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Delineation of a Region in the B2 Bradykinin Receptor That Is Essential

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Delineation of a Region in the B2 Bradykinin Receptor That Is Essential Proc. Nati. Acad. Sci. USA Vol. 91, pp. 4417-4421, May 1994 Biochemistry Delineation of a region in the B2 bradykinin receptor that is essential for high-affinity agonist binding (site-directed mutagenesis/binding site/trnsembrane hellx/neuropeptide receptor/G-protein-coupled receptor) JULIE NARDONE* AND PATRICK G. HOGANt Department of Neurobiology, Harvard Medical School, Boston, MA 02115 Communicated by Edwin J. Furshpan, January 7, 1994 ABSTRACT We have made mutations in the predicted METHODS sixth transmembrane segment of a rat B2 bradykinin recep- Molecular Biology. Isolation of a B2 bradykinin receptor tor and analyzed the variant proteins by expressing them in cDNA from PC12 cells and its subcloning into the expression COS-1 cells. Two amino acid substitutions reduced the vector pLGP3 were described in the preceding paper (15). affinity of the receptor for bradykinin (Phe261 -* Val by Single-stranded plasmid DNA containing uracil (17) was 1600-fold; Thr2" --> Ala by 700-fold) with comparatively prepared by published techniques (18). Site-directed muta- little effect on the affinity for the bradykinin antagonists genesis was performed as described by Kunkel et al. (17). In NPC17731 and D-Arg-[Hyp3,D-Phe7]bradykinin (where Hyp most cases the mutagenesis primer also introduced a trans- is hydroxyproline). Three other substitutions (Gln262 -. Ala, lationally silent mutation that either created or destroyed a Asp2" -- Ala, and Thr269 - Ala) modestly reduced the restriction enzyme recognition site, to facilitate initial iden- affinity for bradykinin and for the antagonist D-Arg-[Hyp3,D- tification of plasmids carrying the desired mutation. The Phe7]bradykinin. Even the most dramatically affected mu- presence of the desired mutation was confirmed by DNA tated receptors were still able to couple, after bradykinin sequencing by the dideoxy chain-termination method (19). binding, to phosphatidylinositol turnover. The data suggest The DNA sequence of the entire coding region was deter- that bradykinin directly contacts the face of the sixth trans- mined for those cDNAs encoding proteins that showed membrane helix formed by the residues Phe261, Gln262, altered binding ofbradykinin. Plasmid DNA was prepared by Thr2'5, Asp2", and Thr269 or that this face of the helix is the standard techniques (20) and was introduced into COS-1 cells site ofintraprotein contacts that serve to stabilize the agonist- by electroporation (21). binding conformation of the receptor. Functional Assays. Equilibrium binding of [3H]bradykinin or [3H]NPC17731 (DuPont/NEN) to COS-1 cells expressing Bradykinin, a by-product of the blood clotting cascade or of mutated receptors was determined as described (15). Kd and plasma leakage into the extravascular space, is a potent B.. were estimated from these measurements by Scatchard mediator of pain and is also involved in inflammation and analysis (22). The ability of unlabeled bradykinin or of other pathological processes (1-3). There is currently inten- unlabeled D-Phe7-substituted kinins to compete for the bind- sive interest in the possible clinical uses of bradykinin ing sites was examined in equilibrium assays (15), and data antagonists (3-7). Bradykinin agonists may also be useful were fitted to a single hyperbola by using a nonlinear least- therapeutically, since there is evidence that kinins exert a squares program, yielding an estimate of IC50. Ki was calcu- cardioprotective effect in myocardial ischemia (8-10). lated by using the standard correction factor (1 + [L]/KL)-1, A first step in the rational design ofbradykinin agonists and where [L] is the concentration of labeled ligand in the antagonists is the identification of residues in the bradykinin experiment, and KL is the Kd for binding ofthe labeled ligand receptor that participate directly in ligand binding. This by the receptor measured in saturation binding experiments. information can then be used to form a more refined picture Levels of inositol phosphates in control cells and in cells of the geometry of the ligand binding site by examining the stimulated for 20 sec with bradykinin were measured accord- interaction with conformationally constrained ligands having ing to Berridge et al. (23, 24) in cultures that had been different substituents, as illustrated by recent studies of incubated for 48 hr in medium containing [3H]inositol. ligand-receptor interactions in the neurokinin 1 receptor (11). A B2 bradykinin receptor that may be responsible for many RESULTS ofthe pharmacological actions ofbradykinin has been cloned Initial Screening of Mutants in the Sixth Transmembrane from human and rat tissues (12-15). It is a member of the Segment. Mutated bradykinin receptors were expressed in family of G-protein-coupled receptors, and there is a high COS-1 cells, and equilibrium high-affinity binding of degree of conservation of the amino acid sequence between [3H]bradykinin was examined with [3H]bradykinin concen- rat and human. trations in the low nanomolar range. Two mutated receptors Here we present an analysis of the effect on ligand binding with the substitutions Thr265 -* Ala (T265A) (Fig. 1) and of amino acid substitutions in the predicted sixth transmem- F261V showed little binding in this range of [3H]bradykinin brane segment (16) of the B2 bradykinin receptor. We show concentrations. Three other mutated receptors-Q262A, that a narrowly delineated region in this transmembrane D268A, and T269A-had an affinity for [3H]bradykinin that segment is essential for formation of a high-affinity binding was decreased by a factor of 5-20 relative to the affinity of site for bradykinin and that specific amino acids in this region the wild-type receptor (Table 1). The remaining mutated interact strongly with bradykinin, but not with two bradyki- receptors-F259A, 1263A, S264A (Fig. 1), F266A, and nin antagonists. L267A-did not differ from the wild-type receptor in affinity The publication costs of this article were defrayed in part by page charge *Present address: Department of Molecular Biology, Massachusetts payment. This article must therefore be hereby marked "advertisement" General Hospital, Boston, MA 02114. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 4417 Downloaded by guest on September 29, 2021 4418 Biochemistry: Nardone and Hogan Proc. NatL Acad Sci. USA 91 (1994) 8- in this assay, since they failed to bind sufficient [3H]brady- 0 co kinin. 0 T265A. While the substitution T265A nearly eliminated x binding of [3H]bradykinin at nanomolar concentrations, it did not affect high-affinity binding of the antagonist E S264A 0.0- [3H]NPC17731 (Fig. 2 and Table 1). The preservation of .0 high affinity for [3H]NPC17731 constitutes direct evidence c 6- 0 0 0 T265A of correct folding and expression of the altered receptor. 0:3 CD The number of [3H]NPC17731 binding sites present at the 0 000. 0 cell surface (average of 3.3 x 105 sites per cell) was 0 4 8 12 16 comparable to the number of receptor sites in COS-1 cells [3H]Bradykinin, nM expressing the wild-type receptor. Competition between unlabeled bradykinin and [3H]NPC17731 for the binding site FIG. 1. Specific binding of [3H]bradykinin to COS-1 cells ex- pressing mutated bradykinin receptors. *, S264A; o, T265A. Kd for demonstrated that bradykinin does bind to the mutated binding to the S264A receptor was 9.1 nM in this experiment. There receptor, with a Ki, 1.3 ,uM, that represents a 700-fold shift was negligible specific binding to the T265A receptor. Nonspecific in affinity relative to the wild-type receptor (Fig. 3 and binding in the presence of 20 pM unlabeled bradykinin, which was Table 1). Similar competition experiments indicated a mod- 110 cpm for S264A and 75 cpm forT265A at the highest concentration est reduction in affinity for D-Arg-[Hyp3,D-Phe7]brady- of [3H]bradykinin, has been subtracted. kinin, with an estimated Ki of 140 nM (Fig. 3 and Table 1). This decrease in affinity, which could be related to the fact for bradykinin (Table 1). In all cases where the mutated that D-Phe7-substituted bradykinin analogues have partial receptors exhibited high-affinity binding, the proteins were agonist activity at B2 receptors, is small in comparison to efficiently expressed. Levels of receptor expression, mea- the effect on [3H]bradykinin binding. sured as the number of sites per cell transformed with DNA, F261V. Like the T265A mutation, the substitution F261V a on were 2.9 x 105 to 2.3 x 106 sites per cell for wild-type receptor had only small effect the binding of [3H]NPC17731 (Kd 14 nM) (Table 1), and did not interfere with expression ofhigh (15) and 7.8 x 104 to 1.3 x 106 sites per cell for mutated levels of receptor (average 3.9 x 105 sites per cell) at the cell receptors. surface. The K, for unlabeled bradykinin binding, determined To probe additional features of the ligand binding site, we examined the ability of the B2 antagonist D-Arg-[Hyp3,D- by competition with [3H]NPC17731, was dramatically shifted to 2.9 a in to for the pM, 1600-fold reduction affinity compared Phe7]bradykinin to compete with [3H]bradykinin wild-type receptor (Fig. 3 and Table 1). The for D-Arg- Two of the Ki binding site of the mutated receptors (Table 1). [Hyp3,D-Phe7]bradykinin was 24 nM (Fig. 3 and Table 1). mutations, Q262A and D268A, that modestly reduced affinity Coupilng to Phosphatidyilnositol Turnover. The mutated for [3H]bradykinin also resulted in a 5- to 20-fold shift in receptors with severely reduced affinity for bradykinin, affinity for the B2 antagonist. Another mutation, T269A, that F261V and T265A, were expressed in COS-1 cells and tested modestly reduced the affinity for bradykinin had a lesser for their ability to couple bradykinin binding to phosphati- effect on the affinity for D-Arg-[Hyp3,D-Phe7]bradykinin, and dylinositol turnover.
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