Introduction

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The ₂-ARs mediate diverse physiological and pharmacological effects of the neurotransmitters/hormones norepinephrine and epinephrine and related synthetic molecules. Three genes encoding human ₂-AR subtypes have been cloned, representing the pharmacologically defined subtypes _2A, _2B, and _2C (Kobilka et al., 1987; Regan et al., 1988; Lomasney et al., 1990). Related ₂-AR genes also have been identified in other species, such as rat, mouse, pig, opossum, and fish (Guyer et al., 1990; Zeng et al., 1990; Lanier et al., 1991; Chen et al., 1992; Link et al., 1992; Svensson et al., 1993; Blaxall et al., 1994). ₂-ARs, like all other members of the GPCR family, consist of a polypeptide chain that is predicted to span the cell membrane seven times. The amino acid sequences within the seven hydrophobic TMs are highly conserved in the three ₂-AR subtypes. These TM regions are predicted to be -helical and to form a pocket crucial for the identification and binding of ligand molecules. Binding of a receptor agonist in this binding cavity either leads to or stabilizes a conformational change in the receptor protein, promoting its coupling with G proteins. The resulting G protein activation initiates a cascade of intracellular biochemical events and physiological responses (Savarese and Fraser, 1992; Scheer et al., 1996).

Several ₂-AR ligands, such as oxymetazoline, chlorpromazine, prazosin, UK 14,304, and dexmedetomidine, display some degree of subtype selectivity in either their binding affinity or functional activity (Marjamäki et al., 1993; Jansson et al., 1994). A comparison of the ligand binding properties of the human ₂-AR subtypes with their species homologues also has revealed some differences. For example, H2A binds the antagonists yohimbine and rauwolscine with significantly higher affinity than its mouse homologue, M2A (Link et al., 1992). Analysis of the primary structures of these two receptors has identified a Cys201-to-Ser201 substitution in the TM5 of M2A. When Ser201 of the M2A was mutated to cysteine, the affinity of the mouse receptor for yohimbine was significantly increased. This suggested that the residue at position 201 in TM5 of _2A-ARs might be exposed in the binding cavity and directly participate in ligand recognition. Site-directed mutagenesis and computer-aided modeling can be used to explore the structural determinants of receptor/ligand interactions, including species differences and subtype selectivity. Mapping of residues exposed in the binding cavity may allow the subsequent synthesis of new therapeutic agents targeted to specific ligand recognition sites.

CEC, which often has been used to discriminate between ₁-AR subtypes in functional assays (Han et al., 1987; Tian et al., 1990), has been shown to inactivate irreversibly H2A and H2C, whereas H2B is relatively resistant to its alkylating effect (Michel et al., 1993). CEC is known to undergo intramolecular cyclization to a reactive aziridinium ion before irreversible receptor inactivation (Vargas et al., 1993). The aziridinium ion presumably forms a covalent bond with the free SH-group of an exposed cysteine residue. The primary structure of H2A has a cysteine in position 201; H2C also has a cysteine in the corresponding position (position 215), whereas the CEC-resistant subtype H2B has a serine in the corresponding position (position 177) (Fig. 1). Such an amino acid substitution might explain the subtype-selective reactivity of CEC at the different human ₂-AR subtypes. To test this hypothesis, we determined the irreversible binding of CEC to the three human ₂-AR subtypes as well as the M2A and constructed and tested a series of mutant _2A-ARs with cysteines located at different positions in this region of TM5.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Amino acid sequence alignment of the fifth hydrophobic TM of human and rodent 2-AR subtypes. The sequences are aligned for maximum homology. The alignment of the entire sequences has been presented elsewhere (Pepperl and Regan, 1994). Boxes, amino acid residue at position 201 or corresponding position.

Computer-aided modeling was used to predict the three-dimensional structure of the H2A. The TM domains of GPCRs usually are presented as fixed -helices, with one side exposed in the binding cavity (Savarese and Fraser, 1992; Baldwin, 1993; Schwartz, 1994). With site-directed mutagenesis, however, the pattern of exposure of residues in TM5 of the dopamine D₂ receptor to a hydrophilic thiol-reactive alkylating agent was shown to be inconsistent with this prediction (Javitch et al., 1995). In our model of the H2A, TM5 was predicted to be -helical, with residues Val197, Ser200, Cys201, and Ser204 forming part of the surface of the ligand-accessible binding site crevice and residues Ile198, Ser199, Ile202, and Gly203 facing the lipid bilayer of the plasma membrane. To map the structure and orientation of the TM5 in the H2A and to test this model, we mutated residues 197-201 and 204, one at a time, to a cysteine. Irreversible binding of CEC was used as a criterion for identifying a sulfhydryl side chain of an introduced cysteine as being exposed in the binding cavity and accessible to CEC. Our results confirmed the -helical structure and predicted rotational orientation of TM5 in H2A.

	Experimental Procedures

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Materials. [³H]RX821002 [2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline] was from Amersham International (Buckinghamshire, UK; specific activity, 52 Ci/mmol). Phentolamine and CEC were from Research Biochemicals (Natick, MA). Cell culture reagents were supplied by GIBCO (Gaithersburg, MD). The 10-mer oligopeptide Tyr-Val-Ile-Ser-Ser-Cys-Ile-Gly-Ser-Phe (TM5 region of H2A: Tyr196 to Phe205) was supplied by the Center for Biotechnology (Turku, Finland). Other chemicals were of analytical grade and were purchased from commercial suppliers.

Reaction of CEC with oligopeptide and mass spectroscopic analysis. The 10-mer oligopeptide Tyr-Val-Ile-Ser-Ser-Cys-Ile-Gly-Ser-Phe was dissolved in 50 mM K⁺-phosphate buffer, pH 7.4, at 21°, and one molar equivalent of CEC was added. The reaction mixture was incubated for 60 min at 37° and analyzed by matrix-assisted laser desorption mass spectrometry (Finnigan MAT, Hemel Hempstead, UK).

Mutagenesis and expression vectors. The cDNA encoding H2A (Kobilka et al., 1987) was inserted into the SmaI site of the vector pALTER-1 (Promega, Madison, WI). Site-directed mutagenesis was performed using the Altered Sites II In Vitro Mutagenesis System (Promega). The mutated DNA fragments were sequenced manually by dideoxy sequencing of double-stranded DNA with Sequenase (United States Biochemical, Cleveland, OH) and confirmed with an ABI377 automated sequencer (Perkin-Elmer Cetus (Norwalk, CT). The WT H2A and the mutated receptor cDNAs were subcloned into the KpnI/BamHI sites of the expression vector pREP4 (InVitrogen, NV Leek, The Netherlands), which also contains the gene for hygromycin B resistance.

Cell culture and transfections. Adherent CHO cells (American Type Culture Collection, Rockville, MD) were cultured in -minimum essential medium supplemented with 2 mM glutamine, 20 mM NaHCO₃, 5% heat-inactivated fetal calf serum, penicillin (50 units/ml), and streptomycin (50 µg/ml). Cells were grown in 5% CO₂ at 37°. The pREP4-based expression constructs were transfected into CHO cells with use of the Lipofectin reagent kit (GIBCO, Paisley, UK). For each transfection, we used 3 µg of plasmid DNA/5 × 10⁴ cells. Hygromycin B (Boehringer-Mannheim Biochemica, Mannheim, Germany)-resistant (550 µg/ml) cell cultures were examined for their ability to bind the ₂-AR antagonist [³H]RX821002. The transfected cells chosen for further experiments were subsequently maintained in 200 µg/ml hygromycin B.

Receptor inactivation and ligand binding. Cells were harvested into chilled phosphate-buffered saline, pelleted, washed, suspended in ice-cold 50 mM K⁺-phosphate buffer, pH 7.4, at 21°, and homogenized with an Ultra-Turrax homogenizer (model T25, Janke & Kunkel, Staufen, Germany; setting, 9500 rpm, twice for 10 sec). The homogenate was used for saturation and competition binding assays or receptor inactivation experiments.

Three-dimensional modeling of H2A binding cavity. The molecular modeling of H2A and the binary complex with CEC will be described in complete detail (V. Cockcroft, A. Marjamäki, H. Frang, M. Pihlavisto, J.-M. Savola, and M. Scheinin, Ligand interaction of serine-cysteine amino acid exchanges in TM5 of _Z-adrenergic receptors, manuscript in preparation.). The structural coordinates of the high-resolution electron cryomicroscopy model of bacteriorhodopsin (Henderson et al., 1990) was used as a three-dimensional template for structural mapping of GPCR sequences.

	Results

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Site-directed mutagenesis and transfections. To examine the structure of the TM5 domain of H2A, amino acid residues from Val197 to Cys201 and Ser204 were mutated to introduce or delete cysteines. The introduced mutations were confirmed and the absence of secondary mutations was verified by dideoxy sequencing of double-stranded DNA.

View this table: [in this window] [in a new window]   TABLE 1 Characterization of CHO cells expressing WT and mutated 2-ARs. The concentration of CEC that inhibited specific [3H]RX821002 binding in competition assays by 50% (IC50) was used to calculate apparent Ki values (inhibition constant) according to the Cheng-Prusoff equation.

Receptor inactivation studies. CEC, an alkylating derivative of clonidine, has been used previously to discriminate between ₁-AR subtypes, but it also has been shown to inactivate ₂-ARs in a subtype-selective manner. Based on our hypothesis, the reactive aziridinium ion derivative of CEC forms a covalent bond with an exposed SH-group of a cysteine residue in the receptor molecule (Fig. 2) and inactivates the receptor by steric blockade of the binding cavity. Covalent bonding of CEC to protein was confirmed by allowing it to react with a synthetic 10-mer oligopeptide corresponding to residues 196-205 of the TM5 region of the H2A and then undergoing mass spectroscopic analysis (Fig. 3). After 1 hr at 37°, the oligopeptide was totally alkylated in a manner consistent with our hypothesis presented in Fig. 2.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   CEC undergoes intramolecular cyclization to a reactive aziridinium ion as reported previously (Vargas et al., 1993). The aziridinium ion then can either react with water or form a covalent adduct with a peptide containing a free SH-group (TM5-SH).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Matrix-assisted laser desorption mass spectra of the oligopeptide Tyr-Val-Ile-Ser-Ser-Cys-Ile-Gly-Ser-Phe (left) and its covalent adduct with CEC after 1 hr at 37° (right). The molecular mass of the peptide increases by 299 units from 1078 to 1377, which is consistent with the reaction scheme presented in Fig. 2. Peaks with masses 38 units higher than the predicted molecular mass (1115.5 and 1415.2) are potassium adducts of peptides. After the reaction (right spectrum), no unreacted oligopeptide remains.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effect of CEC treatment on binding activity of human 2-AR subtypes. CHO cells expressing H2A, H2B, and H2C were incubated in the absence (control) and presence of CEC (1 and 10 µM) for 30 min at 37°, followed by two washes. Residual 2-AR binding was determined by incubation with 2.5 nM [3H]RX821002. Nonspecific binding was defined by 10 µM phentolamine. For comparison purposes, binding assays for all three receptors were carried out simultaneously with the use of the same stock solutions. Results are expressed as percentage of specific [3H]RX821002 binding remaining after treatment with CEC compared with control. Data represent the mean ± standard error of three separate experiments performed in duplicate.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Effect of CEC treatment on binding activity of H2AWT and M2AWT and H2ASer201 and M2ACys201 mutant receptors. Results were obtained as described in the legend for Fig. 4.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Stereo view of the energy-minimized hypothetical model of the binary complex of H2A binding cavity with the aziridinium ion form of chloroethylclonidine. The view direction is (A) from TM1 to TM5 and (B) from above the binding cavity. Only the TM3/TM4/TM5/TM6 end of the receptor cavity is shown. The regions of the helices displayed at the level of the ligand binding site are TM3, 106-117; TM4, 157-170; TM5, 196-209; and TM6, 386-399. Carbon, white; oxygen, red; nitrogen, blue; chlorine, green; and sulfur, yellow. White, hydrogen atoms of the 2-aminoimidazoline moiety of the ligand showing hydrogen bonds with Asp113 (dotted lines). For main chain atoms of the transmembrane helices, TM3, red; TM4, yellow; TM5, orange; and TM6, blue. Labels, amino acid residue numbering of the H2A.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Effect of CEC treatment on binding activity of H2ACys201 and H2ASer201Cys197, H2ASer201Cys198, H2ASer201-Cys199, H2ASer201Cys200, and H2ASer201Cys204 mutant receptors. Results were obtained as described in the legend for Fig. 4.

Ligand binding assays. Saturation isotherms of [³H]RX821002 binding- and LIGAND- (McPherson, 1985) derived K_d (receptor affinity) and B_max (receptor density) values were determined in three separate experiments for each cell line (Table 1). Three-point mutations of H2ASer201, Ser199, Ser200, and Ser204 to cysteine, resulted in 3-5-fold decreases in receptor affinity for the ₂-AR antagonist [³H]RX821002. The expression level of the H2ASer201 mutant used in our experiments was only 156 ± 13 fmol/mg of total cellular protein. Similar results of receptor inactivation by CEC were, however, later obtained in experiments with another batch of H2ASer201 cells, expressing 4736 ± 234 fmol/mg of cell homogenate (inactivation, 7 ± 2%). This indicates that the weak alkylating effect of CEC on H2ASer201 presented in Fig. 5 is not dependent on the receptor expression level.

	Discussion

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In the current study, we were able to demonstrate that an exposed cysteine residue in the binding cavity of ₂-AR is required for the alkylating effect of CEC. Although the apparent binding affinities (apparent K_i value) of CEC were comparable for the WT and mutated H2A-ARs, the alkylating effect of CEC treatment was dependent on the location of a reactive cysteine residue in TM5. True affinity of an irreversible ligand cannot be determined reliably in a conventional competition binding assay, and the apparent affinity of CEC determined in this way actually may represent both reversible and irreversible binding (Michel et al., 1993). Simultaneous inactivation and competitive binding should, however, overestimate the apparent affinity of CEC for ₂-AR subtypes/mutants that become alkylated, indicating the lack of alkylating effects of CEC in our assays is not due to the lack of CEC binding.

We used a receptor model predicting the -helical structure of TM5 in H2A-AR, in which the residues Val197, Ser200, Cys201, and Ser204 were pointing to the binding pocket, whereas the residues Ile198, Ser 199, Ile202, and Gly203 were facing the lipid bilayer. This model was supported by the results obtained through site-directed mutagenesis and CEC inactivation experiments. The primary structures of the TM5 regions of all ₂-AR subtypes contain a cysteine in the position corresponding to Cys209 of H2A (Fig. 1). This cysteine is facing the lipid bilayer of the plasma membrane in our receptor model and thus was not expected to interfere with CEC inactivation experiments. This orientation of Cys209 was supported by the results obtained with H2B, M2A, H2ASer201, H2ASer201Cys198, and H2ASer201Cys199 not containing cysteine residues exposed in the binding cavity and shown to be resistant to the alkylating effect of CEC (Figs. 4, 5, and 7). These results are consistent with an -helical structure of TM5 and provide constraints for the rotational orientation of this helix in relation to the binding cavity.

The ₂-AR is one of the most extensively structurally characterized GPCRs. With site-directed mutagenesis, it has been possible to identify several amino acid residues that are critical for and probably directly involved in ligand binding. The catechol hydroxyl groups of epinephrine seem to interact with two serine residues present in TM5 of ₂-AR (Strader et al., 1989). The two serine residues at corresponding positions of the human _2A-AR (Ser200 and Ser 204) have been shown to participate in hydrogen bond interactions with the catechol hydroxyl groups of catecholamine agonists (Wang et al., 1991). When Ser201 of the mouse _2A-AR was mutated to cysteine, the corresponding amino acid in H2A, this cysteine-to-serine substitution was shown to be critical for the low affinity of the mouse receptor for yohimbine and rauwolscine (Link et al., 1992). Site-directed mutagenesis and analysis of the three-dimensional model of the hamster _1B-AR also indicated that three serine residues, corresponding to positions 200, 201, and 204 in H2A, are important for agonist interactions (Cavalli et al., 1996). When the structural determinants of subtype-selective agonist binding of the hamster _1B-AR were identified, it was shown that mutation of Ala204 to valine (corresponding to position 197 in H2A) in TM5 conferred onto _1B-AR the binding properties of the _1A-AR (Hwa et al., 1995). These results obtained with computer-aided modeling and site-directed mutagenesis from different members of the same receptor family support our model and the location of residues 197, 200, 201, and 204 of H2A in the binding site cavity.

We introduced a new useful approach to mapping of the binding site crevice of human ₂-ARs by using cysteine substitution mutagenesis and irreversible cysteine-specific covalent binding of CEC to the receptor. This method emerges as a very useful tool for structural characterization of the ₂-ARs. Using this method together with three-dimensional modeling, we were able to confirm the predicted -helical structure of TM5 and its orientation in H2A. One of the goals of the current study was to assess whether a so-called molecular yardstick approach could provide distance constraints for determining improved models of GPCRs. This technique, perhaps with some modifications, also could be applicable to structural studies on TM4 and TM6 of the same receptor.

Javitch et al. (1995) and Fu et al. (1996) previously introduced a cysteine-reactive approach using as reactive agents nonspecific polar methanethiosulfonate derivatives to probe another monoamine receptor, the D₂ dopamine receptor. Our method can be seen as a simple development of this approach. We suggest our method has the advantage of introducing recognition specificity by using a thiol-reactive group incorporated into an affinity ligand of the target receptor. This allows the use of significantly lower reagent concentrations and lessens the possibility of indirectly blocking radioligand binding.

Conventional loss-of-function mutagenesis has not always produced definitive answers for the purpose of assigning amino acid residues as being inside the binding cavity. It has been shown that point mutations of positions expected to be outside the binding cavity can have a marked effect on ligand affinities, presumably through conformational control (Fong et al., 1992; Sachais et al., 1993). In addition, several reports exist of residue substitutions at sites at which ligand/receptor contact interaction would be expected on the basis of amino acid conservation patterns that have not shown expected disruption of ligand binding (Befort et al., 1996). Although the receptor modeling presented here is still rather crude, it has been used to devise an experimental approach, gain-of-function mutagenesis, to provide, through covalent bond formation, solid information that will lead to better models of not only the receptors but also binary complexes of ligand and receptor.