The Agonism and Synergistic Potentiation of Weak Partial Agonists by Triethylamine in a1-Adrenergic Receptor Activation: Evidence for a Salt Bridge as the Initiating Process

a1-adrenergic receptor (AR) activation is thought to be initiated by disruption of a constraining interhelical salt bridge (Porter et al., 1996). Disruption of this salt bridge is achieved through a competition for the aspartic acid residue in transmembrane domain three by the protonated amine of the endogenous ligand norepinephrine and a lysine residue in transmembrane domain seven. To further test this hypothesis, we investigated the possibility that a simple amine could mimic an important functional group of the endogenous ligand and break this a1-AR ionic constraint leading to agonism. Triethylamine (TEA) was able to generate concentration-dependent increases of soluble inositol phosphates in COS-1 cells transiently transfected with the hamster a1b-AR and in Rat-1 fibroblasts stably transfected with the human a1a-AR subtype. TEA was also able to synergistically potentiate the second messenger production by weak partial a1-AR agonists and this effect was fully inhibited by the a1-AR antagonist prazosin. However, this synergistic potentiation was not observed for full a1-AR agonists. Instead, TEA caused a parallel rightward shift of the doseresponse curve, consistent with the properties of competitive antagonism. TEA specifically bound to a single population of a1-ARs with a Ki of 28.7 6 4.7 mM. In addition, the site of binding by TEA to the a1-AR is at the conserved aspartic acid residue in transmembrane domain three, which is part of the constraining salt bridge. These results indicate a direct interaction of TEA in the receptor agonist binding pocket that leads to a disruption of the constraining salt bridge, thereby initiating a1-AR activation. a1-ARs are part of a larger family of ARs comprised of three b-, three a2-, and three a1-AR subtypes (a1a-, a1b-, a1d-) (Bylund, 1992). ARs are members of a superfamily of G protein-coupled receptors, all of which share the common structural motif of a single polypeptide chain that transverses the cell membrane using seven a-helical domains. The seven TMDs of the a1-AR form a hydrophilic ligand-binding pocket in which the endogenous agonists epinephrine and norepinephrine bind with the receptor. It is postulated that binding of these agonists changes the a1-AR tertiary protein structure. Spin-labeling studies of the prototypical G proteincoupled receptor rhodopsin have indicated that during the photoactivation process, rigid body movements of TMD three and six take place (Farahbakhsh et al., 1995; Altenbach et al., 1996). Likewise, changes in TMDs six and seven have been observed in bacteriorhodopsin, even though this related seven-TMD receptor does not couple to G proteins (Subramaniam et al., 1993). Site-directed mutagenesis studies of rhodopsin have elucidated an activational mechanism involving the disruption of a salt bridge constraint between Glu113 on TMD three and Lys296, which forms a Schiff’s base with retinal in TMD seven (Robinson et al., 1992). Light-induced isomerization of cis-retinal to the all-trans form breaks this salt bridge, which leads to receptor activation. However, for any G protein-coupled receptor other than rhodopsin, little is known about the agonist-dependent molecular mechanisms of receptor stimulation. Activation of a1-ARs is postulated to be conserved in the rhodopsin paradigm by disruption of a similar salt bridge between a conserved aspartic acid in TMD three and a lysine residue in TMD seven (Porter et al., 1996). We have shown previously through mutagenesis that eliminating the charge of the amino acids that form this a1b-AR salt bridge causes the receptor to become constitutively active. The mechanism of agonist activation is thought to involve a competition for the negative charge of the aspartic acid in TMD three by the This work was supported by an Established Investigator Award from the National American Heart Association (D.M.P.). It was also supported in part by National Institutes of Health Grant RO1-HL52544 (D.M.P.), an unrestricted research grant from Glaxo-Wellcome (D.M.P.), a National American Heart Association Grant-in-Aid (M.T.P.), National Institutes of Health Grant RO1-HL38120 (M.T.P.), and an American Heart Association Postdoctoral Fellowship, Northeast Ohio Affiliate (J.E.P.). ABBREVIATIONS: AR, adrenergic receptor; TMD, transmembrane domain; IP, inositol phosphates; TEA, triethylamine; [I]HEAT, (6)-b([I]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone. 0026-895X/98/040766-06$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 53:766–771 (1998). 766 at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from protonated amine of the endogenous ligand and the positively charged lysine in TMD seven. Hence, the basic amine of the catecholamine ligand disrupts the constraining salt bridge, allowing the a1b-AR to adopt an active conformation. The structure of norepinephrine can be divided into its basic organic components of a phenol ring attached to an ethylamine moiety. Ethylamine contains the positively charged nitrogen of a1-AR agonists known to interact with the conserved aspartic acid in TMD three (Porter et al., 1996). If activation of the a1-AR is initiated through disruption of the salt bridge by the protonated amine of the catecholamine, then simple basic amines may also be able to directly activate the a1-AR. To test this hypothesis, we used TEA as a mimic of norepinephrine to see if this simple amine could initiate a response mediated by a1-AR activation. We found that TEA behaves as an agonist by specifically binding and activating the a1-AR. A synergistic potentiation of weak partial receptor agonists by TEA is consistent with the salt bridge being an initial component for receptor activation. These functional responses of TEA are the result of a direct and competitive effect at the site of the receptor salt bridge that causes a1-AR activation. Experimental Procedures Site-directed mutagenesis. Site-directed mutagenesis was performed on a M13mp19 hamster a1b-AR construct using the oligonucleotide-mediated double primer method (Sambrook et al., 1989) as described previously (Porter et al., 1996). DNA was purified and sequenced by the dideoxy method to verify the mutation. The mutated a1b-AR insert was removed from the phage M13mp19 vector, then subcloned into the eukaryotic expression vector, pMT29. The full length plasmid DNA was again sequenced to verify the mutation. Cell culture and transfection. COS-1 cells (American Type Culture Collection, Rockville, MD) were grown in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum and the transient transfection was performed as previously described using the diethylaminoethyl-dextran method (Porter et al., 1996). Rat-1 fibroblasts stably transfected with the human a1a-AR were grown in Dulbecco’s modified Eagle’s medium plus 500 mg/ml Geneticin (Mediatech, Herndon, VA) supplemented with 5% fetal bovine