic receptor (G_s and G_i (14)), the muscarinic m3 receptor (G_q and G₁₂ (15)), the 5-hydroxytryptamine receptor type 4; Bpa, p-benzoyl-L-phenylalanine. (G_i, G_o, and G_s (16)), the endothelin subtype B receptor (G_i, G_o, and G_q (17, 18)). These different couplings suggest that GPCRs may exist in more than a single active state.

We have previously addressed the question of multiple receptor states by using fluorescence resonance energy transfer (FRET) to monitor ligand binding in parallel with cellular responses. A chimeric green fluorescent neurokinin NK2 receptor, EGFP-NK2R was expressed in HEK293 cells. Two of its neuropeptide agonists, the decapeptide neurokinin A (NKA) and its truncated form, NKA-(4-10), were covalently linked to the fluorophore Texas Red (TR) (19). Kinetic measurements of the associations of TR1-NKA and TR7-NKA-(4-10) to the EGFP-NK2 receptor on living cells were monitored as a diminution of fluorescence emission of EGFP due to FRET.

This analysis showed that the NK2 receptor exists in a minimal number of three receptor conformations, denoted R0, R1, and R2 with intrinsic binding constants specific to each ligand (19). The R0 state corresponded to the inactive state. The R1 state could be populated by the two agonists, was characterized by a rapid association with both ligands, and corresponded to an active conformation triggering calcium responses via heterotrimeric G_q activation. The R2 state was stabilized at equilibrium only by TR1-NKA and was characterized by a slow binding relaxation temporally correlated with a cyclic AMP response unique to TR1-NKA. The R2 state was thus assigned to an active conformation triggering cAMP synthesis putatively through G_s activation (20-22).

In the present study, the phenotype of an EYFP chimeric fluorescent NK2 receptor carrying two point mutations located in the extracellular amino-terminal domain Thr²⁴  Ala and Phe²⁶  Ala has been investigated. These substitutions taken independently have been previously shown to lower the apparent affinity of NKA for the receptor (23, 24), suggesting that Thr²⁴ and Phe²⁶ are part of the binding domain for NKA. In addition, the activation process was reported to be unaffected by the substitutions, as measured by the ability of NKA to trigger calcium responses (23). However, given the demonstration that the NK2 receptor exists in several active states (19), we have now reanalyzed intracellular responses of the mutant receptor that we named Y-NK2mut. We find that the Y-NK2mut receptor is unable to activate cAMP production irrespective of the agonist tested. This shows that Thr²⁴ and Phe²⁶ somehow play a role in the activation process. We have studied the kinetics of interaction between the Y-NK2mut receptor and two of its fluorescent agonists (TR1-NKA and TRC4-NKA) by FRET and compared them to those of the wild-type Y-NK2wt receptor. The experimental data are interpreted in the context of the kinetic model previously used to describe the activation of the EGFP-NK2 receptor. We propose that, in fact, the mutant receptor is affected in its capacity to interconvert between the inactive state R0 and the two active states, behaving like a "constitutively inactive" receptor.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals

Synthetic peptides were obtained from Bachem or Neosystem. Protease inhibitors were obtained from Sigma and Calbiochem. Fluorescent labels and ion chelators were from Molecular Probes, Inc. (Eugene, OR). SR48968 was a kind gift from Sanofi. [³H]SR48968 was purchased from Amersham Biosciences. Geneticin (G418) was from Invitrogen.

Construction of the Mutant Receptor Chimera and Expression in Stable HEK

A NotI-XhoI SP-EYFP fragment, in which SP is the signal peptide and EYFP the enhanced yellow fluorescent protein, was generated as described for the SP-EGFP fragment (25) using pEYFP-C3 (Clontech) as a source of EYFP sequence. The wild-type rat NK2 sequence starting at the amino acid 16 was extracted from the pCEP4-chim1 vector as a XhoI-BamHI fragment (25). The NK2 mutant fragment XhoI-BamHI was amplified by PCR using pCEP4chim1 as a bait with primer 1 (A CCG CTC GAG AGC AAT GCC ACG GGT GTT GCA GCC GCC TCC ATG) (XhoI site underlined) and primer 2 (CG GGA TCC CTA AGC CTG GGC CTT GCA GAT GGG) (BamHI site underlined). The mutated nucleotides are in boldface type in primer 1. The NotI-XhoI SP-EYFP fragment was ligated in frame to the XhoI-BamHI NK2 fragment and inserted in the expression vector pREP9 (Invitrogen) between the NotI and BamHI sites.

HEK 293 cells, grown in minimal essential medium complemented with 10% fetal calf serum and antibiotics (streptomycin/penicillin), were transfected by calcium phosphate precipitation (26) and selected with 600 µg/ml G418. Stable cell lines were passaged twice a week. New stocks were defrosted every 12 weeks.

Fluorescence Microscopy

Cells were grown for 2 days in 24-well plates on 12-mm glass coverslips coated with rat type I collagen. For endosome staining, Texas Red transferrin uptake was done as previously described (27) or cell lines expressing either the Y-NK2wt or the Y-NK2mut receptor were transiently transfected with a pcDNA3 plasmid expressing a Rab5(Q79L)-myc tagged under a cytomegalovirus promoter. On the day of the experiment, cells were washed twice with PBS, fixed in 4% paraformaldehyde-PBS for 15 min at room temperature and then incubated for 15 min in 50 mM NH₄Cl in PBS. Coverslips were mounted onto microscope slides using Möwiol (Calbiochem) and observed either with an inverted microscope (Zeiss Axioplan) equipped with a Hamamatsu CDD camera or with an inverted microscope (Nikon Eclipse TE300) connected to a laser-scanning confocal imaging system (Bio-Rad MRC 1024 ES) using a Plan Apo ×60 1.20 numerical aperture water immersion objective (Nikon).

For epifluorescence measurements, the EYFP was excited at 485 ± 10 nm with a bandpass filter. Emitted fluorescence was detected using a dichroic mirror at 510 nm and a longpass filter of 520 nm. For confocal measurements, excitation was from a 30-milliwatt krypton/argon laser at 10% power. Each stack of two-dimensional images was acquired sequentially in the green channel (PMT2; excitation 488 nm, emission 522 nm) and eventually in the red channel (PMT1; excitation 568 nm, emission 605 nm), before stepping (0.5 µm) the objective in the z axis; typically 10-12 images are taken per cell.

Immunoprecipitations and Immunoblots

Around 5 million cells were resuspended in 800 µl of cold lysis buffer (in 150 mM NaCl, 1.5 mM MgCl₂, 10% glycerol, 5 mM/EDTA, 50 mM Hepes, pH 7.5) supplemented with 1 mM dithiothreitol and one tablet of complete EDTA-free protease inhibitor (for 10 ml of buffer; Roche Molecular Biochemicals). A postnuclear supernatant was prepared as described previously (27). Immunoprecipitations were carried out with a polyclonal rabbit anti-GFP antibody made in our laboratory. The immunoprecipitated complex was prepared as described (27) prior to loading on a 12% SDS-PAGE gel. Gels were blotted onto polyvinylidene difluoride membranes and blots were incubated with a mouse monoclonal anti-GFP antibody (Clontech) at a 1:1000 dilution, followed by incubation with a goat anti-mouse secondary antibody conjugated to horseradish peroxidase (Amersham Biosciences) at a 1:2000 dilution. Detection was achieved using enhanced chemiluminescence (SuperSignal® WestPico; PerbioScience).

Radioligand Binding Experiments

Radioligand binding assays and quantification of receptor sites per cell were performed as described previously (25). Competition experiments were performed at 4 °C with 1 nM [³H]SR48968 for 3 h, and experimental IC₅₀ values were converted to K_i values using the Cheng and Prusoff relationship (28).

Synthesis of Fluorescent Analogs

The peptides NKA (HKTDSFVGLM-NH₂), C7-NKA-(4-10) (DSFCGLM-NH₂) and C4-NKA (HKTCSFVGLM-NH₂) were derivatized with Texas Red as previously described for fluorescent TR1-NKA and TR7-NKA-(4-10) peptides (19).²

Fluorescence Kinetic Measurements

Adherent cells were rinsed with PBS and dissociated from the culture flask in PBS, 5 mM EDTA buffer, pH 7.4. Unless otherwise stated, cell suspensions were at 10⁶ cells/ml in HEPES-bovine serum albumin buffer (137.5 mM NaCl, 1.25 mM MgCl₂, 1.25 mM CaCl₂, 6 mM KCl, 5.6 mM glucose, 10 mM HEPES, 0.4 mM NaH₂PO₄, 1% bovine serum albumin (w/v), pH 7.4) supplemented with protease inhibitors (40 µg/ml bestatin and bacitracin, 20 µg/ml phosphoramidon, 50 µg/ml chymostatin, and 1 µg/ml leupeptin).

All fluorescence measurements on cell suspensions were made on a Fluorolog-2 (SPEX) spectrofluorometer equipped with a 450-watt xenon lamp, one double grating excitation (set at 470 nm) and two single grating emission monochromators (time-based set at 530 and 610 nm). Interaction of TR-modified NKA with the cell surface Y-NK2 receptors was monitored as a decrease of EYFP emission at 530 nm due to fluorescence resonance energy transfer toward the acceptor group TR. Data were acquired with a photon-counting photomultiplier (linear up to 10⁷ counts/s) and stored using the DM3000 software provided with the spectrofluorometer.

Screening of Fluorescent Ligands Using the Association Kinetics Measured by FRET-- Cells were placed in a 1-ml cuvette with magnetic stirring and maintained at 21 °C in the thermostatted cuvette handler of the spectrofluorometer as previously described (25). Time-based recordings were sampled every 300 ms. Ligand was added to the cuvette at around 20-50 s by stopping the recordings for a short time.

Quantitative Analysis of Association by FRET with a Stopped-flow Apparatus-- Quantitative experiments were carried out at 21 °C with an Applied Photophysics SK1E apparatus modified for rapid mixing of living cells. The observation chamber was a Hellma 176.002 (100-µl) quartz circulation cuvette placed in the cuvette holder of the spectrofluorometer. Time-based recordings were sampled every 50 ms as described (19). The range of ligand concentration used in stopped-flow assays was previously determined in equilibrium binding assays, yielding measurement of receptor site occupancy and saturation.

Kinetic Data Analysis

Each trace corresponds to the mean of three consecutive recordings. Fitting was carried out with nonlinear regression, by giving to each triplicate a weighting inversely proportional to the corresponding variance. Traces were analyzed with a single exponential, one exponential plus linear, drift or two exponentials plus linear drift with Kaleidagraph (Synergy Software) (19, 29). The selection of the adequate fitting procedure was based on the comparison of the nonlinear least squares derived for the various fits.

Kinetics of Association-- The single bimolecular interaction data were analyzed with Kaleidagraph, whereas the data corresponding to the model of a bimolecular interaction followed by the interconversion to a second conformation were analyzed with SAS software. The FRET measurements in the stopped-flow system were performed with 2 × 10⁶ cells/ml. Each cell line expressed 750,000 Y-NK2wt receptor sites and 1 million Y-NK2mut receptor sites per cell. Final receptor site concentration in the assay was thus as follows: [Y-NK2wt_T] ~ (750,000 × 2 × 10⁶ × 10³)/6.02 × 10²³ ~ 2.5 nM, and [Y-NK2mut_T] ~ (1,000,000 × 2 × 10⁶ × 10³⁾/6.02 × 10²³ ~ 3 nM.

In the stopped-flow measurements, an exponential drift occurs with a constant rate of 0.025 s¹ at all agonist concentrations, and its amplitude is constant. This is due to the sedimentation of the cells in the cuvette, since it is not observed in recordings performed in a standard cuvette equipped with magnetic stirring. This exponential drift has been taken into account in the kinetic data analysis (and has been afterwards subtracted from the binding traces for fitting of the experimental traces with the simulated traces in the modeling).

Kinetics of Dissociation-- Dissociation curves were analyzed with Kaleidagraph with either one or two exponentials according to the following: [RL](t) = a + be^{k_{off b}t} or [RL](t) = a + be^{k_{off b}t} + ce^{k_{off c}t}. Each relaxation process was characterized by the values of its amplitude (b and c, given as relative amplitudes) and of its dissociation rate constant k_off.

Physiological Responses to Agonists

Calcium Response Analysis-- Adherent cells were loaded with 5 µM Indo-1 in HEPES-bovine serum albumin for 45 min at 37 °C. They were dissociated in PBS and suspended in HEPES-bovine serum albumin buffer. Cell suspension measurements were made at 21 or 37 °C in the 1-ml cuvette on the spectrofluorometer with excitation set at 355 nm and emission detected at 400 nm.

cAMP Measurements-- cAMP measurements were performed as described by Rumenapp et al. (19). Agonists were applied on adherent cells at 21 or 37 °C and at various concentrations for 15 min, unless otherwise stated. Reactions were stopped by the addition of 1 volume of ice-cold 0.2 M HCl.

Kinetic Modeling of Ligand Binding and Responses

Kinetic modeling of ligand binding, and responses was performed as described by Palanche et al. (19). The differential equations for ligand binding are solved in the MATLAB computing environment using a program called STOIC (30). The theoretical model simulates ligand binding for a receptor with one binding site and three conformational states. The conformational states are in freely interconverting equilibria, such that all states are present in defined proportions in the absence of ligand and their equilibria shift as a function of the kinetics of ligand binding.

The entered parameters for each receptor and for each ligand in the simulation are as follows: (i) experimental ligand concentrations, (ii) measured association rate constant k₁ (assuming that they are identical for the three states), (iii) measured dissociation rate constant k_off for each state (all of the experimental dissociation traces have been fitted with two exponentials and correspond to two of the three states, each combination being tested in the modeling), and (iv) intrinsic rates for the transition between two ligand-bound receptor states.

The program simulates and represents as a function of time (i) the theoretical binding and dissociation traces for the indicated concentrations of ligand and (ii) the theoretical proportions of ligand-free and ligand-bound receptor states and allows us to derive values for the intrinsic isomerization constants  between two of the NK2 receptor states.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Construction and Expression of the Fluorescent Y-NK2wt and Y-NK2mut Receptors

Huang et al. (23) have performed an exhaustive site-directed mutagenesis study of the NK2 receptor to identify residues involved in NKA binding. Important residues were mainly located in the amino-terminal domain, the extracellular loops 1 and 2, and in the second hydrophobic transmembrane helix. We have introduced two point mutations in the extracellular extremity of rat NK2 (substitutions Thr²⁴  Ala and Phe²⁶  Ala). Y-NK2wt and Y-NK2mut chimeric receptors were generated by joining the carboxyl terminus of the yellow fluorescent protein EYFP to the amino-terminal part starting at residue Leu¹⁶ of the wild-type and the mutant NK2 (Fig. 1A), as had previously been done for the wild-type NK2 receptor fused to the green fluorescent protein, EGFP (19, 25). Each construct was stably transfected into HEK293 cells. Confocal images of the expressing cells illustrate that the fusion proteins were properly localized at the plasma membrane (Fig. 1B). Intracellular fluorescent signal corresponds mainly to endosomal compartments as measured by Rab5 and Texas Red-transferrin co-localization (data not shown). Each protein had the expected size on polyacrylamide gels (27 kDa of EYFP plus 40 kDa of NK2 receptor) as revealed by immunoblotting with a polyclonal anti-GFP antibody (Fig. 1C).

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Construction and expression of the fluorescent wild-type and mutant Y-NK2 receptors. A, schematic view of the constructed proteins with the fluorescent EYFP fused to the Leu16 on the extracellular part of the rat NK2 receptor. The mutated residues Thr24  Ala and Phe26  Ala are in boldface type. The last shown amino acid, Trp36, corresponds to the amino acid preceding the first transmembrane helix according to the recently determined structure of rhodopsin. B, confocal fluorescence images of HEK 293 cells expressing either the Y-NK2wt or the Y-NK2mut constructs taken in the middle of the cells (2.5 µm from each pole in the z axis). C, immunoblot with a monoclonal anti-GFP antibody following immunoprecipitation of each of the chimeric receptors, Y-NK2wt and Y-NK2mut, by a polyclonal anti-GFP antibody. HEK refers to a control experiment performed on nontransfected cells.

Pharmacological Properties and Physiological Responses to NKA of Activated Y-NK2wt and Y-NK2mut Receptors

Equilibrium Binding-- The number of receptors expressed at the cell surface were estimated from radiolabeled antagonist [³H]SR48968 binding, which is not sensitive to mutations in the amino-terminal domain (23). For each cell line, we have found ~750,000 Y-NK2wt sites and 1,150,000 Y-NK2mut sites per cell. NKA binding affinities were determined from competition experiments performed at equilibrium using 1 nM [³H]SR48968 (Fig. 2A). The apparent inhibition constants for NKA (K_i ± S.D., n = 3) were 4.5 ± 0.5 nM and 45 ± 8 µM for Y-NK2wt and Y-NK2mut receptors, respectively, indicating an apparent 10,000-fold loss of NKA affinity for the mutant, which is in agreement with published data (23, 24).

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Pharmacological properties and physiological responses of Y-NK2wt and Y-NK2mut receptors activated by NKA and TR1-NKA. A and D, measurement of apparent inhibition constants. Displacement of 1 nM [3H]SR48968 by increasing concentrations of NKA (A) or TR1-NKA (D) on cells expressing the Y-NK2mut (black circle) or the Y-NK2wt receptors (white square) after 3 h at 4 °C. Each point corresponds to the mean value of three independent experiments performed in duplicate. The error bars are the S.D. B and E, calcium responses. The extent of intracellular calcium responses induced at 21 °C by increasing concentrations of NKA (B) or TR1-NKA (E) recorded at 400 nm using the calcium probe indo-1 (excitation 355 nm). Each point corresponds to the mean value of the calcium peak of two independent experiments. The error bars are the S.D. C and F, cAMP responses. The amount of cAMP (pmol/wells) produced at 21 °C after 15-min incubation with various concentrations of NKA (C) or TR1-NKA (F). Each point is the mean value of two experiments performed in duplicate.

Responses-- Calcium responses were monitored on cell suspensions using Indo-1 fluorescence variations after agonist addition and quantified as peak response amplitudes. The Y-NK2mut receptor responded to NKA with an EC₅₀ of 3.4 ± 0.2 µM (versus 2.2 ± 0.5 nM for the Y-NK2wt receptor) and a maximal response reaching 70% of the maximal Y-NK2wt receptor response for the same agonist (Fig. 2B). Since we and others reported cAMP production following NK2 receptor activation (19, 20, 31), the capacity of Y-NK2mut to initiate cAMP synthesis was investigated. Unexpectedly, the mutant did not induce any cAMP response even at high concentrations of NKA (up to 100 µM) (Fig. 2C). Previously, we have modeled the activation of the NK2 receptor and proposed that its calcium and cAMP responses are mediated by distinct active states R1 and R2, respectively (Fig. 3), which can be detected by ligand binding kinetic determinations (19). We have shown as well, using FRET, that these states exhibit different affinities for two fluorescent agonists (19). Hence, in order to test further this former proposal, the same type of binding kinetic analysis was undertaken on the Y-NK2mut receptor.

View larger version (7K): [in this window] [in a new window]   Fig. 3.   Three-state kinetic model of the NK2 receptor. The NK2 receptor can oscillate between three states denoted R0, R1, and R2. R0 is the inactive state. The R1 state is coupled to the Gq heterotrimeric protein, and the R2 state is probably coupled to Gs. Intrinsic isomerization constants of the receptor in the absence of ligand are denoted 0 and '0 (e.g. 0 = [R0]/[R1]) and in the presence of ligand are denoted 1 and '1 (e.g. 1 = [R0ligand]/[R1ligand]). K0, K1, and K2 are the intrinsic affinity constants of the ligand for the R0, R1, and R2 states, respectively.

Pharmacological Properties, Physiological Responses, and Kinetics of Association of TR1-NKA to the Y-NK2wt and Y-NK2mut Receptors

Equilibrium Binding and Responses-- TR1-NKA has a TR fluorophore linked to the amino-terminal extremity of NKA and behaves like NKA on the wild-type green EGFP-NK2 receptor in terms of affinities and responses (19). Pharmacological and functional properties of TR1-NKA association to the Y-NK2wt and Y-NK2mut receptors were investigated. The apparent inhibition constants for TR1-NKA were K_i = 4.9 ± 0.2 nM and K_i = 250 ± 20 nM for Y-NK2wt and Y-NK2mut receptors, respectively (Fig. 2D). Calcium responses to TR1-NKA were followed up to the concentration limit (1 µM). EC₅₀ were equal to 0.60 ± 0.1 nM and 180 ± 30 nM for the Y-NK2wt and Y-NK2mut receptors, respectively (Fig. 2E) with a maximal calcium response of the mutant reaching around 30% of the maximal Y-NK2wt receptor response to the same agonist. TR1-NKA interaction with the Y-NK2wt receptor triggered cAMP synthesis with an EC₅₀ of 32 ± 10 nM. There was no detected cAMP accumulation promoted by TR1-NKA association to the Y-NK2mut receptor (Fig. 2F). In summary, TR1-NKA binds to the Y-NK2mut receptor with a better apparent affinity than does nonfluorescent NKA (180-fold) but still with a much lower apparent affinity than to the Y-NK2wt receptor (50-fold). It behaves as a partial agonist in calcium signaling and, like NKA, it does not trigger cAMP production on the Y-NK2mut receptor.

Continuous Monitoring of TR1-NKA Association by FRET-- Cells in suspension were placed in a 1-ml cuvette maintained at 21 °C in the cuvette holder of a spectrofluorometer. Ligands were added to the sample 20-50 s after thermal equilibration. The decrease of the EYFP-emitted fluorescence at 530 nm reflects the occupancy of the cell surface fluorescent receptors by the fluorescent ligand. The binding of TR1-NKA to the Y-NK2mut receptor was different both in terms of amplitude and time course as compared with its binding to the Y-NK2wt receptor. This is illustrated in Fig. 4A with 6 and 170 nM TR1-NKA. We have previously shown that TR7-NKA-(4-10) binds with a single fast step, mainly to the R0 and R1 states of the NK2wt receptor (19). The experimental trace for 100 nM TR1-NKA association to the Y-NK2mut receptor could be superimposed on that obtained for 10 nM TR7-NKA-(4-10) binding to the Y-NK2wt receptor (Fig. 4B). This suggests that TR1-NKA may only bind to the R0 and R1 states of the Y-NK2mut receptor. To refine association time courses and determine binding rate constants, we analyzed the kinetics of TR1-NKA association and dissociation on Y-NK2mut and Y-NK2wt receptors by FRET using a stopped-flow apparatus.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Detection by FRET of the binding of fluorescent NKA analogs to either Y-NK2wt or Y-NK2mut receptors. A, kinetics of TR1-NKA association. Continuous recordings of EYFP fluorescence (at 530 nm) before and after addition (at 50 s) of 6 and 170 nM TR1-NKA to Y-NK2wt (solid line) or Y-NK2mut-expressing cells (dotted line). B, kinetics of TR1-NKA as compared with TR7-NKA-(4-10) associations. Continuous recordings of EYFP fluorescence before and after the addition (at 30 s) of 100 nM TR1-NKA to the Y-NK2mut expressing cells (dotted line) or after the addition of 10 nM (gray solid line) or 100 nM (black solid line) of TR7-NKA-(4-10) to the Y-NK2wt-expressing cells. Each trace corresponds to one representative experiment of at least four independent experiments.

Quantitative Analysis of the Kinetics of the Y-NK2mut Receptor/TR1-NKA Interactions

Ligand binding associations and dissociations were analyzed as previously described (19). The rigorous analysis of association kinetics could not be directly performed with an equation based on the model presented in Fig. 3, because the number of unknown parameters associated with this model is too large to allow their confident simultaneous determination. Binding traces were therefore analyzed either as simple bimolecular reactions or as combinations of bimolecular reactions with interconversions, using equations given below. Parameters derived from the following kinetic data analyses were then incorporated in the three-state model from Fig. 3 in a second step (see Figs. 10-12).

TR1-NKA Association Step-- The binding traces of TR1-NKA to the Y-NK2mut receptor follow a monoexponential time course that can be analyzed as a single bimolecular interaction.

In the presence of a given total concentration of ligand, L_T, the measured fluorescence intensity I can be expressed as follows,

(Eq. 1)

where I₀ and I₁ are the fluorescence intensities associated with R and RL, respectively. Since [R_T], the total concentration of receptors, corresponds to the sum of the concentrations of R and RL, the equation can be rewritten as follows.

(Eq. 2)

Under pseudo-first-order conditions, the concentration of free ligand is equal to the concentration of total ligand, and the concentration of RL is deduced from the following differential equation, assuming that at time 0, [R] = [R_T] and [RL] = 0. 

(Eq. 3)

This gives then the following,

(Eq. 4)

where a represents the FRET amplitude.

Each experimental binding trace (Fig. 5A) recorded under pseudo-first-order conditions ([L_T]  10 × [R_T]) was fitted separately by replacing Equation 4 into Equation 2. TR1-NKA association and dissociation rate constants k₁ and k₂, expressed as means ± S.D. for the family of association curves, were found to be equal to k₁ = 0.8 ± 0.1 × 10⁶ M¹ s¹ and k₂ = 0.10 ± 0.01 s¹. The fluorescence intensities relative to the unliganded or the liganded receptor state are I₀ = 0.93 ± 0.04 and I₁ = 0.75 ± 0.05, respectively. The amplitude of the FRET signal increases with ligand concentration up to a plateau value above 300 nM TR1-NKA. The plot of the amplitude of FRET as a function of TR1-NKA concentration (saturation curve) can be fitted with the empirical Hill equation to yield a dissociation constant K_D = 48 ± 12 nM with a Hill coefficient of 1.1 ± 0.3 (Fig. 5B).

View larger version (54K): [in this window] [in a new window]   Fig. 5.   Real time recordings of TR1-NKA association to the Y-NK2mut receptor and experimentally derived parameters. A, time course of TR1-NKA interaction with the Y-NK2mut receptor. A suspension of 2 × 106 cells/ml is rapidly mixed at 21 °C in the stopped-flow apparatus with buffer containing the desired concentration of fluorescent agonist. Each black trace corresponds to the mean of three consecutive recordings carried out at the indicated ligand concentrations. Data points were acquired every 50 ms. Extinction of EYFP fluorescence at 530 nm (excitation 490 nm) is normalized to the signal amplitude recorded without ligand. The interaction of saturating concentrations (1 µM) of TR1-NKA results in a 15-20% reduction of total sample fluorescence. The gray line is the best fit according to a bimolecular interaction, taking into account the exponential drift observed in control recordings in the absence of ligand. B, saturation of receptor sites by TR1-NKA. Plots of the FRET amplitudes determined from fitting binding traces in A versus molar TR1-NKA concentrations. The black square indicates the FRET amplitude at 2 µM TR1-NKA. C, kinetics of TR1-NKA dissociation. After a 30-min preincubation of cells with 100 nM TR1-NKA, dissociation was initiated by rapid stopped-flow mixing with buffer containing 20 µM SR48968. The solid gray line is the best fit obtained with two exponentials.

TR1-NKA Dissociation-- Dissociation of receptor-ligand complexes obtained at equilibrium with 100 nM TR1-NKA was initiated by rapid mixing with an excess (20 µM) of SR48968 (Fig. 5C). The dissociation relaxation is best represented by a sum of two exponentials (see "Experimental Procedures") revealing a rapid process representing 50% of the FRET amplitude with a dissociation rate constant k_{off b} = 0.30 ± 0.02 s¹ and a slow dissociation step with a rate constant k_{off c} = 0.040 ± 0.001 s¹. This suggests that TR1-NKA, at equilibrium, is bound to two states of the Y-NK2mut receptor.

Quantitative Analysis of the Kinetics of the Y-NK2wt Receptor/TR1-NKA Interactions

Kinetics of TR1-NKA association to the Y-NK2wt receptor were analyzed as previously described (19) according to a bimolecular interaction followed by a rate-limiting isomerization step.

Since the values of k₂ and k₃ are close to each other (19), we derived an equation for the full scheme rather than two equations describing binding and interconversion separately (19). With I₀, I₁, and I₂, the fluorescence intensities associated with R, RL, and R*L, respectively, it follows that the measured fluorescence intensity I can be expressed by the following.

(Eq. 5)

Under pseudo-first-order conditions ([L] ~ [L_T]), the concentrations of RL and R*L are deduced from the following differential equations, assuming that at time 0, [R] = [R_T], [RL] = [R*L] = 0, 

(Eq. 6)

(Eq. 7)

with  = k₂ + k₃ + (k₁[L_T]), ' = k₃,  = k₄  (k₁[L_T]), ' = k₄, and  = k₁[R_T][L_T].

Solution of Equations 6 and 7 yields the following biphasic decay kinetics,

(Eq. 8)

(Eq. 9)

with


The experimental binding traces (Fig. 6A) recorded under pseudo-first-order conditions ([L_T]  10 × [R_T]) were fitted by substituting Equations 8 and 9 into Equation 5. After fitting each curve separately, the rate constants were determined as means ± S.D. for the family of association curves. The final results are k₁ = 1.82 ± 0.02 × 10⁶ M¹ s¹, k₂ = 0.12 ± 0.04 s¹, k₃ = 3.96 ± 0.04 × 10² s¹, and k₄ = 6 ± 4 × 10³ s¹, where k₁ and k₂ are, respectively, the association and the dissociation rate constants for the formation of the RL complex, and k₃ and k₄ are the forward and the backward rate constants, respectively, of the equilibrium between RL and R*L. The relative fluorescence intensities are I₁ = 0.58 ± 0.06 for RL and I₂ = 0.60 ± 0.01 for R*L. In a next step, the theoretical receptor site occupancies for R and R* calculated from Equations 8 and 9 using the above rate constant values were plotted as a function of TR1-NKA concentration (Fig. 6B). This plot can be fitted with the empirical Hill equation to yield for R a dissociation constant equal to 500 ± 90 nM with a Hill coefficient of 1.0 ± 0.1 and for R* a dissociation constant equal to 1.020 ± 0.004 nM, with a Hill coefficient of 1.000 ± 0.003. 

View larger version (41K): [in this window] [in a new window]   Fig. 6.   Real time recordings of TR1-NKA interactions with the Y-NK2wt receptor and experimentally derived parameters. A, time course of TR1-NKA binding to the Y-NK2wt receptor (under the same conditions described in Fig. 5). The addition of saturating concentrations (1 µM) of TR1-NKA results in a 40-50% reduction of total sample fluorescence. The gray line through each data trace is the best fit according to a bimolecular interaction followed by the interconversion to a second conformation. B, maximal receptor state occupancies as a function of TR1-NKA concentrations. The maximal receptor site occupancy for R occurs when d[RL]/dt = 0 and for R* at t = . The rate constant values determined from fitting association traces in A with Equation 5 were thus replaced in the corresponding equation. The plot represents the calculated maximal amount of RL (white square) and R*L (white triangle) as a function of TR1-NKA concentrations. C, kinetics of TR1-NKA dissociation. After a 30-min preincubation of cells with 100 nM TR1-NKA, dissociation was initiated by rapid stopped-flow mixing with buffer containing 20 µM SR48968. The solid gray line is the best fit obtained with two exponentials.

TR1-NKA Dissociation-- Dissociation traces obtained by rapid mixing of 20 µM SR48968 with cells expressing the Y-NK2wt receptor preincubated for 10 min with 100 nM TR1-NKA can be fitted by a two-exponential function (see "Experimental Procedures") comprising a slow phase with a rate constant k_{off b} = 0.0055 ± 0.0002 s¹ and a rapid phase with a rate constant k_{off c} = 0.03 ± 0.01 s¹. At 10 min, the proportion of the rapid to slow dissociation steps is 22/78% (Fig. 6C).

In summary, there is no detectable slow relaxation phase of TR1-NKA binding to the Y-NK2mut receptor in contrast to its binding to the Y-NK2wt receptor. This correlates with a lack of a cAMP response. Therefore, the data strengthen the previous interpretation (19) that the slow binding phase represents binding to an active state R2, which induces cAMP production. We next analyzed the binding properties of other agonists to the Y-NK2mut receptor.

Binding Properties and Responses of TRC4-NKA, an Agonist with Similar Affinities for the Y-NK2wt and the Y-NK2mut Receptors

Continuous Monitoring and Equilibrium Measurements of TRC4-NKA Association-- Other fluorescently labeled analogs of NKA, which carry the TR fluorophore at different positions on the peptide, were screened by FRET at 100 nM by placing the cells in suspension in the 1-ml cuvette maintained at 21 °C in the spectrofluorometer. Among the ligand tested, one peptide, TRC4-NKA, bound to both receptor types with similar binding kinetics as opposed to TR1-NKA (Fig. 7A). This agonist carries the TR on a cysteine that substitutes for an aspartate at position 4. At all TRC4-NKA concentrations tested (Fig. 7B, illustration with 1, 10, and 100 nM), the amplitudes of FRET upon binding to the Y-NK2wt and the Y-NK2mut receptors were identical, indicating that TRC4-NKA has the same affinity for both receptors. This was confirmed by competition experiments against [³H]SR48968 giving K_i = 40 ± 10 and 70 ± 15 nM for the Y-NK2wt and the Y-NK2mut receptors, respectively (Fig. 7C). TRC4-NKA is therefore a ligand that, surprisingly, seems to be unable to discriminate between the mutant and the wild-type receptors in terms of binding properties.

View larger version (49K): [in this window] [in a new window]   Fig. 7.   Detection of TRC4-NKA interactions with either Y-NK2wt or Y-NK2mut receptors. A and B, kinetics of TRC4-NKA as compared with TR1-NKA associations. A, continuous recordings of EYFP fluorescence before and after the addition (at 50 s) of 100 nM TR1-NKA or TRC4-NKA to the Y-NK2wt-expressing (solid line) or Y-NK2mut-expressing (dotted line) cells. Each trace corresponds to one representative experiment of at least four independent experiments. B, continuous recording of EYFP fluorescence before and after the addition (at 20 s) of 1, 10, and 100 nM of TRC4-NKA to the Y-NK2mut-expressing (dotted line) or to the Y-NK2wt-expressing (solid line) cells. Each trace corresponds to one representative experiment of at least four independent experiments. C, measurements of TRC4-NKA apparent inhibition constant. Displacement of 1 nM [3H]SR48968 by increasing TRC4-NKA concentration of Y-NK2mut-expressing (black circle) or YFP-NK2wt-expressing (white square) cells after 3 h at 4 °C. Each point corresponds to the mean value of three independent experiments performed in duplicate. The error bars are the S.D. D, calcium responses; the extent of intracellular calcium responses induced at 21 °C by increasing concentrations of TRC4-NKA recorded at 400 nm using the calcium probe indo-1 (excitation 355 nm). E, cAMP responses; amount of cAMP (pmol/well) produced at 21 °C after a 15-min incubation with various concentrations of TRC4-NKA.

Responses at 21 °C-- Interestingly, the decrease of EYFP fluorescence due to FRET upon binding of TRC4-NKA to both receptors was fast and reached equilibrium rapidly (Fig. 7A). This suggested that TRC4-NKA might not stabilize the slowly equilibrating R2 state of the NK2 receptor and prompted us to test the responses associated with its binding. TRC4-NKA behaved like a partial agonist as compared with TR1-NKA; TRC4-NKA binding to the Y-NK2wt receptor triggered 75% maximal calcium responses as compared with TR1-NKA binding. The maximal calcium response following TRC4-NKA binding to the Y-NK2mut receptor was only 15% compared with TR1-NKA binding to the Y-NK2wt receptor (Fig. 7D). TRC4-NKA binding to either of the Y-NK2 receptors did not induce any cAMP production (Fig. 7E).

All kinetic analyses described above were performed at 21 °C to avoid interference with receptor endocytosis. Hence, all physiological responses were recorded at 21 °C as well. However, at this temperature, the Y-NK2mut calcium responses to TRC4-NKA were very small. To confirm the activation capacity of the Y-NK2mut receptor, physiological responses triggered by all the ligands were reanalyzed at 37 °C.

Calcium Response at 37 °C-- As compared with calcium responses recorded at 21 °C (Figs. 2, B and E, and 7D), the three agonists elicited larger calcium responses at 37 °C (Fig. 8, A-C). Nevertheless, the Y-NK2mut receptor still displayed lower maximal calcium responses than the Y-NK2wt receptor. Concomitantly, the EC₅₀ values for both receptor types were lower at 37 °C than at 21 °C. In particular, for the Y-NK2wt receptor, EC₅₀ values decreased from 2.2 ± 0.5 to 0.5 ± 0.1 nM for NKA and from 0.6 ± 0.1 to 0.18 ± 0.07 nM for TR1-NKA. For the Y-NK2mut receptor, EC₅₀ values decreased from 3.4 ± 0.2 µM to 910 ± 260 nM for NKA and from 180 ± 30 to 130 ± 20 nM for TR1-NKA. At 37 °C, TRC4-NKA activated the Y-NK2wt receptor with an EC₅₀ of 14 ± 4 nM and the Y-NK2mut receptor with an EC₅₀ of 47 ± 10 nM.

View larger version (29K): [in this window] [in a new window]   Fig. 8.   Calcium and cAMP responses promoted at 37 °C by NKA, TR1-NKA, or TRC4-NKA binding to either Y-NK2wt or Y-NK2mut receptors. A-C, calcium responses. Intracellular calcium release was recorded from cells expressing the Y-NK2mut (black circle) or Y-NK2wt (white square) receptor recorded at 37 °C after the addition of increasing concentrations of NKA (A), TR1-NKA (B), and TRC4-NKA (C). Each point corresponds to the mean value of the calcium peak of two independent experiments. The error bars are the S.D. D and E, cAMP responses; the amount of cAMP (pmol/well) synthesized at 37 °C as a function of time upon activation of the Y-NK2wt receptor (D) by 100 µM NKA (white triangle), 1 µM TR1-NKA (white square), or 100 µM forskolin (white circle) or upon activation of the Y-NK2mut receptor (E) by 100 µM NKA (black triangle), 1 µM TR1-NKA (black square), 1 µM TRC4-NKA (black diamond), or without agonist (large cross) or upon activation of the Y-NK2wt receptor by 1 µM TRC4-NKA (white diamond) or without agonist (white circle). Each point was done in duplicate.

cAMP Response at 37 °C-- NKA and TR1-NKA triggered the synthesis of 3 times more cAMP at 37 °C than at 21 °C on the Y-NK2wt receptor (Fig. 8D compared with Fig. 2, C and F). Despite the elevation of temperature, NKA and TR1-NKA still failed to evoke any significant cAMP response on the Y-NK2mut receptor (Fig. 8E). Similarly, TRC4-NKA was unable to stimulate any cAMP production mediated by the two receptor types.

Fast kinetic measurement of TRC4-NKA/receptor interactions were carried out next.

Quantitative Analysis of the Kinetics of the Y-NK2wt and the Y-NK2mut Receptor Interactions with TRC4-NKA

TRC4-NKA Association-- The binding traces of TRC4-NKA to the Y-NK2mut receptor can be analyzed as single pseudo-first-order relaxations according to a bimolecular interaction best fitted by replacing Equation 4 into Equation 2 derived from Scheme 1 (Fig. 9A). The time courses of TRC4-NKA binding to the Y-NK2mut and to the Y-NK2wt receptors were identical. This is illustrated in Fig. 9B with 100 nM TRC4-NKA binding to the two receptors. For both receptors, fitting of the family of association curves allows determination of the association and dissociation rate constants k₁ = 1.0 ± 0.20 × 10⁶ M¹ s¹ and k₂ = 0.067 ± 0.006 s¹ and of the relative fluorescence intensities for ligand-free and ligand-bound receptor states, I₀ = 0.92 ± 0.07 and I₁ = 0.67 ± 0.05. The amplitude of FRET signal increases with ligand concentration up to a plateau value above 200 nM TRC4-NKA. The plot of the amplitude of FRET as a function of TRC4-NKA concentration (saturation curves) can be fitted with the empirical Hill equation to yield a dissociation constant of K_D = 20 ± 3 nM with a Hill coefficient of 1.45 ± 0.45 (Fig. 9C).

View larger version (29K): [in this window] [in a new window]   Fig. 9.   Real time recordings of TRC4-NKA associations with either Y-NK2wt or Y-NK2mut receptors and experimentally derived parameters. A, time course of TRC4-NKA interaction with the Y-NK2mut receptor (under the same conditions described in the legend to Fig. 5). The interaction of saturating concentrations (1 µM) of TRC4-NKA results in a 25-30% reduction of total sample fluorescence. The gray line is the best fit according to a bimolecular interaction, taking into account the exponential drift observed in control recordings in the absence of ligand. B, kinetics of TRC4-NKA interaction with the Y-NK2wt as compared with the Y-NK2mut receptor. Time course of 100 nM TRC4-NKA interaction to the Y-NK2mut (black line) or the Y-NK2wt receptor (gray line). Data points were acquired every 5 ms. C, saturation of receptor sites by TRC4-NKA. Plots of the FRET amplitudes determined from fitting binding traces in A versus TRC4-NKA concentrations. The black square indicates the FRET amplitude at 800 nM TRC4-NKA. D, kinetics of TRC4-NKA dissociation. After a 30-min preincubation of cells with 100 nM TRC4-NKA, dissociation was initiated by rapid stopped-flow mixing of cells expressing either the Y-NK2wt receptor (solid black line) or the Y-NK2mut receptor (dotted black line) with buffer containing 20 µM SR48968. The gray lines are the best fit obtained with two exponentials.

TRC4-NKA Dissociation-- Dissociation of receptor-ligand complexes, obtained at equilibrium with 100 nM TRC4-NKA and initiated by rapid mixing with 20 µM SR48968 (Fig. 9D), is best fitted with a sum of two exponentials (see "Experimental Procedures"). The rapid process with a rate constant of k_{off b} = 0.480 ± 0.001 s¹ represents 25% of the FRET amplitude versus 75% for the slow dissociation step (k_{off c} = 0.0420 ± 0.0001 s¹ for Y-NK2mut and k_{off c} = 0.0510 ± 0.0001 s¹ for Y-NK2wt).

Summary of the Data Used in the Kinetic Modeling of the Y-NK2wt and Y-NK2mut Receptors

The absence of agonist binding to the cAMP-responsive R2 state in the mutant receptor might be explained by two distinct phenotypes: a "binding defect phenotype" or an "isomerization defect phenotype" as described in Ref. 32 and Fig. 3. The binding defect phenotype results from mutations that selectively alter the intrinsic binding affinities (K) of individual conformational states for ligands. The isomerization defect phenotype results from mutations that selectively alter the equilibrium constant () between two given interconvertible conformations of the receptor. In order to discriminate between the two plausible effects of the mutations, we have listed all the binding parameters determined experimentally (Table I) and used a kinetic model of the NK2 receptor preexisting in equilibrium between three states, the inactive R0 state, the calcium-triggering R1 state, and the cAMP-producing R2 state to simulate the binding traces of the two agonists to each receptor. The physiological response constraints that we took into account in the modeling were the following: (i) TRC4-NKA binding to either of the Y-NK2 receptors triggers no cAMP response; (ii) the Y-NK2mut receptor does not signal via cAMP synthesis; and (iii) TRC4-NKA binding to the Y-NK2mut receptor is similar to its binding to the Y-NK2wt receptor but gives lower calcium signaling.

Kinetic Modeling of Agonist Binding to the Y-NK2wt Receptor and Responses

With the aim of determining the parameters of ligand binding and receptor state isomerization corresponding to the three-state model described in the legend to Fig. 3, the experimental kinetic parameters (Table I) were tentatively assigned to resting (R0), calcium-coupled (R1), or cAMP-coupled (R2) states using the STOIC program (30). This program generates binding traces and determines the fractional concentrations of each receptor state as a function of time. We first modeled the parameters of the Y-NK2wt receptor, which simultaneously fit the experimental interaction curves of TR1-NKA and TRC4-NKA (Fig. 10, A and B, respectively) and the proportions of inactive and active states as a function of time (Fig. 10, C and D). The set of parameters given in Table II and Fig. 10E corresponds to the combination yielding the best correlation between agonist binding and biological activity. The intrinsic isomerization constant between R0 and R1 is 0 = [R0]/[R1] = 6.7 and between R1 and R2 is '0 = [R1]/[R2] = 0.63. The intrinsic affinities K0 = (k₁/k_{off R0}), K1 = (k₁/k_{off R1}), and K2 = (k₁/k_{off R2}) of TR1-NKA for each Y-NK2wt receptor state are 376, 16, and 3 nM, respectively (Table II). These isomerization constants and intrinsic affinity values are similar to those determined previously for EGFP-NK2wt (19). TRC4-NKA binding is best simulated when it stabilizes at equilibrium mainly the inactive R0 state and, to a lesser extent, the calcium-triggering R1 state (intrinsic affinity K0 = 54 nM, K1 = 36 nM, and K2 = 450 nM (Fig. 10, E, italic values, and D, red and green lines, respectively). This is in agreement with the partial agonism observed experimentally at 21 °C for the calcium response of TRC4-NKA as compared with TR1-NKA (Fig. 7D versus Fig. 2E).

View larger version (44K): [in this window] [in a new window]   Fig. 10.   Three-state model simulating the experimental binding kinetics of TR1-NKA and TRC4-NKA to the Y-NK2wt receptor. Simulations were carried out using experimentally defined data given in Table I. A and B, simulated kinetics of ligand association as compared with experimental data. Derived kinetic parameters were estimated by manual fitting of TR1-NKA (A) and TRC4-NKA (B) interaction traces in black with the simulation in blue. C and D, simulated kinetics of the proportions of each receptor states. The parameters best fitting the binding data (Table II) were further tested for the corresponding simulated proportions of receptor states (with and without 300 nM ligand (solid and dotted lines, respectively)) as a function of time (red, R0; green, R1; blue, R2) for TR1-NKA (C) and for TRC4-NKA (D). E, summary of the parameters describing TR1-NKA (underlined) and TRC4-NKA (italic type) binding to the Y-NK2wt receptor states. 1, the isomerization constants of the ligand-bound states. 0 values are identical to the previously published ones of the EGFP-NK2 construct (the intrinsic rate for the R0-R1 transition is 4.5 s1, the rate for the R1-R0 transition is 30 s1, the rate for the R1-R2 transition is 0.042 s1, and the rate for the R2-R1 transition is 0.025 s1).