Mutagenesis within Helix 6 of the Human 1-Adrenergic Receptor Identifies Lysine as a Residue Involved in Imparting the High-Affinity Binding State of Agonists

Competition binding isotherms for agonists to G protein-coupled receptors (GPCR) display high and low binding affinities. Mutagenesis of lysine at position 324 in helix 6 of the wild-type (WT) human 1-adrenergic receptor ( 1-AR) generated mutant receptors that had GTP-insensitive single low-affinity binding sites for agonists and reduced potencies of full or partial agonists in stimulating adenylyl cyclase. Unlike the WT 1-AR, intrinsic activities of full and partial agonists in activating the Lys3Ala 1-AR (K324A) mutant were correlated with their binding affinities to the K324A mutant. In assays, such as agonist-mediated phosphorylation and recycling, the K324A mutant and the WT 1-AR behaved similarly. However, in fluorescence resonance energy transfer assays that determined the proximity between the WT 1-AR or the K324A mutant to Gs , there were significant differences. The conceptual framework of the ternary complex model could not adequately account for the behavior of the K324A mutant except under assumptions of low receptor-G protein binding affinities. The single low-affinity binding site of the K324A mutant to isoproterenol was converted by the C-terminal 11-amino-acid peptide of Gs , which acts a GDP-bound Gs mimic, to highand low-affinity sites. Based upon the three-dimensional architecture of the human 1-AR, the distance between Lys 324 and the Asp/Glu-Arg-Tyr motif in helix 3 was the shortest among the various amino acids in helix 6. These findings indicate that Lys lies in a groove between helices 3 and 6, and its mutagenesis generates a mutant receptor with very low binding affinity for the GDPbound isoform of Gs. GPCRs are key bridging molecules between extracellular stimuli such as hormones and neurotransmitters and intracellular signaling cascades. Receptors are multidomain molecules with separate entities for ligand binding, G protein activation, desensitization, and sequestration (Gether et al., 2002). Ligand-activated receptors activate their respective G proteins by promoting the exchange of GTP for GPD bound to the subunit of the heterotrimer (Gether and Kobilka, 1998). GTP for GPD exchange causes the liberation and dissociation of -GTP from complexes that in turn activate the effector enzyme (Bourne, 1997). The observation that agonists, but not antagonists, stabilize an active conformation of the receptor is based on classic theories of receptor activation. This active receptor conformation consists of the agonist, the receptor, and the G protein in a ternary complex that promotes the biological response (De Lean et al., 1980). The proportion of receptors with this active conformation is determined by means of competition radioligand binding assays in which a constant concentration of a radiolabeled antagonist competes with increasing concentrations of the full agonist. These two-site binding isotherms are then analyzed by nonlinear regression to estimate the population of receptors that display high affinity for the agonist versus those with low affinity. The percentage of receptors that display high-affinity equates the active form of the receptor with a ternary complex involving the agonist, the receptor, and the G protein (Stiles et al., 1984). The percentage of receptors with low-affinity equates This work was supported by grants from the Southeast Affiliate of the American Heart Association (to S.W.B.) and the American Lebanese Syrian Associated Charities (to S.W.W.). The confocal microscopy facility is supported by National Institutes of Health grant S10-RR13725. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.025346. ABBREVIATIONS: GPCR, G protein-coupled receptor; 1-AR, 1-adrenergic receptor; HEK, human embryonic kidney; DMEM, Dulbecco’s modified Eagle’s medium; WT, wild type; ICYP, iodocyanopindolol; FITC, fluorescein isothiocyanate; FRET, fluorescence resonance energy transfer; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; GppNHp, guanosine 5 -[ -imido]triphosphate; TCM, ternary complex model. 0026-895X/06/7003-838–850$20.00 MOLECULAR PHARMACOLOGY Vol. 70, No. 3 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 25346/3133109 Mol Pharmacol 70:838–850, 2006 Printed in U.S.A. 838 at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from those receptors that are uncoupled from the G protein and consequently unable to promote the biological response. The role of helix 6 has gained prominence as a pivotal helix in transducing agonist binding to the GPCR into activation of the receptor associated G protein heterotrimer (Farrens et al., 1996). This mechanism proposes that agonist-mediated activation of GPCR leads to breaking and establishing of interactions between the Asp/Glu-Arg-Tyr [i.e., (D/E)RY motif] in helix 3 and helix 6 that applies to rhodopsin, the 2-AR,and other GPCRs (Farrens et al., 1996; Gether et al., 1997; Ballestero et al., 2001). By means of histidine substitutions in helices 3 and 6 of the human 2-AR and the parathyroid receptor, Sheikh et al. (1999) determined the residues in helix 6 that were capable of forming zinc(II) bridges as those involved in agonist-mediated activation of Gs. However, the zinc(II) bridging method failed to identify residues in helix 6 that were involved in regulating the agonist binding affinity to the 2-AR. After the publication of this article, the crystal structure of dark-adapted rhodopsin revealed that helices 3 and 6 were extended -helices that project as an -helix beyond the sequence that is buried in the lipid core of the membrane (Palczewski et al., 2000). The three-dimensional structure of the human 1-AR was modeled based upon the crystal structure of rhodopsin to estimate the distances between the various amino acids in helix 6 relative to the (D/E)RY region in helix 3. Based upon these measurements and additional site-directed mutagenesis in helix 6, we identified a residue in helix 6 that is involved in imparting the high-affinity binding characteristics of the human 1-AR to agonists. Materials and Methods Site-Directed Mutagenesis. The Flag-tagged human 1-AR flanked with HindIII (5 ) and EcoRI (3 ) sites was cloned into the multiple cloning site in the pUC18 plasmid (Delos Santos et al., 2006). Point mutations in helix 6 were generated by site-directed mutagenesis, using the Transformer system (BD Biosciences, San Jose, CA). The sequences of mutated receptors were verified by dideoxy sequencing, followed by cloning of each mutated full-length 1-AR cDNA into the mammalian expression vector pCDNA-3.1 (Invitrogen, Carlsbad CA). Cell Culture and Transient Transfections. HEK-293 cells were cultured in DMEM supplemented with 10% fetal bovine serum until they were 90% confluent. The WT 1-AR or its point-mutants in pCDNA 3.1 were transiently transfected into HEK-293 cells using the Cytofectene reagent (Bio-Rad Laboratories, Hercules, CA) as follows. Plasmid DNA (5 g) was diluted into 200 l of DMEM and then mixed with an equal volume of DMEM containing 12 l of Cytofectene at room temperature for 30 min. Then 4 ml of DMEM was added, and the DNA-lipid complex was layered over the cells for 5 h at 37°C. Then, an equal volume of DMEM 10% FBS was added to each culture dish, and the cells were cultured for an additional