Spontaneous Human B2 Bradykinin Receptor Activity Determines the Action of Partial Agonists as Agonists or Inverse Agonists

Abstract

In this report, we show that desensitization regulates ligand-independent, spontaneous activity of the human B2 bradykinin (BK) receptor, and the level of spontaneous receptor activity determines the action of the BK antagonists and partial receptor agonists NPC17731 and HOE140 as agonists or inverse agonists. Spontaneous receptor activity was monitored by measuring basal cellular phosphoinositide (PI) hydrolysis as a function of the density of the receptor in transiently transfected HEK293 cells. Minimal spontaneous activity of the wild-type B2 receptor was detected in these cells. Mutating a cluster of serines and threonines within the fourth intracellular domain of the receptor, which is critical for agonist-promoted desensitization, significantly increased the spontaneous receptor activity. BK, the natural B2 receptor ligand and, consequently, a full agonist, stimulated PI hydrolysis at high and low levels of spontaneous receptor activity. On the other hand, the partial agonists NPC17731 and HOE140 were stimulatory, or agonists, at the lower level of receptor activity but inhibitory, or inverse agonists, at the higher level of activity. These results show that receptors are desensitized in response to their spontaneous activity. Furthermore, these results, which refute traditional theories, show that the capacity of a drug to modulate a receptor response is not intrinsic to the drug but is also dependent on the cellular environment in which the drug acts. In this report, we show that desensitization regulates ligand-independent, spontaneous activity of the human B2 bradykinin (BK) receptor, and the level of spontaneous receptor activity determines the action of the BK antagonists and partial receptor agonists NPC17731 and HOE140 as agonists or inverse agonists. Spontaneous receptor activity was monitored by measuring basal cellular phosphoinositide (PI) hydrolysis as a function of the density of the receptor in transiently transfected HEK293 cells. Minimal spontaneous activity of the wild-type B2 receptor was detected in these cells. Mutating a cluster of serines and threonines within the fourth intracellular domain of the receptor, which is critical for agonist-promoted desensitization, significantly increased the spontaneous receptor activity. BK, the natural B2 receptor ligand and, consequently, a full agonist, stimulated PI hydrolysis at high and low levels of spontaneous receptor activity. On the other hand, the partial agonists NPC17731 and HOE140 were stimulatory, or agonists, at the lower level of receptor activity but inhibitory, or inverse agonists, at the higher level of activity. These results show that receptors are desensitized in response to their spontaneous activity. Furthermore, these results, which refute traditional theories, show that the capacity of a drug to modulate a receptor response is not intrinsic to the drug but is also dependent on the cellular environment in which the drug acts. G protein-coupled receptor bradykinin GPCR kinase intracellular domain phosphoinositide wild-type GPCR1 constitute the largest family of receptors and mediate responses to numerous agonists including hormones, neurotransmitters, and sensory stimuli. Receptor activation by the agonist ligand is considered the first step in receptor signal transduction. However, the recent discovery that GPCR exhibit ligand-independent, spontaneous, and mutation-induced activity has introduced a new function that needs to be considered in cellular signaling and ligand action (1Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (757) Google Scholar) and that can lead to disease (2Coughlin S.R. Curr. Opin. Cell Biol. 1994; 6: 191-197Crossref PubMed Scopus (121) Google Scholar, 3Milano C.A. Allen L.F. Rockman H.A. Dolber P.C. McMinn T.R. Chien K.R. Johnson T.D. Bond R.A. Lefkowitz R.J. Science. 1994; 264: 582-586Crossref PubMed Scopus (664) Google Scholar, 4Arvanitakis L. Geras-Raaka E. Varma A. Gershengorn M.C. Cesarman E. Nature. 1997; 385: 347-350Crossref PubMed Scopus (577) Google Scholar). GPCR have evolved as the most common targets for therapeutic drugs (5Stadel J.M. Wilson S. Bergsma D.J. Trends Pharmacol. Sci. 1997; 18: 430-437Abstract Full Text PDF PubMed Scopus (141) Google Scholar). Drug efficacy is defined as the capacity of a drug to activate or inactivate a receptor response and is fundamental to pharmacology and drug development. Classically, this parameter has been considered an intrinsic property of the drug and independent of the cellular environment in which the drug acts (6Ariëns E.J. Arch. Int. Pharmacodyn. Ther. 1954; 99: 32-49PubMed Google Scholar, 7Stephenson R.P. Br. J. Pharmacol. 1956; 11: 379-393Crossref PubMed Scopus (965) Google Scholar, 8Furchgott R.F. Harper N.J. Simmonds A.B. Advances in Drug Research. Academic Press, New York1996: 21-55Google Scholar). Partial agonists, or mixed agonists/antagonists, are prevalent among GPCR drugs and are defined as ligands that elicit a submaximal receptor response and block the response to the natural receptor ligand, a full agonist, or any other ligand of higher efficacy. It has been predicted that in the absence of a more efficacious agonist, the efficacy and behavior of a partial agonist should be directly dependent upon the level of spontaneous receptor activity (9Kenakin T. Pharmacol. Rev. 1996; 48: 413-463PubMed Google Scholar). In other words, if the partial agonist ligand elicits a response that is higher than the spontaneous activity, then the ligand should behave as an agonist, whereas if the ligand elicits a lower response, then the ligand should block the spontaneous activity and, consequently, behave as an inverse agonist. Desensitization is classically defined as the fading of a receptor response to persistent agonist stimuli and manifests itself in numerous biological processes. Agonist-promoted desensitization of GPCR is common (10Hausdorf W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1088) Google Scholar), and the mechanism for this process has been described in considerable detail (11Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar, 12Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (858) Google Scholar). Desensitization in response to spontaneous receptor activity, or basal desensitization, should also occur but has not been investigated to any significant extent. However, this process represents one cellular mechanism which may regulate spontaneous receptor activity and, consequently, the action of partial receptor agonists. In this study, we used the human B2 bradykinin (BK) receptor expressed in HEK293 cells to show that receptors are subject to desensitization in response to their spontaneous activity. Furthermore, the level of basal desensitization and, consequently, spontaneous receptor activity determines the efficacy and behavior of two potent and selective BK antagonists and partial B2 receptor agonists as either agonists or inverse agonists at the receptor. [prolyl-3,4-3H]NPC17731 (53.5 Ci/mmol) and myo- [3H]inositol (10–20 Ci/mmol) were obtained from NEN Life Science Products. All other chemicals were obtained as previously described (13Fathy D.B. Mathis S.A. Leeb T. Leeb-Lundberg L.M.F. J. Biol. Chem. 1998; 273: 12210-12218Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Mutations and transfections of the human B2 receptor were done as described previously (13Fathy D.B. Mathis S.A. Leeb T. Leeb-Lundberg L.M.F. J. Biol. Chem. 1998; 273: 12210-12218Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Mutations were made using a polymerase chain reaction-ligation-polymerase chain reaction protocol, and HEK293 and A10 cells were transfected with varying amounts of receptor DNA (0.01–0.2 μg/106 cells) using the calcium phosphate precipitate method and LipofectAMINE, respectively. Expression of receptor constructs was assayed by radioligand binding on intact cells essentially as described previously (14Tropea M.M. Munoz C.M. Leeb-Lundberg L.M.F. Can. J. Physiol. Pharmacol. 1992; 70: 1360-1371Crossref PubMed Scopus (13) Google Scholar). In short, cells were incubated in Leibovitz'sl-15 medium, pH 7.4, 0.1% bovine serum albumin, including the protease inhibitors bacitracin (140 μg/ml) and 1,10-phenanthroline (1 mm) and a saturating concentration of [3H]NPC17731 (3–5 nm) at 4 °C for 60–90 min. The assays were terminated by dilution in ice-cold phosphate-buffered saline, 0.3% bovine serum albumin and rapid vacuum filtration on Whatman GF/C filters previously soaked in 1% polyethyleneimine. Activities of receptor constructs were assayed by monitoring PI hydrolysis and mobilization of intracellular Ca2+ essentially as described previously (13Fathy D.B. Mathis S.A. Leeb T. Leeb-Lundberg L.M.F. J. Biol. Chem. 1998; 273: 12210-12218Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 15Mathis S.A. Criscimagna N.L. Leeb-Lundberg L.M.F. Mol. Pharmacol. 1996; 50: 128-139PubMed Google Scholar). PI hydrolysis was assayed in HEK293 cells prelabeled with 1 μCi/mlmyo-[3H]inositol in Dulbecco's modified Eagle's medium, 5% heat-inactivated horse serum. Following washing, the cells were incubated in Leibovitz's l-15 medium containing 50 mm LiCl in the absence and presence of ligands for 30 min at 37 °C. The cytosolic free Ca2+ signal from single fura-2 labeled A10 cells was acquired as the ratio of Ca2+-bound fura-2/Ca2+-free fura-2, denoted as F340/F380, and processed by an integrated digital imaging fluorescence microscopy system. The average of the net peak ratio values for wild-type (WT) and B2ASer/Thr were 2.44 and 2.15, respectively. Aligning the matching frames from individual traces allowed for the averaging of normalized responses from multiple experiments. As small variations occurred from one experiment to the next in the amount of elapsed time between ligand additions, artificial breaks were inserted in the traces so that both marker frames could be matched. To analyze ligand-independent, spontaneous activity of the human WT B2 receptor, basal cellular PI hydrolysis was assayed as a function of the density of the receptor in transiently transfected HEK293 cells (Fig. 1 B). The slope of this function, which may be considered an index of the level of spontaneous receptor activity and which we term index of basal activity, or IB, was very low (0.03), indicating that the WT receptor was virtually inactive in the absence of a ligand in these cells. In the presence of 1 μm BK, the slope, which we term index of maximal agonist-promoted activity, or IA, was 0.86, a 23-fold increase over the ligand-independent B2 receptor activity (Fig.1 C). BK activation of the WT receptor also was observed by monitoring the downstream mobilization of intracellular Ca2+ in transiently transfected A10 cells. As shown in Fig.1 D, 0.1 μm BK elicited a rapid and transient increase in the intracellular free Ca2+ concentration in these cells, which returned to nearly base-line levels by 5 min. The absence of any residual receptor-mediated Ca2+ mobilization and the lack of effect of the BK antagonist HOE140 (1 μm) after approximately 5 min of agonist exposure indicated that the WT receptor was desensitized at this time point as described previously (15Mathis S.A. Criscimagna N.L. Leeb-Lundberg L.M.F. Mol. Pharmacol. 1996; 50: 128-139PubMed Google Scholar). To determine whether the low level of ligand-independent, spontaneous activity of the WT B2 receptor was due to desensitization in response to spontaneous receptor activity, or basal desensitization, we mutated receptor residues critical for agonist-promoted desensitization. This mechanism involves the GRK-mediated phosphorylation of specific clusters of serines and threonines in intracellular domain three and/or four (IC-IV) of the receptor (11Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar, 12Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (858) Google Scholar). As shown in Fig. 1 A, the human B2 receptor IC-IV contains several serines and threonines, including Ser316, Ser339, Thr342, Thr345, Ser346, and Ser348, which are conserved among known B2 receptors and which may serve as GRK substrates. Furthermore, BK stimulates serine and threonine phosphorylation of the human B2 receptor (16Blaukat A. AbdAlla S. Lohse M.J. Muller-Esterl W. J. Biol. Chem. 1996; 271: 32366-32374Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). To screen for the role of these residues in the regulation of ligand-independent receptor activity, alanine mutants of each individual conserved residue were assayed for basal PI hydrolysis at approximately equal receptor densities (∼100 fmol/106 cells). As shown in Fig.2 (left panel), individual mutation of Ser339, Thr342, Thr345, and Ser348 yielded small but significant increases (1.4–1.5-fold) in basal PI hydrolysis, whereas mutation of Ser316 and Ser346 had essentially no effect. Interestingly, alanine mutation of Thr342, Thr345, Ser346, and Ser348 (Ser/Thr cluster) as a cluster to yield B2ASer/Thr resulted in an increase (5.1-fold) in basal PI hydrolysis, which was much higher than that seen with each individual mutation alone. Furthermore, expression of increasing amounts of B2ASer/Thr yielded an IB of 0.19 which was 6-fold greater than that of the WT B2 receptor which was 0.03 (Fig. 1 B). These results show that the human WT B2 receptor is phosphorylated and desensitized in response the spontaneous receptor activity and that this process regulates the spontaneous activity of the receptor. The phosphorylation apparently involves primarily Ser339, Thr342, Thr345, and Ser348 in IC-IV. Considering the effect of mutating these residues as a cluster, the full manifestation of receptor desensitization may require phosphorylation of more than one of these residues. Our identification of putative residues phosphorylated in spontaneous B2 receptor desensitization is in good agreement with a very recent study of phosphorylated residues in IC-IV of the unstimulated rat B2 receptor using mass spectrometry (17Soskic V. Nyakatura E. Roos M. Muller-Esterl W. Godovac- Zimmermann J. J. Biol. Chem. 1999; 274: 8539-8545Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The BK-stimulated B2 receptor activity was sensitive to mutation of the same conserved residues as the ligand-independent, spontaneous receptor activity (Fig. 2, right panel). Small but significant increases (1.4–1.8-fold) in BK-stimulated PI hydrolysis were observed by individually mutating Ser339, Thr342, Thr345, and Ser348, whereas a significantly larger BK stimulation (2.4-fold) was observed by mutating these residues as a cluster. Interestingly, truncation of IC-IV at residue 313 resulted in a construct that bound BK but did not elicit either a spontaneous or BK-stimulated response. In the experiment described in the figure, the alanine mutant of Ser316 exhibited an apparently elevated activity, but this activity was due to a higher level of expression of this mutant in this representative experiment. Addition of 1 μm BK to B2ASer/Thr yielded an IA of 1.5, which was approximately 2-fold greater than that of the WT B2 receptor which was 0.86 (Fig. 1 C). The equilibrium dissociation constants of BK binding to the WT B2 receptor (0.104 ± 0.025 nm; n = 2) and B2ASer/Thr (0.123 ± 0.026 nm;n = 2) were approximately the same, indicating that the elevated stimulation of B2ASer/Thr was not due to an increase in the affinity of BK for the receptor. The importance of the Ser/Thr cluster in receptor desensitization was also observed in BK stimulation of intracellular Ca2+ mobilization. As shown in Fig. 1 D, stimulation of B2ASer/Thr with BK resulted in a response which was biphasic with a second phase characterized by a plateau of elevated free Ca2+, and this response remained elevated for ≫5 min. That the second phase was due to sustained agonist-stimulated receptor activity was evident from the immediate and large decrease in the free Ca2+ level following addition of HOE140 (1 μm) after approximately 5 min of agonist stimulation. Thus, desensitization of ligand-dependent and -independent B2 receptor activity involves the same cluster of serines and threonines in IC-IV. This cluster is located between two acidic residues, Glu337 and Glu350, which are conserved among B2 receptors and which have been shown to be important for GRK recognition (18Onorato J.J. Palczewski K. Regan J.W. Caron M.G. Lefkowitz R.J. Benovic J.L. Biochemistry. 1991; 30: 5118-5125Crossref PubMed Scopus (188) Google Scholar). Based on the observation of ligand-independent, spontaneous, and mutation-induced GPCR activity, current models state that these receptors spontaneously isomerize between inactive and activated conformational states termed R and R*, respectively, and that R* associates with a G protein to form R*G which triggers the intracellular signal (1Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (757) Google Scholar, 9Kenakin T. Pharmacol. Rev. 1996; 48: 413-463PubMed Google Scholar). R* is also believed to be a substrate for GRK and, consequently, subject to desensitization (19Pei G. Samama P. Lohse M. Wang M. Codina J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2699-2702Crossref PubMed Scopus (140) Google Scholar). In this model, full agonists act by selectively stabilizing, and therefore maximally shifting, the equilibrium toward the activated R* state and, consequently, R*G, whereas inverse agonists act by stabilizing the inactive R state. This model predicts that a full agonist behaves as an agonist independently of the spontaneous receptor activity. The only exception would be if the spontaneous activity is as high as the agonist-stimulated activity in which case the agonist should have no effect. Indeed, BK dose-dependently stimulated the activity of both the WT B2 receptor and B2ASer/Thr (Fig.3A). Thus, BK acts as an agonist independently of the level of spontaneous activity and receptor desensitization. The higher maximal BK response on B2ASer/Thr was expected, since this construct is subject to less desensitization than the WT B2 receptor (Fig. 3 A). NPC17731 and HOE140 are two structurally similar, potent, and selective BK peptide antagonists (20Kyle D.J. Martin J.A. Burch R.M. Carter S. J. L. J.A. R.F. L.F. J. Chem. 1991; PubMed Scopus Google Scholar, K. U. W. G. S. G. W. J. Br. J. Pharmacol. 1991; PubMed Scopus Google Scholar). results have been the action of these Partial by HOE140 was observed on the rat WT B2 receptor transiently expressed in cells U. AbdAlla S. K. Muller-Esterl W. Biochemistry. 1996; PubMed Scopus Google Scholar), whereas inverse by HOE140 and NPC17731 was observed on the B2 receptor in of rat cells L.M.F. Mathis S.A. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). by HOE140 was also observed on the human WT B2 receptor transiently expressed in cells J. J.L. T. Mol. Pharmacol. 1999; PubMed Scopus Google Scholar). The absence and presence of partial by HOE140 was observed in B2 receptor from and M. M. J.L. E. Br. J. Pharmacol. 1994; PubMed Scopus Google Scholar). Thus, the efficacy and behavior of HOE140 and/or NPC17731 on the the B2 receptor. that NPC17731 dose-dependently stimulated the activity of the human WT B2 receptor expressed in HEK293 cells. The same effect was observed with HOE140 (Fig. 3 C). these ligands act as agonists on this receptor in this system. In NPC17731 and HOE140 dose-dependently the basal activity of B2ASer/Thr (Fig. and C). these ligands act as inverse agonists on this receptor in this system. These results the apparently action of BK antagonists in the and that the specific action of NPC17731 and HOE140 is by the level of spontaneous activity and basal desensitization of the B2 receptor. This is by the that in rat cells, in which the B2 receptor is subject to very desensitization (14Tropea M.M. Munoz C.M. Leeb-Lundberg L.M.F. Can. J. Physiol. Pharmacol. 1992; 70: 1360-1371Crossref PubMed Scopus (13) Google Scholar), HOE140 and NPC17731 behave as inverse agonists L.M.F. Mathis S.A. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). These also show that inverse is a parameter than the of of agonist. The action of NPC17731 and HOE140 either as agonists by B2 receptor activity or as inverse agonists by basal receptor activity is in with current two receptor state as in these models a ligand either the R* state or the inactive R state but not both (1Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (757) Google Scholar). previously a model of agonist binding to the B2 receptor, which an receptor state L.M.F. Mathis S.A. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). This model was on the of BK to the of three receptor binding R R* the of the equilibrium state was by as the G protein-coupled state of the receptor. Thus, in this model to R* in In the model, the state R* was to be a activated state of the receptor not to the G protein L.M.F. Mathis S.A. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). This model can results by that NPC17731 and HOE140 Thus, the activity of the spontaneously is due to a high level of desensitization, these ligands would as agonists by their to the R On the other hand, the activity of the spontaneously is high due to a low level of desensitization, these ligands would as inverse agonists by their to the Thus, the intrinsic parameter of these drugs may be their to R*, and drug efficacy and behavior on the activity of R* to that of It may be that in the model, the level of maximal stimulation of the WT B2 receptor by NPC17731 and HOE140 should equal that of their maximal of As shown in Fig. and this was not the However, this may be if R* is also a substrate for GRK and, consequently, subject to desensitization. the maximal stimulation of the WT B2 receptor should the maximal of the less desensitized In results several and critical of regulation of ligand-independent GPCR activity and the action of partial receptor agonist. these results a GPCR which upon mutation to an increase in spontaneous receptor activity. this mutation is from previously A. Cotecchia S. J. 1997; PubMed Scopus Google Scholar), in that the activity of a spontaneously of activated receptors by of desensitization than by to the receptor in an inactive state. However, an effect of this mutation on the equilibrium for the between the inactive and activated receptor states be Considering that desensitization on the state and by variations the activity of receptors and in both desensitization and this mechanism the with the to GPCR activity independently of a receptor this mechanism directly the action of receptor ligands and, the which has been in the NPC17731 and In results, which directly refute theories, show that the capacity of a drug to modulate a receptor response is not intrinsic to the drug but is also dependent on the cellular environment in which the drug acts. P. S. M. Mol. Pharmacol. 1996; 50: Google Scholar) that the of several inverse agonists at the receptor were following agonist to desensitization. However, the spontaneous receptor activity was not monitored in this study and, consequently, could not be to the inverse agonist efficacy. results show that inverse is a parameter than the of of agonist. results are with the of a activated receptor state which is by NPC17731 and HOE140 and which is higher in activity than the inactive R state and lower in activity than the activated R* state.