Biomedical subjects

J A Ballesteros

Publications and source records attributed to J A Ballesteros.

At least 19 recordsLinked to original sources

PubMed2001

The first transmembrane segment of the dopamine D2 receptor: accessibility in the binding-site crevice and position in the transmembrane bundle.

The binding site of the dopamine D2 receptor, like that of homologous G-protein-coupled receptors (GPCRs), is contained within a water-accessible crevice formed among its seven transmembrane segments (TMs). Using the substituted-cysteine-accessibility method (SCAM), we are mapping the residues that contribute to the surface of this binding-site crevice. We have now mutated to cysteine, one at a time, 21 consecutive residues in TM1. Six of these mutants reacted with charged sulfhydryl reagents, whereas bound antagonist only protected N52(1.50)C from reaction. Except for A38(1.36)C, none of the substituted cysteine mutants in the extracellular half of TM1 appeared to be accessible. Pro(1.48) is highly conserved in opsins, but absent in catecholamine receptors, and the high-resolution rhodopsin structure showed that Pro(1.48) bends the extracellular portion of TM1 inward toward TM2 and TM7. Analysis of the conversation of residues in the extracellular portion of TM1 of opsins showed a pattern consistent with alpha-helical structure with a conserved face. In contrast, this region in catecholamine receptors is poorly conserved, suggesting a lack of critical contacts. Thus, in catecholamine receptors in the absence of Pro(1.48), TM1 may be straighter and therefore further from the helix bundle, consistent with the apparent lack of conserved contact residues. When examined in the context of a model of the D2 receptor, the accessible residues in the cytoplasmic half of TM1 are at the interface with TM7 and with helix 8 (H8). We propose the existence of critical contacts of TM1, TM7, and H8 that may stabilize the inactive state of the receptor.

Amino Acid Substitution↗

PubMed2001

Activation of the beta 2-adrenergic receptor involves disruption of an ionic lock between the cytoplasmic ends of transmembrane segments 3 and 6.

The movements of transmembrane segments (TMs) 3 and 6 at the cytoplasmic side of the membrane play an important role in the activation of G-protein-coupled receptors. Here we provide evidence for the existence of an ionic lock that constrains the relative mobility of the cytoplasmic ends of TM3 and TM6 in the inactive state of the beta(2)-adrenergic receptor. We propose that the highly conserved Arg-131(3.50) at the cytoplasmic end of TM3 interacts both with the adjacent Asp-130(3.49) and with Glu-268(6.30) at the cytoplasmic end of TM6. Such a network of ionic interactions has now been directly supported by the high-resolution structure of the inactive state of rhodopsin. We hypothesized that the network of interactions would serve to constrain the receptor in the inactive state, and the release of this ionic lock could be a key step in receptor activation. To test this hypothesis, we made charge-neutralizing mutations of Glu-268(6.30) and of Asp-130(3.49) in the beta(2)-adrenergic receptor. Alone and in combination, we observed a significant increase in basal and pindolol-stimulated cAMP accumulation in COS-7 cells transiently transfected with the mutant receptors. Moreover, based on the increased accessibility of Cys-285(6.47) in TM6, we provide evidence for a conformational rearrangement of TM6 that is highly correlated with the extent of constitutive activity of the different mutants. The present experimental data together with the recent high-resolution structure of rhodopsin suggest that ionic interactions between Asp/Glu(3.49), Arg(3.50), and Glu(6.30) may constitute a common switch governing the activation of many rhodopsin-like G-protein-coupled receptors.

Adrenergic beta-Agonists↗

PubMed2001

A conserved Asn in transmembrane helix 7 is an on/off switch in the activation of the thyrotropin receptor.

The thyrotropin (TSH) receptor is an interesting model to study G protein-coupled receptor activation as many point mutations can significantly increase its basal activity. Here, we identified a molecular interaction between Asp(633) in transmembrane helix 6 (TM6) and Asn(674) in TM7 of the TSHr that is crucial to maintain the inactive state through conformational constraint of the Asn. We show that these residues are perfectly conserved in the glycohormone receptor family, except in one case, where they are exchanged, suggesting a direct interaction. Molecular modeling of the TSHr, based on the high resolution structure of rhodopsin, strongly favors this hypothesis. Our approach combining site-directed mutagenesis with molecular modeling shows that mutations disrupting this interaction, like the D633A mutation in TM6, lead to high constitutive activation. The strongly activating N674D (TM7) mutation, which in our modeling breaks the TM6-TM7 link, is reverted to wild type-like behavior by an additional D633N mutation (TM6), which would restore this link. Moreover, we show that the Asn of TM7 (conserved in most G protein-coupled receptors) is mandatory for ligand-induced cAMP accumulation, suggesting an active role of this residue in activation. In the TSHr, the conformation of this Asn residue of TM7 would be constrained, in the inactive state, by its Asp partner in TM6.

Animals↗

PubMed2001

The TXP motif in the second transmembrane helix of CCR5. A structural determinant of chemokine-induced activation.

CCR5 is a G-protein-coupled receptor activated by the chemokines RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein 1alpha and 1beta, and monocyte chemotactic protein 2 and is the main co-receptor for the macrophage-tropic human immunodeficiency virus strains. We have identified a sequence motif (TXP) in the second transmembrane helix of chemokine receptors and investigated its role by theoretical and experimental approaches. Molecular dynamics simulations of model alpha-helices in a nonpolar environment were used to show that a TXP motif strongly bends these helices, due to the coordinated action of the proline, which kinks the helix, and of the threonine, which further accentuates this structural deformation. Site-directed mutagenesis of the corresponding Pro and Thr residues in CCR5 allowed us to probe the consequences of these structural findings in the context of the whole receptor. The P84A mutation leads to a decreased binding affinity for chemokines and nearly abolishes the functional response of the receptor. In contrast, mutation of Thr-82(2.56) into Val, Ala, Cys, or Ser does not affect chemokine binding. However, the functional response was found to depend strongly on the nature of the substituted side chain. The rank order of impairment of receptor activation is P84A > T82V > T82A > T82C > T82S. This ranking of impairment parallels the bending of the alpha-helix observed in the molecular simulation study.

Amino Acid Sequence↗

PubMed2001

Structural mimicry in G protein-coupled receptors: implications of the high-resolution structure of rhodopsin for structure-function analysis of rhodopsin-like receptors.

The availability of a high-resolution structure of rhodopsin now allows us to reconsider research attempts to understand structure-function relationships in other G protein-coupled receptors (GPCRs). A comparison of the rhodopsin structure with the results of previous sequence analysis and molecular modeling that incorporated experimental results demonstrates a high degree of success for these methods in predicting the helix ends and protein-protein interface of GPCRs. Moreover, the amino acid residues inferred to form the surface of the binding-site crevice based on our application of the substituted-cysteine accessibility method in the dopamine D(2) receptor are in remarkable agreement with the rhodopsin structure, with the notable exception of some residues in the fourth transmembrane segment. Based on our analysis of the data reviewed, we propose that the overall structures of rhodopsin and of amine receptors are very similar, although we also identified localized regions where the structure of these receptors may diverge. We further propose that several of the highly unusual structural features of rhodopsin are also present in amine GPCRs, despite the absence of amino acids that might have thought to have been critical to the adoption of these features. Thus, different amino acids or alternate microdomains can support similar deviations from regular alpha-helical structure, thereby resulting in similar tertiary structure. Such structural mimicry is a mechanism by which a common ancestor could diverge sufficiently to develop the selectivity necessary to interact with diverse signals, while still maintaining a similar overall fold. Through this process, the core function of signaling activation through a conformational change in the transmembrane segments that alters the conformation of the cytoplasmic surface and subsequent interaction with G proteins is presumably shared by the entire Class A family of receptors, despite their selectivity for a diverse group of ligands.

Amino Acid Sequence↗

PubMed2000

Agonist-induced conformational changes at the cytoplasmic side of transmembrane segment 6 in the beta 2 adrenergic receptor mapped by site-selective fluorescent labeling.

The environmentally sensitive, sulfhydryl-reactive, fluorescent probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene-diamine (IANBD) was used as a molecular reporter of agonist-induced conformational changes in the beta(2) adrenergic receptor, a prototype hormone-activated G protein-coupled receptor. In the background of a mutant beta(2) adrenergic receptor, with a minimal number of endogenous cysteine residues, new cysteines were introduced in positions 269(6.31), 270(6.32), 271(6.33), and 272(6.34) at the cytoplasmic side of transmembrane segment (TM) 6. The resulting mutant receptors were fully functional and bound both agonists and antagonist with high affinities also upon IANBD labeling. Fluorescence spectroscopy analysis of the purified and site-selectively IANBD-labeled mutants suggested that the covalently attached fluorophore was exposed to a less polar environment at all four positions upon agonist binding. Whereas evidence for only a minor change in the molecular environment was obtained for positions 269(6.31) and 270(6.32), the full agonist isoproterenol caused clear dose-dependent and reversible increases in fluorescence emission at positions 271(6.33) and 272(6.34). The data suggest that activation of G protein-coupled receptors, which are activated by "diffusible" ligands, involves a structural rearrangement corresponding to the cytoplasmic part of TM 6. The preferred conformations of the IANBD moiety attached to the inserted cysteines were predicted by employing a computational method that incorporated the complex hydrophobic/hydrophilic environment in which the cysteines reside. Based on these preferred conformations, it is suggested that the spectral changes reflect an agonist-promoted movement of the cytoplasmic part of TM 6 away from the receptor core and upwards toward the membrane bilayer.

Adrenergic beta-Agonists↗

PubMed2000

The forgotten serine. A critical role for Ser-2035.42 in ligand binding to and activation of the beta 2-adrenergic receptor.

Previous work in the beta(2)-adrenergic receptor demonstrated critical interactions between Ser-204 and Ser-207 in the fifth membrane-spanning segment and the meta-OH and para-OH, respectively, of catecholamine agonists (Strader, C. D., Candelore, M. R., Hill, W. S., Sigal, I. S., and Dixon, R. A. (1989) J. Biol. Chem. 264, 13572-13578). Using the substituted cysteine accessibility method in the beta(2)-adrenergic receptor, we have found that in addition to Ser-204 and Ser-207, Ser-203 is also accessible on the surface of the binding-site crevice and is occluded by bound agonist. Mutation of Ser-203 to Ala, Val, or Cys reduced the binding affinity and adenylyl cyclase-activating potency of agonists containing a meta-OH, whereas their affinities and potencies were largely preserved by mutation of Ser-203 to Thr, which maintained an OH at this position. Thus both Ser-203 and Ser-204 appear to interact with the meta-OH of catecholamines, perhaps through a bifurcated H bond. Furthermore, the removal of the OH at position 203 led to a significant loss of affinity of antagonists with nitrogen in their heterocyclic ring structure. The greatest effect was seen with pindolol, a partial agonist, suggesting that a H bond between the heterocyclic ring and Ser-203 may play a role in partial agonism. In contrast, the affinities of antagonists such as propranolol or alprenolol, which have cyclic structures without H-bonding capability, were unaltered after mutation of Ser-203.

Adrenergic beta-2 Receptor Antagonists↗

PubMed2000

The fourth transmembrane segment of the dopamine D2 receptor: accessibility in the binding-site crevice and position in the transmembrane bundle.

The binding site of the dopamine D2 receptor, like that of homologous G-protein-coupled receptors (GPCRs), is contained within a water-accessible crevice formed among its seven transmembrane segments (TMSs). Using the substituted-cysteine-accessibility method (SCAM), we are mapping the residues that contribute to the surface of this binding-site crevice. We have mutated to cysteine, one at a time, 21 consecutive residues in the fourth TMS (TM4). Eleven of these mutants reacted with charged sulfhydryl-specific reagents, and bound antagonist protected nine of these from reaction. For the mutants in which cysteine was substituted for residues in the cytoplasmic half of TM4, treatment with the reagents had no effect on binding, consistent with these residues being inaccessible and with the low-resolution structure of the homologous rhodopsin, in which TM3 and TM5 occlude the cytoplasmic half of TM4. Although hydrophobicity analysis positions the C-terminus of TM4 at 4.64, Pro-Pro and Pro-X-Pro motifs, which are known to disrupt alpha-helices, occur at position 4.59 in a number of homologous GPCRs. The SCAM data were consistent with a C-terminus at 4.58, but it is also possible that the alpha-helix extends one additional turn to 4.62 in the D2 receptor, which has a single Pro at 4.59. In homologous GPCRs, the high degree of sequence variation between 4.59 and 4.68 is more characteristic of a loop domain than a helical segment. This region is shown here to be very conserved within functionally related receptors, suggesting an important functional role for this putative nonhelical domain. This inference is supported by observed ligand-specific effects of mutations in this region and by the predicted spatial proximity of this segment to known ligand binding sites in other TMs.

Amino Acid Sequence↗

PubMed2000

Serine and threonine residues bend alpha-helices in the chi(1) = g(-) conformation.

The relationship between the Ser, Thr, and Cys side-chain conformation (chi(1) = g(-), t, g(+)) and the main-chain conformation (phi and psi angles) has been studied in a selection of protein structures that contain alpha-helices. The statistical results show that the g(-) conformation of both Ser and Thr residues decreases their phi angles and increases their psi angles relative to Ala, used as a control. The additional hydrogen bond formed between the O(gamma) atom of Ser and Thr and the i-3 or i-4 peptide carbonyl oxygen induces or stabilizes a bending angle in the helix 3-4 degrees larger than for Ala. This is of particular significance for membrane proteins. Incorporation of this small bending angle in the transmembrane alpha-helix at one side of the cell membrane results in a significant displacement of the residues located at the other side of the membrane. We hypothesize that local alterations of the rotamer configurations of these Ser and Thr residues may result in significant conformational changes across transmembrane helices, and thus participate in the molecular mechanisms underlying transmembrane signaling. This finding has provided the structural basis to understand the experimentally observed influence of Ser residues on the conformational equilibrium between inactive and active states of the receptor, in the neurotransmitter subfamily of G protein-coupled receptors.

Animals↗

PubMed1999

Electrostatic and aromatic microdomains within the binding-site crevice of the D2 receptor: contributions of the second membrane-spanning segment.

The binding-site of the dopamine D2 receptor, like that of other homologous G protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Using the substituted cysteine accessibility method (SCAM), we previously mapped the residues in the third, fifth, sixth, and seventh membrane-spanning segments that contribute to the surface of this binding-site crevice. We have now mutated to cysteine, one at a time, 22 consecutive residues in the second membrane-spanning segment (M2) and expressed the mutant receptors in HEK 293 cells. Eleven of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and 9 of these 11 were protected from reaction by a reversible dopamine antagonist, sulpiride. We infer that the side chains of the residues at the 11 reactive loci (D80, L81, V83, V87, P89, W90, V91, V92, L94, E95, V96) are on the water-accessible surface of the binding-site crevice and that 9 of these are occluded by bound antagonist. The pattern of accessibility suggests an alpha-helical conformation. A broadening of the angle of accessibility near the binding site is consistent with the presence of a kink at Pro89. On the basis of the enhanced rates of reaction of positively charged sulfhydryl reagents, we infer the presence of an electrostatic microdomain composed of three acidic residues in M2 and the adjacent M3 that could attract and orient cationic ligands. Furthermore, based on the enhanced reactivity of the hydrophobic cation-containing reagent, we infer the presence of an aromatic microdomain formed between M2, M3, and M7.

Amino Acid Substitution↗

PubMed1999

The role of a conserved proline residue in mediating conformational changes associated with voltage gating of Cx32 gap junctions.

We have explored the role of a proline residue located at position 87 in the second transmembrane segment (TM2) of gap junctions in the mechanism of voltage-dependent gating of connexin32 (Cx32). Substitution of this proline (denoted Cx32P87) with residues G, A, or V affects channel function in a progressive manner consistent with the expectation that a proline kink (PK) motif exists in the second transmembrane segment (TM2) of this connexin. Mutations of the preceding threonine residue T86 to S, A, C, V, N, or L shift the conductance-voltage relation of wild-type Cx32, such that the mutated channels close at smaller transjunctional voltages. The observed shift in voltage dependence is consistent with a reduction in the open probability of the mutant hemichannels at a transjunctional voltage (Vj) of 0 mV. In both cases in which kinetics were examined, the time constants for reaching steady state were faster for T86N and T86A than for wild type at comparable voltages, suggesting that the T86 mutations cause the energetic destabilization of the open state relative to the other states of the channel protein. The structural underpinnings of the observed effects were explored with Monte Carlo simulations. The conformational space of TM2 helices was found to differ for the T86A, V, N, and L mutants, which produce a less bent helix ( approximately 20 degrees bend angle) compared to the wild type, which has a approximately 37 degrees bend angle. The greater bend angle of the wild-type helix reflects the propensity of the T86 residue to hydrogen bond with the backbone carbonyl of amino acid residue I82. The relative differences in propensity for hydrogen bonding of the mutants relative to the wild-type threonine residue in the constructs we studied (T86A, V, N, L, S, and C) correlate with the shift in the conductance-voltage relation observed for T86 mutations. The data are consistent with a structural model in which the open conformation of the Cx32 channel corresponds to a more bent TM2 helix, and the closed conformation corresponds to a less bent helix. We propose that the modulation of the hydrogen-bonding potential of the T86 residue alters the bend angle of the PK motif and mediates conformational changes between open and closed channel states.

Animals↗

PubMed1999

Dopamine D4/D2 receptor selectivity is determined by A divergent aromatic microdomain contained within the second, third, and seventh membrane-spanning segments.

Conserved features of the sequences of dopamine receptors and of homologous G-protein-coupled receptors point to regions, and amino acid residues within these regions, that contribute to their ligand binding sites. Differences in binding specificities among the catecholamine receptors, however, must stem from their nonconserved residues. Using the substituted-cysteine accessibility method, we have identified the residues that form the surface of the water-accessible binding-site crevice in the dopamine D2 receptor. Of approximately 80 membrane-spanning residues that differ between the D2 and D4 receptors, only 20 were found to be accessible, and 6 of these 20 are conservative aliphatic substitutions. In a D2 receptor background, we mutated the 14 accessible, nonconserved residues, individually or in combinations, to the aligned residues in the D4 receptor. We also made the reciprocal mutations in a D4 receptor background. The combined substitution of four to six of these residues was sufficient to switch the affinity of the receptors for several chemically distinct D4-selective antagonists by three orders of magnitude in both directions (D2- to D4-like and D4- to D2-like). The mutated residues are in the second, third, and seventh membrane-spanning segments (M2, M3, M7) and form a cluster in the binding-site crevice. Mutation of a single residue in this cluster in M2 was sufficient to increase the affinity for clozapine to D4-like levels. We can rationalize the data in terms of a set of chemical moieties in the ligands interacting with a divergent aromatic microdomain in M2-M3-M7 of the D2 and D4 receptors.

Amino Acid Sequence↗

PubMed1998

A cluster of aromatic residues in the sixth membrane-spanning segment of the dopamine D2 receptor is accessible in the binding-site crevice.

The binding site of the dopamine D2 receptor, like that of other homologous G protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Using the substituted-cysteine accessibility method, we previously mapped the residues in the third, fifth, and seventh membrane-spanning segments that contribute to the surface of this binding-site crevice. We have now mutated to cysteine, one at a time, 22 consecutive residues in the sixth membrane-spanning segment (M6) and expressed the mutant receptors in HEK 293 cells. Ten of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and all but one were protected from reaction by a reversible dopamine antagonist, sulpiride. Thus, we infer that the side chains of the residues at the reactive loci (V378, F382, W386, P388, F389, F390, T392, H393, I394, and I397) are on the water-accessible surface of the binding-site crevice. The pattern of accessibility is consistent with an alpha-helical conformation with a wide angle of accessibility near the binding site itself and a narrower stripe continuing toward the cytoplasmic portion of the binding-site crevice. This pattern of accessibility is consistent with the presence of a proline kink which could bend the extracellular portion of M6 into the binding-site crevice where it would be more broadly accessible than the cytoplasmic portion of the membrane-spanning segment. Four highly conserved aromatic residues and a histidine are clustered together on the water-accessible surface of the binding-site crevice. They define an interconnected "aromatic cluster" that may be involved in ligand binding and receptor activation.

Amino Acid Sequence↗

PubMed1998

A proposed structure for transmembrane segment 7 of G protein-coupled receptors incorporating an asn-Pro/Asp-Pro motif.

Transmembrane segment (TMS) 7 has been shown to play an important role in the signal transduction function of G-protein-coupled receptors (GPCRs). Although transmembrane segments are most likely to adopt a helical structure, results from a variety of experimental studies involving TMS 7 are inconsistent with it being an ideal alpha-helix. Using results from a search of the structure database and extensive simulated annealing Monte Carlo runs with the new Conformational Memories method, we have identified the conserved (N/D)PxxY region of TMS 7 as the major determinant for deviation of TMS 7 from ideal helicity. The perturbation consists of an Asx turn and a flexible "hinge" region. The Conformational Memories procedure yielded a model structure of TMS 7 which, unlike an ideal alpha-helix, is capable of accommodating all of the experimentally derived geometrical criteria for the interactions of TMS 7 in the transmembrane bundle of GPCRs. In the context of the entire structure of a transmembrane bundle model for the 5HT2a receptor, the specific perturbation of TMS 7 by the NP sequence suggests a structural hypothesis for the pattern of amino acid conservation observed in TMS 1, 2, and 7 of GPCRs. The structure resulting from the incorporation of the (N/D)P motif satisfies fully the H-bonding capabilities of the 100% conserved polar residues in these TMSs, in agreement with results from mutagenesis experiments. The flexibility introduced by the specific structural perturbation produced by the (NP/DP) motif in TMS 7 is proposed to have a significant role in receptor activation.

Amino Acid Sequence↗

PubMed1997

Agonists induce conformational changes in transmembrane domains III and VI of the beta2 adrenoceptor.

Agonist binding to G protein-coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that agonist binding to the beta2 adrenoceptor induces a conformational change around 125Cys in transmembrane domain (TM) III and around 285Cys in TM VI. A series of mutant beta2 adrenoceptors with a limited number of cysteines available for chemical derivatization were purified, site-selectively labeled with the conformationally sensitive, cysteine-reactive fluorophore IANBD and analyzed by fluorescence spectroscopy. Like the wild-type receptor, mutant receptors containing 125Cys and/or 285Cys showed an agonist-induced decrease in fluorescence, while no agonist-induced response was observed in a receptor where these two cysteines were mutated. These data suggest that IANBD bound to 125Cys and 285Cys are exposed to a more polar environment upon agonist binding, and indicate that movements of transmembrane segments III and VI are involved in activation of G protein-coupled receptors.

Adrenergic beta-Agonists↗

PubMed1997

Structural instability of a constitutively active G protein-coupled receptor. Agonist-independent activation due to conformational flexibility.

Mutations in several domains can lead to agonist-independent, constitutive activation of G protein-coupled receptors. However, the nature of the structural and molecular changes that constitutively turn on a G protein-coupled receptor remains unknown. Here we show evidence that a constitutively activated mutant of the beta2 adrenergic receptor (CAM) is characterized by structural instability and an exaggerated conformational response to ligand binding. The structural instability of CAM could be demonstrated by a 4-fold increase in the rate of denaturation of purified receptor at 37 degrees C as compared with the wild type receptor. Spectroscopic analysis of purified CAM labeled with the conformationally sensitive and cysteine-reactive fluorophore, N,N'dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)ethylenediamine, further indicated that both agonist and antagonist elicit more profound structural changes in CAM than in the wild type protein. We propose that the mutation that confers constitutive activity to the beta2 adrenergic receptor removes some stabilizing conformational constraints, allowing CAM to more readily undergo transitions between the inactive and the active states and making the receptor more susceptible to denaturation.

Adrenergic beta-Agonists↗

PubMed1996

Residues in the seventh membrane-spanning segment of the dopamine D2 receptor accessible in the binding-site crevice.

The binding site of the dopamine D2 receptor, like that of other homologous G-protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Using the substituted-cysteine accessibility method, we previously mapped the residues that form the surface of the binding-site crevice in the third and fifth membrane-spanning segments (M3 and M5). We have now mutated to cysteine, one at a time, 26 consecutive residues in and flanking the seventh membrane-spanning segment (M7) and expressed the mutant receptors in HEK 293 cells. Nine of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and were protected from reaction by a reversible dopamine antagonist, sulpiride. Thus, we infer that the side chains of these residues are in the water-accessible surface of the binding-site crevice. The pattern of accessibility of the cysteine-substitution mutants is consistent with M7 being a kinked alpha-helix.

Amino Acid Sequence↗

PubMed1996

Contribution of a helix 5 locus to selectivity of hallucinogenic and nonhallucinogenic ligands for the human 5-hydroxytryptamine2A and 5-hydroxytryptamine2C receptors: direct and indirect effects on ligand affinity mediated by the same locus.

An important determinant of the neurobehavioral responses induced by a drug is its relative receptor selectivity. The molecular basis of ligand selectivity of hallucinogenic and nonhallucinogenic compounds of varying structural classes for the human 5-hydroxytryptamine (5-HT)2A and 5-HT2C receptors was investigated with the use of reciprocal site-directed mutagenesis. Because these two closely related receptor subtypes differ in the amino acid present at position 5.46 (residues 242 and 222 in the sequences, respectively), the effects of corresponding substitutions in the 5-HT2A[S5.46(242)-->A] and 5-HT2C[A5.46(222)-->S] receptors were studied in tandem. By studying both receptors, the direct and indirect effects of mutations on affinity and selectivity can be distinguished. The ergolines studied, mesulergine (selective for the 5-HT2C receptor) and d-lysergic acid diethylamide (selective for the 5-HT2A receptor), reversed their relative affinity with mutations in each receptor, supporting a direct role of this locus in the selectivity of these ligands. However, interchange mutations in either receptor led to decreased or unchanged affinity for (+/-)-1-)(2,5-dimethoxy-4-iodophenyl)-2-aminopropane and ketanserin, which have higher affinity for the 5-HT2A receptor, consistent with little contribution of this locus to the selectivity of these ligands. The indoleamines studied were affected differently by mutations in each receptor, suggesting that they bind differently to the two receptor subtypes. Mutation of this locus in the 5-HT2A receptor decreased the affinity of all indoleamines, whereas the interchange mutation of the 5-HT2C receptor did not affect indoleamine affinity. These results are consistent with a direct interaction between this side chain and indoleamines for the 5-HT2A receptor but not for the 5-HT2C receptor. Furthermore, this analysis shows that the higher affinity of 5-HT and tryptamine for the 5-HT2C receptor than for the 5-HT2A receptors is not due to the difference at this locus. The hallucinogens studied [d-lysergic acid diethylamide, psilocin, bufotenin, and (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane] fell into different classes in this analysis. For the classes of ligand studied, the side-chain difference at this position directly determines relative ligand selectivity only for ergolines and may contribute to the specific effects of hallucinogens in this class.

Amino Acid Sequence↗