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 wildtype 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.GPCR 1 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 (1) and that can lead to disease (2-4).GPCR have evolved as the most common targets for therapeutic drugs (5). 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 (6 -8). 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 (9). 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 t...

In order to identify agonist-and antagonist-binding epitopes in the human B1 and B2 bradykinin (BK) receptors, we exploited the ability of these receptors to discriminate between peptide ligands that differ only by the absence (B1) and presence (B2) of a C-terminal Arg. This was done by constructing chimeric proteins in which specific domains were exchanged between these receptors as recently described by us (Leeb, T., Mathis, S. A., and LeebLundberg, L. M. Seven-transmembrane domain GPCR 1 constitute by far the largest family of plasma membrane receptors. These receptors bind ligands of widely diverse origins, and are unsurpassed as therapeutic targets. Consequently, much effort has been devoted to mapping of the binding sites for agonist and antagonist ligands in these receptors (1, 2). Even though peptides are the most common class of ligands for GPCR, few peptide GPCR have been investigated thus far, and in most of those cases the identity of the peptide-binding epitopes remains elusive.Receptors for kinins, pro-inflammatory peptides 8 -10 amino acids in length, have been classified into two subtypes, termed B1 and B2 (3), and are members of the GPCR superfamily (4, 5). These receptor subtypes, although only 36% identical, discriminate between peptide agonists that differ only in their C-terminal residue; BK binds to the B2 receptor, whereas the C-terminally truncated carboxypeptidase fragments desArg 9 -BK and des-Arg 10 -Lys-BK, or des-Arg 10 -KD, bind to the B1 receptor. Several high affinity B2 receptor-selective decapeptide antagonists structurally derived from BK have been developed, including NPC17731 and HOE140 (6 -8). Interestingly, the fact that the des-Arg 10 analogs of these peptides act as high affinity B1 receptor-selective antagonists emphasizes the significance of the C-terminal Arg in receptor subtype selectivity (9 -11).In the B2 receptor, extensive analysis of most of the TMs and a significant amount of the ECs by alanine-scanning mutagenesis has yielded no information about residues important for antagonist binding and has identified only a few residues important for agonist binding (12-15). We recently developed a novel, potentially more effective strategy for mapping the binding sites in kinin receptors, which is based on the identification of receptor epitopes that enable these receptors to discriminate between ligands (11). This strategy involves the exchange of individual TMs between the B1 and B2 receptor subtypes and the subsequent exchange of non-conserved residues that are possible candidates for discriminatory action. This approach is intrinsically more reliable than alanine-scanning mutagenesis as it yields in sequence both loss-of-function and gain-of-function mutations.In an initial study, we used this strategy to identify specific residues in TM-VI of the human WT B1 and B2 receptors that are partially responsible for enabling these receptor subtypes to discriminate between peptide agonists (11). In the present study, we analyzed the role of TM-III in peptide ligand discrimi...

In order to investigate the molecular basis for the ability of the human B1 and B2 bradykinin (BK) receptor subtypes to discriminate between subtype-selective ligands, we constructed chimeric proteins in which the sixth transmembrane domains (TM-VI) of these receptors were exchanged. The pharmacological profiles of the constructs were analyzed by radioligand binding in particulate preparations of transiently transfected HEK293 cells using the agonist [3H]des-Arg10-kallidin and the antagonist [3H]NPC17731. The ability of these constructs to transmit an intracellular signal was measured in transiently transfected A10 cells, a vascular smooth muscle cell line, by single cell Ca2+ imaging. Substitution of B1 TM-VI into the B2 receptor (B2(B1VI)) dramatically reduced the affinity of the B2-selective agonist BK, whereas the affinity of the B2-selective antagonist NPC17731 was unaltered. High affinity BK binding was fully regained when two residues, Tyr259 and Ala263, near the extracellular surface of TM-VI in B2(B1VI), were replaced with the corresponding residues in the wild-type B2 receptor, which are Phe259 and Thr263. The construct B1(B2VI), produced by substitution of B2 TM-VI into the B1 receptor, did not support high affinity binding of the B1-selective agonist des-Arg10-kallidin. In contrast to BK and des-Arg10-kallidin, the binding of the less subtype-selective agonist kallidin showed little sensitivity to TM-VI exchange. These results show that TM-VI in the human B1 and B2 BK receptor subtypes, although only 36% identical, are structurally compatible. Furthermore, this domain contributes significantly to the ability of these receptors to discriminate between the subtype-selective agonists BK and des-Arg10-kallidin.