Design, Synthesis and Pharmacological Evaluation of 5-Hydroxytryptamine1a Receptor Ligands to Explore the Three-Dimensional Structure of the Receptor

  1. Marı́a L. López-Rodrı́guez1,
  2. Bruno Vicente1,
  3. Xavier Deupi2,
  4. Sergio Barrondo3,
  5. Mireia Olivella2,
  6. M. José Morcillo4,
  7. Bellinda Behamú1,
  8. Juan A. Ballesteros5,
  9. Joan Sallés3 and
  10. Leonardo Pardo2
  1. 1Departamento de Quı́mica Orgánica I, Facultad de Ciencias Quı́micas, Universidad Complutense, Madrid, Spain (M.L.L.-R., B.V., B.B.); 2Laboratori de Medicina Computacional, Unitat de Bioestadı́stica, Facultat de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain (X.D., M.O., L.P.);3Departamento de Farmacologı́a, Universidad del Pais Vasco, Vitoria, Spain (S.B., J.S.); 4Facultad de Ciencias, Universidad Nacional de Educación a Distancia, Madrid, Spain (M.J.M.); and 5Novasite Pharmaceuticals, Inc., San Diego, California (J.A.B.)
  1. Dr. Leonardo Pardo, Unitat de Bioestadı́stica, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain. E-mail: leonardo.pardo{at}uab.es
 
Next Section

Abstract

In this work, we evaluate the structural differences of transmembrane helix 3 in rhodopsin and the 5-hydroxytryptamine 1A (5-HT1A) receptor caused by their different amino acid sequence. Molecular dynamics simulations of helix 3 in the 5-HT1A receptor tends to bend toward helix 5, in sharp contrast to helix 3 in rhodopsin, which is properly located within the position observed in the crystal structure. The relocation of the central helix 3 in the helical bundle facilitates the experimentally derived interactions between the neurotransmitters and the Asp residue in helix 3 and the Ser/Thr residues in helix 5. The different amino acid sequence that forms helix 3 in rhodopsin (basically the conserved Gly3.36Glu3.37 motif in the opsin family) and the 5-HT1A receptor (the conserved Cys3.36Thr3.37 motif in the neurotransmitter family) produces these structural divergences. These structural differences were experimentally checked by designing and testing ligands that contain comparable functional groups but at different interatomic distance. We have estimated the position of helix 3 relative to the other helices by systematically changing the distance between the functional groups of the ligands (1 and2) that interact with the residues in the receptor. Thus, ligand 1 optimally interacts with a model of the 5-HT1A receptor that matches rhodopsin template, whereas ligand 2 optimally interacts with a model that possesses the proposed conformation of helix 3. The lack of affinity of1 (Ki > 10,000 nM) and the high affinity of 2 (Ki = 24 nM) for the 5-HT1A receptor binding sites, provide experimental support to the proposed structural divergences of helix 3 between the 5-HT1A receptor and rhodopsin.

G protein-coupled receptors (GPCRs) are membrane proteins that transmit extracellular signals of neurotransmitters, peptides, and glycoproteins through heterotrimeric G proteins bound in the interior of the cell (Ji et al., 1998). The GPCR family possesses highly conserved motifs in the transmembrane region (Ballesteros and Weinstein, 1995; Horn et al., 1998), which suggests a common transmembrane structure. Recently, the detailed three-dimensional (3-D) structure of the GPCR rhodopsin (RHO) was determined at 2.8-Å resolution (Palczewski et al., 2000). This structure has confirmed that RHO and probably the RHO family of GPCRs are formed by a highly organized heptahelical transmembrane bundle. This structural homology between RHO and the other GPCRs probably does not extend to the extracellular domain, for which there is very little homology, and is highly structured in RHO, blocking the access of the extracellular ligand to the core of the receptor (Bourne and Meng, 2000).

The amino acid residues involved in ligand binding have been primarily identified by pharmacological and mutagenesis studies [for review, seevan Rhee and Jacobson (1996)]. In particular, agonists and antagonists of the neurotransmitter subfamily of GPCRs bind with their protonated amine to the conserved Asp3.32 (seeMaterials and Methods for the receptor-numbering scheme), in transmembrane helix (TMH) 3 (Strader et al., 1988). The hydroxyl groups present in the chemical structure of many neurotransmitters seem to hydrogen bond (Strader et al., 1989; Liapakis et al., 2000) a series of conserved Ser/Thr residues (5.42, 5.43, and 5.46), in TMH 5. Moreover, mutagenesis experiments on the α2- (Suryanarayana et al., 1991), β2- (Suryanarayana and Kobilka, 1993), 5-HT1A (Guan et al., 1992), and 5-HT1B (Glennon et al., 1996) receptors have shown that Asn7.39, in TMH 7, is important in conferring specificity to a series of ligands such as pindolol and propanolol.

The publication of the crystal structure of RHO has provided the arrangement of the TMHs in the cell membrane (Palczewski et al., 2000). The central TMH 3 is near TMH 5 in its cytoplasmic end and far from TMH 5 in its extracellular end, which hinders the binding of the small neurotransmitter molecules between Asp3.32 and the implicated Ser/Thr5.42,5.43,5.46 residues, located at the extracellular side. This finding was previously noted in the translation of the electron density maps of frog RHO (Unger et al., 1997) into an α-carbon template (Baldwin et al., 1997). Thus, the following factor should be taken into account. Wide ranges of extracellular ligands, from small neurotransmitters to large peptides and hormones, are recognized by the different GPCR subfamilies. Each subfamily has probably developed specific structural motifs that allow the receptor to accommodate the different extracellular ligands. Interestingly, RHO possesses two nonconserved successive Gly residues at positions 89 (Gly2.56) and 90 (Gly2.57). This specific motif of the opsin family induces a significant distortion of TMH 2, which bends strongly toward TMH 1 (Palczewski et al., 2000). In contrast, the chemokine family of GPCR possesses in this region of TMH 2 a conserved Thr2.56XPro2.58 motif, where X is any amino acid. We have recently shown that this TxP motif in CCR5 bends TMH 2 toward the center of the bundle and away from TMH 1 (Govaerts et al., 2001a). Moreover, this structural singularity is important for chemokine-induced functional response (Govaerts et al., 2001a). Thus, the presence of specific and conserved residues among the families of GPCR may result in structural differences among them. The similarities and differences between RHO and other GPCRs have recently been reviewed in detail (Ballesteros et al., 2001).

In this work, we aim to evaluate the structural differences of TMH 3 in RHO and the 5-HT1A receptor (5-HT1AR) caused by their different amino acid sequence. The conformation of TMH 3 in the neurotransmitter family of GPCR changes the location of Asp3.32 and in consequence where the extracellular ligand is placed. Thus, we aim to estimate the position of TMH 3 relative to the other helices, primarily TMH 5 and 7 where ligands bind, in the inactive conformation of the 5-HT1AR. Several approaches have been developed to elucidate intermolecular distances between helices: double revertant mutant constructs (Zhou et al., 1994), spin labeling (Yang et al., 1996), zinc site engineering (Elling et al., 1995, 1999), and Cys crosslinking (Yu et al., 1995). We have developed a new approach in which the distance between the functional groups of the ligand that interact with the residues in the receptor is systematically varied. This procedure has allowed us to discern between conformations of the receptor obtained computationally. Antagonists are preferred over agonists to explore the inactive form of the receptor. Recently, we have reported the pharmacological characterization of EF-7412 as an antagonist in vivo in pre- and postsynaptic 5-HT1AR sites (Lopez-Rodriguez et al., 2001a,b). We have designed, synthesized, and pharmacologically evaluated a new set of compounds, using EF-7412 as a template, to discern between computer models of the 5-HT1AR.

Previous SectionNext Section

Materials and Methods

Residue Numbering Scheme.

Each transmembrane residue is numbered with the helix number (from 1 to 7) in which it is located plus its relative position to the most conserved residue in the helix, arbitrarily labeled 50 (Ballesteros and Weinstein, 1995). Therefore, the most conserved TMH 3 residue is designated with the index number 3.50 (Arg3.50). The Asp preceding the Arg in the (D/E)RY motif is designated Asp3.49, and the Tyr after the Arg is designated Tyr3.51.

Molecular Dynamics Simulations of TMH 3 in RHO and the 5-HT1AR.

The peptide corresponding to the residues from 3.22 to 3.54 in TMH 3 of RHO (Ace-PTGCYFEGFFATLGGEIALWSLVVLAIERYVVV-NMe), and the 5-HT1AR (Ace-QVTCDLFIALDVLCCTSSILHLCAIALDRYWAI-NMe), were built in the standard α-helix conformation (backbone dihedral angles φ and ψ of −58 and −47 degrees). All ionizable residues in the helices were considered uncharged. The structures obtained were placed in a rectangular box containing methane molecules (2693 and 3095 for RHO and 5-HT1AR, respectively) to mimic the hydrophobic environment of the TMHs. A similar procedure has recently been employed to mimic the membrane in molecular dynamics simulations of the thyrotropin receptor (Govaerts et al., 2001b) and TMH 2 of the CCR5 receptor (Govaerts et al., 2001a). The sizes of the boxes were 74.5 × 43.5 × 41.0 Å for RHO, and 76.5 × 45.5 × 41.5 Å for 5-HT1AR. The systems were energy-minimized (500 steps), heated (from 0 to 300°K in 15 ps), equilibrated (from 15 to 500 ps) and the production run (from 500 to 1000 ps) was carried out at constant volume using the particle mesh Ewald method to evaluate electrostatic interactions. Structures were collected every 5 ps during the last 500 ps of simulation (100 structures per simulation). The molecular dynamics simulations were run with the Sander module of AMBER 5 (http://www.amber.ucsf.edu/amber/amber.html), the all-atom force field (Cornell et al., 1995), SHAKE bond constraints in all bonds, a 2-fs integration time step, and constant temperature of 300°K coupled to a heat bath.

The logistic regression model (Hosmer and Lemeshow, 1989) was used to fit the binary dependent variable (RHO, 5-HT1AR) to the independent variables: the torsional angles (φ and Ψ) of the residues spanning from 3.33 to 3.48 (32 variables) obtained during the molecular dynamics trajectory (a total of 200 structures). In contrast to the standard regression analysis, the dependent variable in the logistic regression is discrete, taking only two possible values (RHO and 5-HT1AR). The stepwise method was employed to select the independent variables in the model. Thus, only the torsional angles φ and Ψ that better classify the structures as RHO or 5-HT1AR are included in the regression equation. The odds ratio is a function of the coefficient of the independent variable in the regression equation and measures how many times it is more likely to be RHO or 5-HT1AR with a decrease or an increase of 1° in the torsional angles (independent variables). The larger the value of the odds ratio, the more predictive the independent variable is. Independent variables with odds ratio of 1 indicates no predictive power. Calculations were performed with SAS 6.11 (SAS Institute, Cary, NC).

A Molecular Model of the 5-HT1AR.

The 3-D model of the transmembrane domain of the 5-HT1AR was constructed by computer-aided model building techniques from the crystal structure of RHO (Palczewski et al., 2000) (PDB access number1F88). Conserved residues Asn55 (residue number in the PDB file of RHO) and Asn54 (residue number in the human 5-HT1AR sequence) [Asn1.50 in the generalized numbering scheme (Ballesteros and Weinstein, 1995)]; Asp83 and Asp82 (Asp2.50); Arg135 and Arg134(Arg3.50); Trp161 and Trp161 (Trp4.50); Pro215 and Pro207(Pro5.50); Pro267 and Pro360 (Pro6.50); and Pro303 and Pro397(Pro7.50) were employed in the alignment of RHO and human 5-HT1AR transmembrane sequences. All ionizable residues in the helices were considered uncharged with the exception of Asp2.50, Asp3.32, Asp3.49, Arg3.50, and Glu6.30. SCWRL-2.1 was employed to add the side chains of the nonconserved residues based on a backbone-dependent rotamer library (Dunbrack and Cohen, 1997). This computer model, which maintains the position of the TMHs as in RHO, is denoted 5-HT1ARRHO. TMH 3 was then replaced by the most representative structure of the geometries obtained during the molecular dynamics trajectory of TMH 3 in 5-HT1AR (see above). This representative structure was selected by automatically clustering the collected geometries into conformationally related subfamilies with the program NMRCLUST (Kelley et al., 1996). The backbone of the highly conserved E/DR3.50Y motif superimposed the structures. This computer model, which changes relative to 5-HT1ARRHO the position of TMH 3 at the extracellular side, is denoted 5-HT1ARMD.

The initial structure of the complex between (±)-2-[4-[4-(6-hy droxy-2-pyridyl)piperazin-1-yl]butyl]-1,3-dioxoperhydropyrrolo[1,2- c]imidazole (1) and (±)-2-[4-[4-(m-(acetylamino)phenyl)piperazin- 1-yl]butyl]-1,3-dioxoperhydropyrrolo[1,2-c]imidazole (2) and the 5- HT1AR was obtained from the previously reported structure of the complex between EF-7412 and the 5-HT1AR (Lopez-Rodriguez et al., 2001b). Subsequently, the complete systems were energy-minimized (5000 steps). Energy minimizations were run with the Sander module of AMBER 5 (http://www.amber.ucsf.edu/amber/amber.html), the all-atom force field (Cornell et al., 1995), and a 13-Å cutoff for nonbonded interactions. Parameters for ligands 1 and 2 were adapted from the force field of Cornell et al. (1995) using RESP point charges (Cieplak et al., 1995).

Chemistry.

Derivative 1 was synthesized by the following procedure: 2.0 ml of triethylamine (1.5 g, 14.6 mmol) was added to a suspension of 2.5 g (9 mmol) of 2-(4-bromobutyl)-1,3-dioxoperhydropyrrolo[1,2-c]imidazole (Lopez-Rodriguez et al., 1996) and 2.7 g (15 mmol) of 1-(6-hydroxy-2-pyridyl)piperazine (Pavia et al., 1987) in 19 ml of acetonitrile. The mixture was refluxed for 20 to 24 h (thin-layer chromatography). Then, the solvent was evaporated under reduced pressure and the residue was resuspended in water and extracted with dichloromethane (3 × 100 ml). The combined organic layers were washed with water and dried over MgSO4. After evaporation of the solvent, the crude oil was purified by column chromatography (dichloromethane) to afford 1.1 g (33%) of 1, which was converted into the hydrochloride salt. Derivative 2 was synthesized by the following procedure: 0.11 ml (1.6 mmol) of acetyl chloride was added dropwise to a solution of 600 mg (1.6 mmol) of 2-[4-[4-(m-aminophenyl)piperazin-1-yl]butyl]-1,3-dioxoperhydropyrrolo[1,2-c]imidazole (Lopez-Rodriguez et al., 2001a) in 20 ml of pyridine at 0°C. After stirring at room temperature for 1.5 h (thin-layer chromatography), the mixture was diluted with 50 ml of methylene chloride and washed with a saturated aqueous solution of CuSO4, water, and brine (25 ml). The organic layer was dried (Na2SO4) and the solvent evaporated under reduced pressure to afford 668 mg (67%) of 2, which was converted to the hydrochloride salt. The new compounds were characterized by IR and1H- and 13C-NMR spectroscopy and gave satisfactory combustion analyses (C, H, N).

Radioligand Binding Assays.

The 5-HT1Areceptor binding studies were performed by a modification of a procedure described previously (Clark et al., 1990). The cerebral cortices of male Sprague-Dawley rats (Rattus norvegicus albinus) weighing 180 to 200 g were homogenized in 10 volumes of ice-cold Tris buffer (50 mM Tris-HCl, pH 7.7 at 25°C) and centrifuged at 28,000g for 15 min. The membrane pellet was washed twice by resuspension and centrifugation. After the second wash, the resuspended pellet was incubated at 37°C for 10 min. Membranes were then collected by centrifugation and the final pellet was resuspended in 50 mM Tris-HCl, 5 mM MgSO4, and 0.5 mM EDTA buffer, pH 7.4 at 37°C. Fractions of the final membrane suspension (about 1 mg of protein) were incubated at 37°C for 15 min with 0.6 nM [3H]8-hydroxy-2-dipropylaminotetralin (133 Ci/mmol), in the presence or absence of several concentrations of the competing drug, in a final volume of 1.1 ml of assay buffer (50 mM Tris-HCl, 10 nM clonidine, 30 nM prazosin, pH 7.4 at 37°C). Incubation was terminated by rapid vacuum filtration through Whatman GF/B filters, presoaked in 0.05% poly(ethylenimine), using a Brandel cell harvester. The filters were then washed with the assay buffer and dried. The filters were placed in poly(ethylene) vials to which 4 ml of a scintillation cocktail (Aquasol) was added, and the radioactivity bound to the filters was measured by liquid scintillation spectrometry. The data were analyzed by an iterative curve-fitting procedure (Prism; GraphPad Software, San Diego, CA), which provided IC50, Ki, andr2 values for test compounds;Ki values were calculated from the Cheng and Prusoff equation (Cheng and Prusoff, 1973). The protein concentrations of the rat cerebral cortex were determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard. Nonspecific binding was determined with 10 μM 5-HT. Competing drug, nonspecific, total and radioligand bindings were defined in triplicate.

Previous SectionNext Section

Results and Discussion

Amino Acid Composition of TMH 3 in the Opsin and Neurotransmitter Families of GPCR.

We analyze in this section the amino acid sequence of TMH 3 in the opsin and neurotransmitter families that might cause structural differences in the helix. These differences are relevant because the crystal structure of RHO (Palczewski et al., 2000) is an appropriate template to model the 3-D structure of receptors for neurotransmitters and the conformation of TMH 3 in the neurotransmitter family changes the location of Asp3.32, the anchoring point of both agonists and antagonists (Strader et al., 1988;van Rhee and Jacobson, 1996). The intracellular side of TMH 3 contains in both cases the highly conserved E/DR3.50Y motif. The protonation of E/D3.49 is thought to be important in G-protein coupling (Arnis et al., 1994; Oliveira et al., 1994; Scheer et al., 1996). We assume that this common E/DR3.50Y motif, in the compact cytoplasmatic surface, is hold in similar position in both families. Thus, the location of the amino acid 3.32 in the opsin and the neurotransmitter families, relative to the E/DR3.50Y motif, will depend on the amino acid composition of the residues spanning from 3.33 to 3.48.

Table 1 shows the statistical analysis of the conservation pattern in this continuous stretch of residues from 3.33 to 3.48 of all GPCR sequences denoted as (Rhod)opsin (245 entries) and Amine (288 entries) in GPCRDB (Horn et al., 1998), as of December 2001. Ser or Thr or Cys residues are present in 6 of 16 positions (3.35, 3.36, 3.37, 3.39, 3.44, and 3.47) more than 50% of the time in the neurotransmitter family, in sharp contrast to the opsin family, which contains only two positions (3.33 and 3.42). Ser, Thr, and Cys residues play a special role in α-helices because they can form an intrahelical hydrogen bond between the side chain OHγ (or SHγ) and the i-3 or i-4 carbonyl oxygen of the preceding turn (Gray and Matthews, 1984). This additional hydrogen bond to the peptide carbonyl oxygen can produce significant changes in the curvature of the helix (Ballesteros et al., 2000; Govaerts et al., 2001a). It is important to note that Ser, Thr, and Cys are not present simultaneously in the opsin and the neurotransmitter families in the 3.33–3.48 range (Table 1). Besides, there are two amino acid sites in the TMH 3 domain of the opsin family at which a Gly is present in more than 50% of the receptors: Gly3.35 (50.0%) and Gly3.36 (98.8%). Gly is most often located in loop regions and acts as helix-breaker in soluble proteins (O'Neil and DeGrado, 1990). In contrast, Gly residues are frequently detected in the transmembrane segments of membrane proteins (Senes et al., 2000) which suggests a structural role. The absence of the side chain in Gly probably adds flexibility to Gly-containing helices (Kumar and Bansal, 1998). The neurotransmitter family possesses Cys residues at these 3.35 and 3.36 positions. Thus, the different attributes of the amino acids forming TMH 3 in the opsin and the neurotransmitter families can produce significant structural deviations among them.

View this table:
Table 1

Statistical analysis of the conservation pattern in a continuous stretch of residues from 3.33 to 3.48 in TMH 3 of all GPCR sequences denoted as (Rhod)opsin and Amine in GPCRDB.

Molecular Dynamics Simulations of TMH 3 in RHO and the 5-HT1AR.

To obtain a rough idea of the possible consequences that the different amino acid sequences that form TMH 3 in rhodopsin and the 5-HT1AR might have on the structure, we performed a molecular modeling exercise using the 3-D structure of rhodopsin as the template (Palczewski et al., 2000). Figure 1, a (view parallel to the membrane with the extracellular side at the top) and b (perpendicular to the membrane from the extracellular side), show the result of superimposing the structures computed during the molecular dynamics trajectory (see Materials and Methods for computational details) of the amino acid sequence that form TMH 3 in RHO (orange) and the 5-HT1AR (green) on TMH 3 of RHO. The backbone of the highly conserved E/DR3.50Y motif superimposed the computed structures and the helix bundle of RHO. This procedure hypothesizes that the common E/DR3.50Y motif is located in similar positions in rhodopsin and the 5-HT1AR. Visual inspection of the helix axes of the computed structures in Fig. 1, a and b, reveal that TMH 3 in RHO and the 5-HT1AR behaves differently. The conformational space explored by the extracellular part of TMH 3 in the 5-HT1AR is precisely toward TMH 5. In contrast, the energetically available structures of RHO are distant to TMH 5, basically within the position of TMH 3 in the crystal structure.

Figure 1
View larger version:
Figure 1

a and b, the α-carbon traces of the transmembrane helix bundle of RHO (Palczewski et al., 2000) are depicted as tube ribbons in red for TMHs 3 and 5 and white for the other TMHs. The views are parallel to the membrane with the extracellular side at the top (a) and perpendicular to the membrane from the extracellular side (b). The helix axes of the structures computed during the molecular dynamics trajectory of the amino acid sequence that form TMH 3 in RHO (orange) and the 5-HT1AR (green) are displayed. c, the representative structure of the geometries obtained during the molecular dynamics trajectory of TMH 3 in the 5-HT1AR (green helix axes) is shown in green. d and e, detailed view of the transmembrane helix bundle of 5-HT1ARRHOcomplexed with ligand 1 (d) and 5-HT1ARMD complexed with ligand 2 (e). The Cα traces of the extracellular part (top) of TM 3, 5, and 7 are only shown. Nonpolar hydrogens are not depicted to offer a better view of the recognition pocket. Figures were created using MolScript ver. 2.1.1 (Kraulis, 1991) and Raster3D ver. 2.5 (Merritt and Bacon, 1997).

The logistic regression model (see Materials and Methods for computational details) was employed to characterize the amino acid positions in RHO and the 5-HT1AR that most influence the structural differences observed in TMH 3. The binary dependent variable (RHO, 5-HT1AR) was fitted to the torsional angles (Φ and Ψ) of the residues spanning from 3.33 to 3.48. Table 2 shows the torsional angles selected in the stepwise procedure and the odds ratio of the included variables. The torsional angles Φ of the residue at position 3.35 (Φ3.35); Φ and Ψ at positions 3.36 and 3.37 (Φ3.36, Φ3.37, Ψ3.36, and Ψ3.37); and Ψ at positions 3.39, 3.43, and 3.46 (Ψ3.39, Ψ3.43, and Ψ3.46), properly classify 100% of the input conformations of RHO and the 5-HT1AR. However, the predictive power of the selected torsions is not the same. The variables Φ3.36, Φ3.37, Ψ3.36, and Ψ3.37possess the highest odds ratio (Table 2) and thus the highest classification power. A logistic regression model with only these four independent variables already classifies 93% of the input conformations. Remarkably, Gly3.36 is highly conserved in the opsin family (98.8%) but not in the neurotransmitter family that contains Cys (56.9%). Substitution of Gly3.36 in RHO with more bulky residues promotes partial agonist activity of 11-cis-retinal (Han et al., 1997). Thr3.37 is present 85.1% of the time in the neurotransmitter family and is absent in the opsin family (Glu 34.3%, Ile 21.8%). It is important to note that Thr3.37 is not present in the 5-HT6 and muscarinic receptor subfamilies (seeConclusions). Substitution of Thr3.37in the α1B-adrenergic receptor by Ala produces epinephrine and norepinephrine to behave as partial agonists (Cavalli et al., 1996). The same authors concluded that Thr3.37 might play a role in preserving the receptor structure and function rather than directly interacting with the agonist (Cavalli et al., 1996).

View this table:
Table 2

Torsional angles and its odds ratio of the selected variables in the stepwise logistic regression between the binary dependent variable (RHO, 5-HT1AR) and the torsional angles (Φ and Ψ) obtained during the molecular dynamics trajectory of the residues spanning from 3.33 to 3.48.

We propose that the different structural properties of Gly3.36Glu3.37 in the opsin family and Cys3.36Thr3.37in the neurotransmitter family produce different TMH 3 orientations. This results in structural divergences between the neurotransmitter family of GPCR and RHO template (Palczewski et al., 2000). The absence of Thr3.37 in the muscarinic receptors also suggests structural divergences relative to the other members of the neurotransmitter family. Incorporation into the RHO template (white and red transmembrane helix bundle in Fig. 1c) of a representative conformation of TMH 3 (green transmembrane helix in Fig. 1c; seeMaterials and Methods for computational details) results in a significant displacement of Asp3.32 toward TMH 5, without modifying the more compact cytoplasmatic surface. This relocation facilitates the experimentally derived interactions between the neurotransmitters and the Ser/Thr residues in TMH 5. In particular, Ser5.42 and Thr5.43 are important in the binding of agonists to the 5-HT1AR (Ho et al., 1992). The magnitude of the relocation might be estimated from the structures depicted in Fig. 1c. Thus, the distances between the α-carbon positions of the implicated Asp3.32 and Ser5.42 and Thr5.43 residues in the RHO template (5-HT1ARRHO, see methods) are 14.6 and 15.9 Å, respectively. These distances decrease to 12.6 and 14.1 Å if the obtained conformation of TMH 3 from the 5HT1AR is incorporated into the RHO template (5-HT1ARMD).

It must be stressed that there may be other structural variations that could facilitate the binding of neurotransmitter to their receptors. We must be open to the possibility that the different sequence of the other transmembrane helices might also cause structural differences as well. However, the conservation of functionally important sequence motifs within the rhodopsin-like GPCR family has been interpreted to mean that the basic characteristics of the rhodopsin fold are similar in the different receptor subtypes. We propose that structural adaptation of a receptor to its cognate ligand is necessary in some domains of the transmembrane region while still maintaining a similar overall rhodopsin fold. We hypothesize structural differences only in TMH 3, whereas the other transmembrane helices remain unchanged relative to the RHO template.

Design and Test of 5-HT1AR Ligands That Interact with Asp3.32 and Asn7.39 to Discern between the Conformation of TMH 3.

We aim to provide experimental support to the proposed conformation of TMH 3 by designing and testing 5-HT1AR ligands that contain comparable functional groups but differ in the interatomic distance between them. The rationale behind this approach is that by varying the distance between the functional groups of the ligand that interact with the side chains of the receptor, we will be able to discern between the computer models of TMH 3. EF-7412 (see Table 3), a recent pharmacologically characterized antagonist in vivo in pre- and postsynaptic 5-HT1AR sites (Lopez-Rodriguez et al., 2001a), will be used as a template. It was proposed that EF-7412 forms an ionic interaction with Asp3.32throughout the protonated amine of the piperazine ring, hydrogen bonds with Asn7.39 throughout them-NHSO2Et group, and hydrogen bonds with Thr3.37, Ser5.42, and Thr5.43 throughout the hydantoin moiety of the ligand (Lopez-Rodriguez et al., 2001b). A first approach would be to change the distance between the protonated amine of the piperazine ring and the hydantoin moiety of the ligand to assess the conformation of TMH 3 relative to TMH 5. However, the flexibility of the –CH2 chain connecting both groups would impede to obtain any reliable conclusion. Nevertheless, the bending of TMH 3 toward TMH 5 also modifies the position of TMH 3 relative to TMH 7 at the extracellular site. Thus, we have designed 5-HT1AR ligands that intended to interact with Asp3.32 and Asn7.39, to discriminate the conformation of TMH 3 relative to TMH 7. Remarkably, these two positions have also been used to elucidate intermolecular distances by zinc site engineering experiments: substitution of Asp3.32 for His and Asn7.39for Cys in the β2-adrenergic receptor results in a mutant that is activated by free zinc ions (Elling et al., 1999).

View this table:
Table 3

In vitro binding data of compounds EF-7412, 1, and 2

Table 3 shows the chemical structures of compounds 1 and2. These ligands replace them-NHSO2Et group of EF-7412 with a common –NHCO group that optimally interacts with the side chain of Asn7.39. This –NHCO group is located at different positions in the structure relative to the protonated amine. The interatomic distance between the nitrogen of the protonated amine and the centroid of the –NHCO group for compounds 1 and2 are 6.4 and 8.5 Å, respectively. Structures 1and 2 were optimized (see Materials and Methodsfor computational details) inside 5-HT1ARRHO and 5-HT1ARMD models, respectively. Figure 1, d and e, show 1 and 2, respectively, in the binding pocket. The unique N-H group of the protonated amine of both ligands interacts with one of the Oδ atoms of Asp at the optimized distance between heteroatoms of 2.5 Å in both cases. Moreover, the N-H moiety of the common –NHCO group acts as a hydrogen bond donor in the hydrogen bond interaction with the Oδ1 atom of Asn, at the optimized distances between heteroatoms of 2.8 Å in both ligands, and the CO moiety of –NHCO group acts as a hydrogen bond acceptor in the hydrogen bond interaction with the Nδ2-H moiety of Asn, at the optimized distances between heteroatoms of 2.8 or 2.9 Å for ligands 1 or2, respectively. Moreover, the proposed recognition of the extracellular ligands involves the hydrogen bonds between both CO groups of the hydantoin moiety of the ligand and Thr3.37 (2.9 Å in both ligands), Ser5.42 (3.5 Å) and Thr5.43 (3.5 Å). Thus, 1 interacts optimally with 5-HT1ARRHO, which matches RHO template, whereas 2 optimally interacts with 5-HT1ARMD, which possesses the proposed conformation of TMH 3. It is important to note that the interaction of the –NHCO group of ligand 2 with Asn7.39 would benefit from a more bent conformation of TMH 3, which located the helix closer to TMH 5 and farther from TMH 7 at the extracellular part. This more extreme conformation was energetically accessible during the molecular dynamics trajectory of TMH 3 (see above). However, this conformational subfamily was not selected as the most representative in the automatic clustering procedure with the program NMRCLUST and was not used in the construction of 5-HT1ARMD(see Materials and Methods).

Table 3 shows the in vitro affinity of compounds 1(Ki > 10,000 nM) and 2(Ki = 24 nM) for the 5-HT1AR binding sites. The lack of affinity of1, which was designed to match RHO template (5-HT1ARRHO), and the high affinity of 2, which was designed to interact with a modified template of RHO (5-HT1ARMD), provides experimental support to the proposed structural divergences of TMH 3 between the 5-HT1AR and RHO.

Previous SectionNext Section

Conclusions

We have presented in this study a structural analysis of the conformation of TMH 3 in RHO and the 5-HT1AR in the context of the crystal structure of RHO (Palczewski et al., 2000). This analysis is relevant because the structure of RHO is normally used as a template to model the class A family of GPCRs and the conformation of TMH 3 in the neurotransmitter family changes the location of Asp3.32, the anchoring point of both agonists and antagonists (Strader et al., 1988; van Rhee and Jacobson, 1996). The different amino acid sequence of TMH 3 in RHO (basically the conserved Gly3.36Glu3.37 motif in the opsin family) and the 5-HT1AR (the conserved Cys3.36Thr3.37 motif in the neurotransmitter family) produces significant structural divergences. Molecular dynamics simulations of the amino acid sequence that forms TMH 3 in the 5-HT1AR tends to bend toward TMH 5, in sharp contrast to the amino acid sequence that forms TMH 3 in RHO, which is properly located within the position observed in the crystal structure. The relocation of the central TMH 3 facilitates the experimentally derived interactions between the neurotransmitters and the Asp residue in TMH 3 and the Ser/Thr residues in TMH 5.

We have designed two new ligands (1 and 2) that are thought to interact, in addition to other residues in the 5-HT1AR, with Asp3.32 in TMH 3 and Asn7.39 in TMH 7. Ligand 1interacts optimally with a model of the 5-HT1Areceptor that matches rhodopsin template, whereas ligand 2interacts optimally with a model that possesses the proposed conformation of helix 3. The lack of affinity of 1(Ki > 10,000 nM) and the high affinity of 2 (Ki = 24 nM) for the 5-HT1AR binding sites provides experimental support to the proposed structural divergences of helix 3 between the 5-HT1AR and RHO. The significant difference in affinity (Ki > 10000 nM versus Ki = 24 nM) between these similar compounds that contain comparable functional groups led us to suggest that the 5-HT1AR binding sites are not flexible and the extracellular ligand must be accommodated in the binding site in an optimal manner.

Statistical analysis of the conservation pattern at the 3.37 position shows that Thr (85.1%) is present in all the neurotransmitter family of GPCRs apart from the 5-HT6 receptor which contains Ser (2.4%) and the muscarinic receptors which contains Asn (10.8%). All these polar side chains can form intrahelical hydrogen bonds with the backbone and bend helices (Ballesteros et al., 2000). There is more degree of variability across neurotransmitter receptors at the 3.36 locus. Cys (56.9%) is present in the α-adrenergic, dopamine (with the exception of D1), histamine (with the exception of H1), and serotonin (with the exception of 5-HT2 and 5-HT4) subfamilies of receptors; Ser (28.5%) is present in the D1, H1, 5-HT2, and muscarinic receptors; Thr (1.7%) is present in the 5-HT4 receptor; and Val (12.1%) is present in the β-adrenergic subfamily of receptors. The side chains of both Ser and Thr can form hydrogen bonds with the backbone (Ballesteros et al., 2000), the side chain of Cys can also form hydrogen bonds with the backbone but of less strength, and the nonpolar side chain of Val cannot form hydrogen bonds. We have shown recently that the impairment of CCR5 receptor activation caused by the T82V, T82C, and T82S mutations parallels with the bending of the α-helix caused by these residues (Govaerts et al., 2001a). Thus, the presence of Thr, Ser, Cys, or Val alters to a greater or lesser degree the conformation of the helix. The wide range of bending and twisting that can result from the presence of these residues in TMH 3 has recently been illustrated (Ballesteros et al., 2001). These findings suggest that there might be some degree of variability in TMH 3 across the neurotransmitter family. Importantly, there are conservation patterns among subfamilies at the 3.36 and 3.37 positions. D1, H1, and 5-HT2 receptors contain SerThr, 5-HT4 receptors contain ThrThr, β-adrenergic receptors contain ValThr, 5-HT6 receptors contain CysSer, muscarinic receptors contain SerAsn, and all the others contain CysThr. These findings might serve to model the complexes between the neurotransmitter family and their ligands. These models are important because they provide the tools for guiding the design and synthesis of new ligands with predetermined affinities and selectivity.

Previous SectionNext Section

Acknowledgments

Computer facilities were provided by the Centre de Computació i Comunicacions de Catalunya.

Previous SectionNext Section

Footnotes

  • This work was supported in part by grants from Dirección General de Investigación Cientı́fica y Tecnológica (PB97-0282), Comisión Interministerial de Ciencia y Tecnologica (SAF98–0064-C02 and SAF99-0073), Comunidad de Madrid (08.5/0079/2000), Universidad del Pais Vasco (G15/98), and Fundació La MaratóTV3 (0014/97).

  • Abbreviations:
    GPCRs
    G protein coupled receptors
    3-D
    three-dimensional
    RHO
    rhodopsin
    TMH
    transmembrane helix
    5-HT1AR
    5-hydroxytryptamine1A receptor
    EF-7412
    2-[4-[4(m-ethylsulfonamido)-phenyl)piperazin-1-yl[butyl]-1,3-dioxoperhydropyrrolo[1,2-c]imidazole
    • Received September 27, 2001.
    • Accepted March 21, 2002.
Previous Section
 

References

  1. ↵
    1. Arnis S,
    2. Fahmy K,
    3. Hofmann KP,
    4. Sakmar TP
    (1994) A conserved carboxylic acid group mediates light-dependent proton uptake and signaling by rhodopsin. J Biol Chem 269:23879–23881.
    Abstract/FREE Full Text
  2. ↵
    1. Baldwin JM,
    2. Schertler GFX,
    3. Unger VM
    (1997) An alpha-carbon template for the transmembrane helices in the rhodopsin family of G-protein-coupled receptors. J Mol Biol 272:144–164.
    CrossRefMedlineWeb of Science
  3. ↵
    1. Ballesteros JA,
    2. Deupi X,
    3. Olivella M,
    4. Haaksma EEJ,
    5. Pardo L
    (2000) Serine and threonine residues bend α-helices in the χ1= g− conformation. Biophys J 79:2754–2760.
    Medline
  4. ↵
    1. Ballesteros JA,
    2. Shi L,
    3. Javitch JA
    (2001) Structural mimicry in G protein-coupled receptors: implications of the high-resolution structure of rhodopsin for structure-function analysis of rhodopsin-like receptors. Mol Pharmacol 60:1–19.
    Abstract/FREE Full Text
  5. ↵
    1. Ballesteros JA,
    2. Weinstein H
    (1995) Integrated methods for modeling G-protein coupled receptors. Methods Neurosci 25:366–428.
    CrossRef
  6. ↵
    1. Bourne HR,
    2. Meng EC
    (2000) Structure. Rhodopsin sees the light. Science (Wash DC) 289:733–734.
    FREE Full Text
  7. ↵
    1. Cavalli A,
    2. Fanelli F,
    3. Taddei C,
    4. De Benedetti PG,
    5. Cotecchia S
    (1996) Amino acids of the alpha1B-adrenergic receptor involved in agonist binding: differences in docking catecholamines to receptor subtypes. FEBS Lett 399:9–13.
    CrossRefMedlineWeb of Science
  8. ↵
    1. Cheng YC,
    2. Prusoff WH
    (1973) Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol 22:3099–3108.
    CrossRefMedlineWeb of Science
  9. ↵
    1. Cieplak P,
    2. Cornell WD,
    3. Bayly C,
    4. Kollman PA
    (1995) Application of the multimolecule and multiconformational RESP methodology to biopolymers: charge derivation for DNA, RNA and proteins. J Comput Chem 16:1357–1377.
    CrossRefWeb of Science
  10. ↵
    1. Clark RD,
    2. Weinhardt KK,
    3. Berger J,
    4. Fischer LE,
    5. Brown CM,
    6. MacKinnon A,
    7. Kilpatrick AT,
    8. Spedding M
    (1990) 1,9-Alkano-bridged 2,3,4,5-tetrahydro-1H-3-benzazepines with affinity for the α2-adrenoceptor and the 5-HT1A receptor. J Med Chem 33:633–641.
    CrossRefMedline
  11. ↵
    1. Cornell WD,
    2. Cieplak P,
    3. Bayly CI,
    4. Gould IR,
    5. Merz KM, Jr,
    6. Ferguson DM,
    7. Spellmeyer DC,
    8. Fox T,
    9. Caldwell JW,
    10. Kollman PA
    (1995) A second generation force field for the simulation of proteins, nucleic acids and organic molecules. J Am Chem Soc 117:5179–5197.
    CrossRef
  12. ↵
    1. Dunbrack RLJ,
    2. Cohen FE
    (1997) Bayesian statistical analysis of protein sidechain rotamer preferences. Protein Sci 6:1661–1681.
    MedlineWeb of Science
  13. ↵
    1. Elling CE,
    2. Nielsen SM,
    3. Schwartz TW
    (1995) Conversion of antagonist-binding site to metal-ion site in the tachykinin NK-1 receptor. Nature (Lond) 374:74–77.
    CrossRefMedline
  14. ↵
    1. Elling CE,
    2. Thirstrup K,
    3. Holst B,
    4. Schwartz TW
    (1999) Conversion of agonist site to metal-ion chelator site in the β2-adrenergic receptor. Proc Natl Acad Sci USA 96:12322–12327.
    Abstract/FREE Full Text
  15. ↵
    1. Glennon RA,
    2. Dukat M,
    3. Westkaemper RB,
    4. Ismaiel AM,
    5. Izzarelli DG,
    6. Parker EM
    (1996) The binding of propanolol at 5-hydroxytryptamine1Dβ T355N mutant receptors may involve formation of two hydrogen bonds to asparagine. Mol Pharmacol 49:198–206.
    Abstract
  16. ↵
    1. Govaerts C,
    2. Blanpain C,
    3. Deupi X,
    4. Ballet S,
    5. Ballesteros JA,
    6. Wodak S,
    7. Vassart G,
    8. Pardo L,
    9. Parmentier M
    (2001a) The TxP motif in the second transmembrane helix of CCR5: a structural determinant in chemokine-induced activation. J Biol Chem 276:13217–13225.
    Abstract/FREE Full Text
  17. ↵
    1. Govaerts C,
    2. Lefort A,
    3. Costagliola S,
    4. Wodak S,
    5. Ballesteros JA,
    6. Pardo L,
    7. Vassart G
    (2001b) A conserved Asn in TM7 is a on/off switch in the activation of the TSH receptor. J Biol Chem 276:22991–22999.
    Abstract/FREE Full Text
  18. ↵
    1. Gray TM,
    2. Matthews BW
    (1984) Intrahelical hydrogen bonding of serine, threonine and cysteine residues within alpha-helices and its relevance to membrane-bound proteins. J Mol Biol 175:75–81.
    CrossRefMedlineWeb of Science
  19. ↵
    1. Guan X-M,
    2. Peroutka SJ,
    3. Kobilka BK
    (1992) Identification of a single amino acid residue responsible for the binding of a class of β-adrenergic receptor antagonists to 5-HT1A receptors. Mol Pharmacol 41:695–698.
    Abstract
  20. ↵
    1. Han M,
    2. Lou J,
    3. Nakanishi K,
    4. Sakmar TP,
    5. Smith SO
    (1997) Partial agonist activity of 11-cis-retinal in rhodopsin mutants. J Biol Chem 272:23081–23085.
    Abstract/FREE Full Text
  21. ↵
    1. Ho BY,
    2. Karschin A,
    3. Branchek T,
    4. Davidson N,
    5. Lester HA
    (1992) The role of conserved aspartate and serine residues in ligand binding and in function of the 5-HT1A receptor: a site-directed mutation study. FEBS Lett 312:259–262.
    CrossRefMedlineWeb of Science
  22. ↵
    1. Horn F,
    2. Weare J,
    3. Beukers MW,
    4. Hörsch S,
    5. Bairoch A,
    6. Chen W,
    7. Edvardsen Ø,
    8. Campagne F,
    9. Vriend G
    (1998) GPCRDB: an information system for G protein coupled receptors. Nucleic Acids Res 26:277–281.
  23. ↵
    1. Hosmer DW,
    2. Lemeshow S
    (1989) Applied Logistic Regression. (John Wiley & Sons, Inc. New York).
  24. ↵
    1. Ji TH,
    2. Grossmann M,
    3. Ji I
    (1998) G protein-coupled receptors. I. Diversity of receptor-ligand interactions. J Biol Chem 273:17299–17302.
    FREE Full Text
  25. ↵
    1. Kelley LA,
    2. Gardner SP,
    3. Sutcliffe MJ
    (1996) An automated approach for clustering an ensemble of NMR-derived protein structures into conformationally-related subfamilies. Protein Eng 9:1063–1065.
    FREE Full Text
  26. ↵
    1. Kraulis J
    (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structure. J Appl Cryst 24:946–950.
  27. ↵
    1. Kumar S,
    2. Bansal M
    (1998) Geometrical and sequence characteristics of α-helices in globular proteins. Biophys J 75:1935–1944.
    MedlineWeb of Science
  28. ↵
    1. Liapakis G,
    2. Ballesteros JA,
    3. Papachristou S,
    4. Chan WC,
    5. Chen X,
    6. Javitch JA
    (2000) The forgotten serine. A critical role for Ser-2035.42 in ligand binding to and activation of the β2-adrenergic receptor. J Biol Chem 275:37779–37788.
    Abstract/FREE Full Text
  29. ↵
    1. Lopez-Rodriguez ML,
    2. Morcillo MJ,
    3. Fernandez E,
    4. Porras E,
    5. Orensanz L,
    6. Beneytez ME,
    7. Manzanares J,
    8. Fuentes JA
    (2001a) Synthesis and structure-activity relationships of a new model of arylpiperazines. 5. Study of the physicochemical influence of the pharmacophore on 5-HT1A/α1-adrenergic receptor affinity: synthesis of a new derivative with mixed 5-HT1A/D2 antagonist properties. J Med Chem 44:186–197.
    CrossRefMedline
  30. ↵
    1. Lopez-Rodriguez ML,
    2. Morcillo MJ,
    3. Fernandez E,
    4. Rosado ML,
    5. Pardo L,
    6. Schaper K-J
    (2001b) Synthesis and structure-activity relationships of a new model of arylpiperazines. 6. Study of the 5-HT1A/α1-adrenergic receptor affinity by classical Hansch analysis, artificial neural networks and computational simulation of ligand recognition. J Med Chem 44:198–207.
    CrossRefMedline
  31. ↵
    1. Lopez-Rodriguez ML,
    2. Rosado ML,
    3. Benhamú B,
    4. Morcillo MJ,
    5. Sanz AM,
    6. Orensanz L,
    7. Beneytez ME,
    8. Fuentes JA,
    9. Manzanares J
    (1996) Synthesis and structure-activity relationships of a new model of arylpiperazines. 1. 2-[[4-(o-Methoxyphenyl)piperazin-1-yl]methyl]-1,3-dioxoperhydroimidazo[1,5-a]pyridine: a selective 5-HT1A receptor agonist. J Med Chem 39:4439–4450.
    CrossRefMedline
  32. ↵
    1. Lowry OH,
    2. Rosebrough NJ,
    3. Farr AL,
    4. Randall RJ
    (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275.
    FREE Full Text
  33. ↵
    1. Merritt EA,
    2. Bacon DJ
    (1997) Raster3D: photorealistic molecular graphics. Methods Enzymol 277:505–524.
    MedlineWeb of Science
  34. ↵
    1. O'Neil KT,
    2. DeGrado WF
    (1990) A thermodynamic scale for the helix-forming tendencies of the commonly occurring amino acids. Science (Wash DC) 250:646–651.
    Abstract/FREE Full Text
  35. ↵
    1. Oliveira L,
    2. Paiva ACM,
    3. Sander C,
    4. Vriend G
    (1994) A common step for signal transduction in G-protein coupled receptors. Trends Pharmacol Sci 15:170–172.
    CrossRefMedline
  36. ↵
    1. Palczewski K,
    2. Kumasaka T,
    3. Hori T,
    4. Behnke CA,
    5. Motoshima H,
    6. Fox BA,
    7. LeTrong I,
    8. Teller DC,
    9. Okada T,
    10. Stenkamp RE,
    11. et al.
    (2000) Crystal structure of rhodopsin: a G protein-coupled receptor. Science (Wash DC) 289:739–745.
    Abstract/FREE Full Text
  37. ↵
    1. Pavia ML,
    2. Taylor CP,
    3. Hershenson FM,
    4. Lobbestael SJ
    (1987) 6-Alkoxy-N,N-disubstituted-2-pyridinamines as anticonvulsant agents. J Med Chem 30:1210–1214.
    CrossRefMedline
  38. ↵
    1. Scheer A,
    2. Fanelli F,
    3. Costa T,
    4. De Benedetti PG,
    5. Cotecchia S
    (1996) Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation. EMBO (Eur Mol Biol Organ) J 15:3566–3578.
    MedlineWeb of Science
  39. ↵
    1. Senes A,
    2. Gerstein M,
    3. Engelman DM
    (2000) Statistical analysis of amino acid patterns in transmembrane helices: the GxxxG motif occurs frequently and in association with β-branched residues at neighboring positions. J Mol Biol 296:921–936.
    CrossRefMedlineWeb of Science
  40. ↵
    1. Strader CD,
    2. Candelore MR,
    3. Hill WS,
    4. Dixon RA,
    5. Sigal IS
    (1989) A single amino acid substitution in the beta-adrenergic receptor promotes partial agonist activity from antagonists. J Biol Chem 264:16470–16477.
    Abstract/FREE Full Text
  41. ↵
    1. Strader CD,
    2. Sigal IS,
    3. Candelore MR,
    4. Rands MR,
    5. Hill WS,
    6. Dixon RAF
    (1988) Conserved aspartic acid residues 79 and 113 of the β-adrenergic receptor have different roles in receptor function. J Biol Chem 263:10267–10271.
    Abstract/FREE Full Text
  42. ↵
    1. Suryanarayana S,
    2. Daunt DA,
    3. Zastrow MV,
    4. Kobilka BK
    (1991) A point mutation in the seventh hydrophobic domain of the α2-adrenergic receptor increases its affinity for a family of β receptor antagonists. J Biol Chem 266:15488–15492.
    Abstract/FREE Full Text
  43. ↵
    1. Suryanarayana S,
    2. Kobilka BK
    (1993) Amino acid substitutions at position 312 in the seventh hydrophobic segment of the β2-adrenergic receptor modify ligand-binding specificity. Mol Pharmacol 44:111–114.
    Abstract
  44. ↵
    1. Unger VM,
    2. Hargrave PA,
    3. Baldwin JM,
    4. Schertler GF
    (1997) Arrangement of rhodopsin transmembrane alpha-helices. Nature (Lond) 389:203–206.
    CrossRefMedline
  45. ↵
    1. van Rhee AM,
    2. Jacobson KA
    (1996) Molecular architecture of G protein-coupled receptors. Drug Devel Res 37:1–38.
  46. ↵
    1. Yang K,
    2. Farrens DL,
    3. Attenbach C,
    4. Farahbakhsh ZT,
    5. Hubbell WL,
    6. Khorana HG
    (1996) Structure and function in rhodopsin. Cysteines 65 and 316 are in proximity in a rhodopsin mutant as indicated by disulfide formation and interactions between attached spin labels. Biochemistry 35:14040–14046.
    CrossRefMedline
  47. ↵
    1. Yu H,
    2. Kono M,
    3. McKee TD,
    4. Oprian DD
    (1995) A general method for mapping tertiary contacts between amino acid residues in membrane-embedded proteins. Biochemistry 34:14963–14969.
    CrossRefMedline
  48. ↵
    1. Zhou W,
    2. Flanagan C,
    3. Ballesteros JA,
    4. Konvicka K,
    5. Davidson JS,
    6. Weinstein H,
    7. Millar RP,
    8. Sealfon SC
    (1994) A reciprocal mutation supports helix 2 and helix 7 proximity in the gonadotropin-releasing hormone receptor. Mol Pharmacol 45:165–170.
    Abstract
« Previous | Next Article »Table of Contents

This Article

  1. doi: 10.1124/mol.62.1.15 Molecular Pharmacology July 1, 2002 vol. 62 no. 1 15-21
  1. AbstractFree
  2. » Full Text
  3. Full Text (PDF)

Classifications

Responses

  1. Submit a response
  2. No responses published

Navigate This Article

Current Issue

  1. November 2009, 76 (5)
  1. Current Issue
  1. Alert me to new issues of Molecular Pharmacology