©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Related Contribution of Specific Helix 2 and 7 Residues to Conformational Activation of the Serotonin 5-HT Receptor (*)

Stuart C. Sealfon (1) (2)(§), Ling Chi (1), Barbara J. Ebersole (3) (4), Vladimir Rodic (1), Daqun Zhang (5), Juan A. Ballesteros (5), Harel Weinstein (4) (5)

From the (1)Fishberg Research Center in Neurobiology and the Departments of (2)Neurology, (3)Anesthesiology, (4)Pharmacology, and (5)Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York 10029

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A conserved helix 2 Asp is required for the proper function of many G-protein-coupled receptors. To reveal the structural basis for the role of this residue, the additive effects of mutations at this locus and at a conserved helix 7 locus were investigated in the 5-HT receptor. All mutant receptors studied retained high affinity agonist and antagonist binding. Whereas an Asp Asn mutation in helix 2 eliminated coupling, interchanging the residues at the two positions by a second mutation of Asn Asp in helix 7 restored receptor function. These data suggest that these residues are adjacent in space and interact. The loss of function observed with Ala at either position is consistent with each side chain forming hydrogen bonds. Molecular dynamics simulations were performed on three-dimensional computational models of agonist-receptor complexes of both the wild-type receptor and the Asp Asn mutant receptor. Consonant with the lack of coupling observed for the mutant construct, introducing the mutation into the computational model produced a conformational change in a direction opposite to that seen from computational simulations of activation of the wild-type receptor model. These results implicate both loci in a common hydrogen-bonding network underlying receptor activation by agonist.


INTRODUCTION

Members of the G-protein-coupled receptor (GPCR)()superfamily contain seven putative transmembrane helix (TMH) domains and activate intracellular heterotrimeric G-proteins. The cloning of more than 200 distinct members of this gene family has revealed a high degree of homology in the TMH regions in which certain amino acid sequence motifs are highly conserved among the different receptors (for review, see Ref. 1). Sequences resistant to alteration during an evolutionary process that has generated receptors for ligands as diverse as neurotransmitters and glycoprotein hormones are likely to constitute structural elements that mediate universal receptor functions. One structural template that is potentially shared by most, if not all, GPCRs is an interhelical network of side chain interactions underlying the activation of these receptors. The highly conserved amino acids are therefore reasonable candidates for having a key functional role in the agonist-induced rearrangement of the TMHs that constitutes the mechanism of receptor activation.

The functional roles of highly conserved GPCR residues are being explored with site-directed mutagenesis, and many have been shown to be required for proper receptor function. However, little is currently understood about the structural basis for the disruptive effect of mutations at these loci. For example, the conserved TMH-2 Asp has been studied in a large number of GPCRs, and mutations of this residue have been found to alter signal transduction(2, 3, 4, 5, 6, 7, 8, 9, 10, 11) ; agonist affinity(2, 3, 6, 8, 10, 12, 13, 14, 15) ; and allosteric modulation by sodium(15, 16, 17, 18) , pH (16), or guanyl nucleotides(2, 3, 4, 8, 10, 11) . When viewed together, the varying functional effects of mutations at this site in different GPCRs suggest that this residue is involved in receptor rearrangement among different conformational states. While this rearrangement is likely to involve interactions of this residue with side chains in other helices, there is no direct data from any receptor that conserves this TMH-2 Asp to illuminate the nature of this interaction or to identify candidate interaction sites in other helices.

The absence of fine resolution structural data on any GPCR makes it especially difficult to propose molecular mechanisms that could explain why specific mutations produce the particular effects that are observed in various assays of receptor function. Although a 9-Å projection density map showing the helical nature of the transmembrane domains and their arrangement has recently been obtained for rhodopsin (19), the low resolution does not permit inferences about side chain proximities and helix-helix interactions in this or any other GPCR.

In an attempt to generate the experimental basis for understanding receptor activity at a detailed molecular level, we have begun to study the functional effects of mutation of the conserved TMH-2 locus and the additive effects of concomitant mutations at other receptor sites. To interpret the results from such studies in a structural context, we have developed techniques for modeling both receptor structure and conformational behavior in response to mutation and/or complexing with ligand, an approach referred to as dynamic modeling(28) . The evaluation of these models by molecular dynamics simulations can generate experimentally testable hypotheses that relate structural details to functional mechanisms. For example, the reciprocal mutation studies of the gonadotropin-releasing hormone (GnRH) receptor integrated with computational modeling of GPCRs led to the proposal of a spatial proximity of the TMH-2 locus, which contains an Asp in nearly all GPCRs, to a specific locus in TMH-7(20) . In these studies, a functionally disruptive mutation in one helix is corrected by the introduction of a second mutation in the other helix. Restoration of function by the second mutation indicates that the two residues share a microenvironment. This explanation for their interrelated functional roles is consistent with the network of interactions observed in the three-dimensional model of the GnRH receptor(20) . However, the residues present in the GnRH receptor at the two loci studied in TMH-2 and TMH-7 diverge from the conservation pattern found in most other GPCRs. Therefore, we were interested in the ability of our approach to elucidate the relationship between the two loci in a neurotransmitter receptor having the pattern found in >95% of GPCRs, i.e. Asp in TMH-2 and Asn in TMH-7.

In this study, the role of the conserved TMH-2 Asp and its relationship to the TMH-7 Asn in the activation of the serotonin 5-HT receptor was investigated by complementary site-directed mutagenesis experiments. From the pharmacological characterization of the mutant receptors, we are able to demonstrate a function-restoring reciprocal mutation involving these two loci. In parallel, we have evaluated the effects of mutation of TMH-2 Asp Asn in a three-dimensional molecular model of the 5-HT receptor. Computational simulation studies of the wild-type 5-HT receptor model have characterized the pattern of structural rearrangements induced by agonist binding in the helix bundle and have suggested a mechanism of receptor activation(21, 22, 23) . Similar molecular dynamics simulations for the mutant receptor are shown here to lead to a very different structural rearrangement of the receptor regions presumed to be involved in coupling to G-proteins. Evaluation of the results of site-directed mutagenesis in the context of these dynamic simulations of a receptor model provides a structural hypothesis for the functional effects of these mutations. Our results suggest that a common hydrogen-bonding network involving the two loci is required for receptor activation by agonist.


EXPERIMENTAL PROCEDURES

DNA Constructs and Transfection

A cDNA clone encoding the human 5-HT receptor was generously provided by Dr. Alan G. Saltzman(24) . The insert was digested with SmaI and ASP700 and ligated into SmaI-digested pALTER (Promega, Madison, WI). Mutations were introduced with oligonucleotides following the manufacturer's protocol and were confirmed by sequencing. For expression, the insert was subcloned into the XbaI and EcoRV sites of pcDNAI/Amp (Invitrogen, San Diego, CA) for expression. The mutations were reconfirmed by sequencing the mutation site in the expression vector. COS-1 cells (American Type Culture Collection, Rockville, MD) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Each 100-mm plate, seeded 24 h earlier with 3 10 cells, was transfected with 8 µg of DNA construct and 48 µl of Lipofectamine (Life Technologies, Inc.).

Phosphatidylinositol Hydrolysis Assay

24 h following the transfection in 100-mm plates, the cells were harvested with trypsin and seeded in 2 12-well plates. 32 h later, the medium was replaced by serum-free medium containing 0.5 µCi/ml myo-[H]inositol (DuPont NEN). 16 h later, the cells were washed and incubated with the desired concentrations of 5-HT in the presence of 20 mM LiCl for 45 min at 37 °C. Cell extracts, in 10 mM formic acid, were applied to a Dowex ion-exchange column before elution by buffer containing 1 M ammonium formate and 0.1 M formic acid. The procedure essentially followed published protocols(25) . After scintillation counting, the data were plotted using Kaleidagraph (Synergy Software, Reading, PA) and fitted to the following equation: E = E/(1 + (EC/D)), where E is the maximum stimulation, D is the concentration of the agonist, and EC represents the agonist concentration that produces half-maximal stimulation.

Ligand Binding Assay

Three days following transfection, cells were harvested, and cell pellets were stored at -70 °C. For preparation of membranes, all procedures were carried out at 4 °C. Pellets were thawed and homogenized in 20 ml of 50 mM Tris-HCl (pH 7.4) (buffer A) at 25 °C with a Polytron homogenizer (setting 6 for 8 s). An additional 20 ml of buffer A was added, and the homogenates were centrifuged at 35,000 g for 15 min. The membrane pellets were resuspended in buffer A with a Teflon/glass homogenizer (10 strokes by hand). Each binding incubation tube contained 70-180 µg of membrane protein, [H]Ketanserin (DuPont NEN), unlabeled drug as required, and buffer A in a final volume of 1.0 ml. For saturation binding studies, eight concentrations (0.05-10 nM) of [H]Ketanserin (specific activity of 85 Ci/mmol) were tested in duplicate. For competition binding studies, the concentration of [H]Ketanserin was 1.7-2.0 nM, and 10 concentrations of competing ligands were tested in duplicate. Nonspecific binding was determined in the presence of 10 µM methysergide. Incubations were carried out for 1 h at 37 °C and were terminated by rapid filtration through Whatman GF/C glass fiber filters (Brandel cell harvester) followed by washing with 10 ml of buffer A at 4 °C. The radioactivity retained on the filters was quantitated by liquid scintillation spectrometry. Protein content was measured by the method of Lowry et al. (26).

Specific binding was determined by subtracting the amount of [H]Ketanserin bound in the presence of methysergide from the amount bound in the absence of methysergide. For saturation studies, B and K values for [H]Ketanserin were obtained by a fit of the data to the following equation: specific binding = B/(1 + (K/D)), where D is the free concentration of [H]Ketanserin. For competition studies, specific binding was expressed as a percent of specific binding in the absence of the competing ligands and fit to the following equation: % binding = 100 - (100/(1 + (IC/[5-HT]))), where IC is the concentration of competing drug that produces 50% specific binding of [H]Ketanserin. IC values were converted to K values by the method of Cheng and Prusoff (27) by using the K values for [H]Ketanserin determined for each construct from saturation binding studies. Curve fitting of data was carried out with Kaleidagraph.

Locus Numbering Scheme

The positions of various amino acids in the receptor sequence and the structural changes in the various receptor constructs are identified throughout with a numbering scheme that facilitates the comparison among different receptors. This consensus numbering scheme has been defined in detail elsewhere(28) . Briefly, each locus is identified by a TMH number followed by the position relative to the most conserved residue in that helix, which is assigned the number 50. The conserved Asp in TMH-2 of the 5-HT receptor, for example, is numbered 2.50, and Asn, located one residue before the conserved Pro in TMH-7, is numbered 7.49. The original numbering is retained in parentheses, and amino acid mutations are indicated by an arrow. Thus, the 5-HT construct containing a mutation of Asn in TMH-7 to Ala is written Asn Ala.

Molecular Modeling of 5-HTReceptor Mutants and Computational Simulations of Interactions with Ligands

The model of the transmembrane helix bundle of the 5-HT receptor and its development have been reported previously(21, 29) . A primary objective in the early development of this model was to reflect the pharmacological data on the structure-activity relations of 5-HT receptor ligands. The relative position of the helices in this particular model of the 5-HT receptor differs from that presented in other published GPCR models, but it is one of the arrangements compatible with the information currently available for rhodopsin(30) . In this model of the 5-HT receptor, the relative positions of the TMH-2 Asp locus and the TMH-7 Asn locus proposed in the GnRH receptor model (20) are preserved. The close agreement between the results from computational explorations of the effects of ligand binding to this receptor model and quantitative pharmacological data (21, 22) supports its use to construct the molecular model of an Asp Asn mutation in TMH-2.

Mutation of Asp Asn was introduced in the molecular model of the 5-HT receptor described previously (21). The construct was energy-optimized, and a molecular dynamics run was carried out for 100 ps with the CHARMM program (31) using the same protocol as described previously(21) . The equilibrium structure of the mutant receptor was obtained from the energy-minimized average structure over the final 60-100-ps period of the simulation. 5-HT was positioned in the binding site of the mutant receptor in the same manner and using the same parameters for molecular dynamic simulations as reported previously(21) . Simulation of the ligand-receptor complex was carried out for 210 ps, and the equilibrated structure of the agonist-receptor complex was obtained from the energy-minimized average over the final 110-210-ps period of the simulation. The changes produced by ligand binding in the structure of the receptor model were determined as described (21, 22, 23) by calculating the root mean square deviations of the positions of the -carbons in the average structure of the complexes relative to the equilibrated receptor structures.


RESULTS

Agonist-stimulated Inositol Phosphate Accumulation

The wild-type and mutant receptors were expressed in COS-1 cells, and the concentration-response curves for 5-HT-stimulated inositol 1-phosphate accumulation were determined. Representative concentration-response curves are shown in Fig. 1, and all results are summarized in . The EC value measured for 5-HT on the wild-type receptor was 2.2 ± 4 10M, with a maximal PI accumulation of 5.2 ± 0.7-fold over basal levels. Mutation of Asp Asn eliminated detectable PI accumulation, confirming a report of this mutation in the rat receptor(10) . Introducing the second mutation in helix 7, Asn Asp, interchanges the residues at the two positions and restores agonist-induced PI hydrolysis. The efficacy of activation of this reciprocal mutant receptor was apparently reduced, as indicated by a maximal stimulation of only 3 ± 0.6-fold over basal levels and an increased EC value for 5-HT (1.94 ± 0.36 10M) relative to the wild-type receptor (see ``Discussion''). Introducing only the single mutation of Asn Asp caused an intermediate increase in the EC value for 5-HT relative to the wild-type receptor (9.5 ± 0.4 10M). The requirement for a polar residue at either position was explored with substitution by Ala at position 2.50(120) or 7.49(376). No 5-HT-stimulated PI hydrolysis was observed following expression of either construct ().


Figure 1: Concentration-response curves of PI hydrolysis with 5-HT stimulation for the wild-type and mutant constructs. A concentration-response curve representative of three experiments is shown. The Asp Ala and Asn Ala receptors showed no response over basal levels at any 5-HT concentration, and for clarity, these data are not plotted. Although a lower maximal response was obtained with the single TMH-7 Asn Asp mutation in the experiment shown, the E values with this construct were, on average, comparable to those obtained with the wild-type receptor (Table I).



Radioligand Binding of Wild-type and Mutant Receptors

The wild-type and mutant 5-HT receptors expressed transiently in COS-1 cells bound [H]Ketanserin with high affinity. Representative saturation isotherms for three of the constructs are shown in Fig. 2A; data for all constructs are shown in . The affinity of the mutant receptors for [H]Ketanserin varied by <2-fold compared with the wild type. In contrast, B values, calculated from the maximal specific binding of [H]Ketanserin, were dependent on the particular construct. The lowest levels of expression were obtained with the Asn Asp mutant, and highest levels with the Asp Asn mutant. The expression level for a given construct was found to be highly consistent in separate preparations.


Figure 2: Saturation and competition binding to the wild-type and mutant 5-HT receptors. A, saturation binding of [H]Ketanserin to the wild-type and mutant 5-HT receptors. Incubations and analyses were carried out as described under ``Experimental Procedures.'' At 10 nM [H]Ketanserin, nonspecific binding was 39% of total binding for the wild type (WT), 27% for Asp Asn, and 59% for the double mutant. Inset, representation of data according to the method of Scatchard (35). Duplicate points from one experiment are shown. These results were replicated in one additional experiment. B, competition by 5-HT and mianserin for [H]Ketanserin binding to the wild-type and mutant 5-HT receptors. Incubations and analyses were carried out as described under ``Experimental Procedures.'' Opensymbols are competition with mianserin, and closedsymbols are competition with 5-HT. , wild-type receptor; , Asp Asn receptor; , double mutant receptor. The concentrations of [H]Ketanserin for 5-HT and mianserin competition were 0.9 and 1.1 nM, respectively. Nonspecific binding was 15% of total binding for the wild type, 8% for Asp Asn, and 27% for the double mutant. Duplicate points from one experiment are shown. These results were replicated in one additional experiment.



The affinities of 5-HT for the wild-type and mutant receptors and of 2,5-dimethoxy-4-iodoamphetamine (DOI) LSD, mianserin, haloperidol, and 5-methoxygramine for the wild-type, Asp Asn, and double mutant receptors were determined in competition binding experiments for sites labeled with [H]Ketanserin. Fig. 2B shows representative competition curves, and all data are summarized in . The affinity of 5-HT for four of the mutant receptors varied by <2-fold. For the Asp Ala receptor mutant, the affinity of 5-HT was 5 times lower than for the wild-type receptor. The affinities of the other agonists and antagonists for the receptor mutants studied were close to values measured for the wild-type receptor.

Molecular Dynamics Simulations of the Asp Asn Mutant Receptor

The structure of the Asp Asn receptor mutant obtained from the modeling and molecular dynamics equilibration procedures differs from that of the wild-type 5-HT receptor model reported previously(21) . The major difference between the models of the wild-type and mutant receptors is localized in TMH-5 and TMH-6. The segments of helices 5 and 6 that connect to the third intracellular loop were found in the previously reported simulations of ligand-receptor complexes of the wild-type 5-HT receptor to be the sites most affected by the binding of agonists(21, 22, 23) . In particular, the average structure of the ligand-receptor complex in which 5-HT is positioned in the binding site of the wild-type receptor showed a large rearrangement of these segments of TMH-5 and TMH-6, pointing away from each other in the helix bundle (Fig. 3A). It is noteworthy, therefore, that the structure of the uncomplexed Asp Asn receptor model differs from that of the wild-type uncomplexed receptor mainly in the same region. However, the departure is in a direction opposite to that found in the simulation of wild-type receptor activation by agonist (Fig. 3B). Binding 5-HT to the mutant receptor model induces a small outward movement in that same region of the helix 5 and 6 segments, but this is insufficient to correct the paradoxical inward displacement in the uncomplexed Asp Asn mutant relative to the wild type. Thus, the outward movement resulting from 5-HT binding to the mutant does not have the structural characteristics of the ``activated'' wild-type receptor model. These results obtained from simulations of a receptor model are remarkably consonant with the experimental finding that this mutant does not mediate PI hydrolysis. No significant conformational rearrangement in the intracellular portions of the other TMH domains was observed upon binding of agonist to either receptor model. For comparison, the response of the wild-type receptor model to the binding of the antagonist 5-hydroxygramine is shown in Fig. 3C. With antagonist in the binding pocket, no significant displacement is observed in any of the domains of the 5-HT receptor model that were affected by agonist binding (21-23).


Figure 3: Time-averaged structures from molecular dynamics simulations of uncomplexed and ligand-bound 5-HT receptor models. The -carbon trace of the helices is viewed from the side parallel to the membrane with the outside of the cell at the top of the figure. A, effects of complexing 5-HT with the wild-type receptor (see Ref. 21 for details). Significant conformational changes (see arrows) are observed only in the segments of helices 5 and 6 toward the intracellular side. Black lines, uncomplexed wild-type receptor; gray lines, wild-type receptor complexed with 5-HT; curved arrows, conformational change induced by 5-HT binding. B, comparison of the effects of complexing 5-HT with the wild-type receptor and with the Asp Asn receptor mutant. Because there was no significant change in the position of the other helices, only TMH-5 and TMH-6 are shown. Note that the helices of the unbound mutant receptor are positioned differently than those of the unbound wild-type receptor and that the mutant receptor shows a smaller conformational change upon 5-HT binding (compare black and grayarrows). Black lines and curved arrows, wild-type receptor uncomplexed and complexed with 5-HT; gray lines and curved arrows, mutant receptor uncomplexed and complexed with 5-HT. C, effects of complexing the antagonist 5-hydroxygramine with the wild-type receptor (gray lines) (see Ref. 21 for details). No significant conformational changes are observed relative to the unbound receptor (black lines).




DISCUSSION

If two protein loci are independent in their contribution to a measured property, the effect of a mutation at either locus should be additive(32, 33) . The effects of mutations at the TMH-2 and TMH-7 positions studied are not additive. Mutation of the conserved TMH-2 Asp Asn in the 5-HT receptor eliminates coupling; this effect is reversed by introducing a second mutation of Asn Asp in TMH-7. This restoration of function, albeit to a lower maximal level, by the second mutation cannot be due to any beneficial effect of this mutation by itself because, in fact, the TMH-7 mutation alone does not enhance function. Thus, the two loci cannot be independent, and these results therefore support the hypothesis that these residues are adjacent in space and have a shared network of interactions(20) . The finding that the Ala mutations at each of these loci decrease or eliminate receptor-mediated PI hydrolysis implicates both side chains of the wild-type receptor in the process of receptor activation.

The altered coupling observed with these mutations cannot be attributed to disruption of the binding pocket because the affinities for agonists and antagonists do not vary significantly among all the constructs studied. Furthermore, the functional differences caused by the mutations are not due to differences in the levels of expression obtained because the construct with the highest level of expression, Asp Asn, mediates no detectable PI hydrolysis, whereas the receptor with the lowest expression, Asn Asp, has an E value equivalent to that of the wild-type receptor. Thus, the observed decrease in or absence of agonist-mediated PI hydrolysis must represent an alteration in the capacity of each mutant receptor studied to mediate coupling to signal transduction.

The results of molecular dynamics simulations of the effects of agonist binding to the wild-type and mutant receptors suggest a possible mechanism for the loss of coupling observed with the TMH-2 Asp Asn mutation. As previously reported, simulation of agonist binding to a computational model of the wild-type receptor resulted in a conformational rearrangement affecting only the distal segments of helices 5 and 6 that connect to the third intracellular loop(21, 23) . Simulations of complexes of the wild-type receptor interacting with ligands that are known to produce a range of responses differing in their pharmacological potencies resulted in a structural change in the same direction(22) . We find that in a model in which mutation of Asp Asn is introduced, the same segments of helices 5 and 6 are distorted in a direction opposite to that seen with activation of the wild-type receptor. Simulation of the effect of agonist binding to this mutant model produces only a small movement of these helices toward the positioning achieved by the wild-type receptor-ligand complex (Fig. 3B). Thus, the molecular models of the wild-type receptor and the Asp Asn mutant receptor manifest different conformational rearrangements of the helices adjacent to a domain implicated in G-protein coupling(1) . These findings parallel the results of mutagenesis, which show that this mutation causes a loss of detectable coupling to PI turnover.

It is notable that the structure of the mutant receptor model itself is quite different from that of the wild-type receptor model in the segments of the two helices that are proximal to the third intracellular loop. Furthermore, whereas antagonists induce essentially no conformational change in the wild-type receptor model (Fig. 3C), the agonist induces a conformational rearrangement of the mutant receptor model that is different from that of the wild-type receptor (Fig. 3B). Therefore, the results from computational simulations with these models suggest that agonist still induces a conformational change in the mutant receptor, but that the specific helix rearrangements achieved may not support the same mechanism of coupling to the G-proteins mediating activation of phospholipase C. These simulation results are interesting in view of the variable effects on signal transduction obtained with mutation of this locus in different receptors(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 34) . In studies of this locus in the -adrenergic receptor, for example, distinct coupling pathways are differentially affected. Whereas an -adrenergic receptor having a mutation of Asp Asn was found to be uncoupled from potassium currents, coupling to calcium currents was preserved(4) . Our modeling results suggest that the mutant receptor undergoes an agonist-induced conformational change, albeit different from that of the wild-type receptor, and may thus sustain coupling to some G-proteins. Thus, the selective preservation of coupling reported with mutation of this site in the -adrenergic receptor may result from the induction of a different conformational state that has an altered G-protein selectivity.

The modeling and mutagenesis results are consistent with the hypothesis that a favorable network of hydrogen bonding involving the side chains at the two loci is required for proper agonist-induced conformational changes in the receptor. A hydrogen-bonding network involving Asn in the wild-type receptor may connect to Asp. Thus, substitution by Ala at either position eliminates the capacity of each locus to form hydrogen bonds and thereby leads to a loss of the interactions required for receptor activation. In the Asp Asn mutant receptor, Asn eliminates the charge and one of the groups that can serve as a hydrogen-bond acceptor; these properties are reinstated by the double mutant. The Asn Asp receptor is relatively good at mediating PI hydrolysis, which can be attributed to protonation of one of the Asp side chains in the vicinity of an already negatively charged Asp. This restores the hydrogen-bonding relationship to that found in the wild-type receptor and thereby allows the network of interactions involving these loci to persist in this mutant.

Although the double mutant receptor has improved function relative to the single mutant Asp Asn receptor, it is significantly impaired in its capacity to mediate signal transduction. The partial restoration of function observed with the reciprocal mutation is important in implicating both loci in a common hydrogen-bonding network mediating receptor activation. However, the decreased efficacy of 5-HT at the double mutant receptor provides additional insight into the functional structure of the receptor. This decreased function in comparison to the wild-type receptor indicates that the two loci are not functionally interchangeable. The functional asymmetry of the two loci most likely reflects differences in the interactions of the two side chains at these loci with other receptor groups. One additional contact site of the Asp side chain suggested by three-dimensional models of GPCRs is the highly conserved Asn in TMH-1, a hypothesis that is being tested experimentally.

The results obtained with mutation of these loci have interesting parallels and contrasts relative to those reported for the GnRH receptor(20) . In both receptors, a single mutation at the TMH-2 locus disrupts function, and functionality is restored by a second, interchanging mutation at the TMH-7 locus. The demonstration of a restoration of function by reciprocal mutations involving these two loci in both the GnRH receptor and the 5-HT receptor suggests that the structures of the two receptors and their mechanisms of activation are likely to be similar. However, the precise mutations that are functionally accommodated at each position differ in the two receptors. For example, having an Asn in TMH-2 and TMH-7 of the GnRH receptor is tolerated, whereas having an Asp in both positions leads to a loss of high affinity binding and coupling. In the 5-HT receptor, the opposite pattern is seen, with Asp in both positions being well tolerated, whereas Asn in both positions causing elimination of detectable coupling. The reason for the differences in the residues preferred at each position across receptors must reside in differences in the microenvironments of the two loci in different receptors. With the structural basis for the adjacencies of these loci in the receptor molecule clarified by the results of reciprocal mutations and dynamic models, the specific side chains contributing to the unique microenvironment of each locus in the two receptors can be putatively identified. The hypotheses generated can be probed experimentally to elucidate other contacts made by each locus in each wild-type receptor.

Mutation of TMH-2 Asp Asn has previously been studied in the rat 5-HT receptor, and a loss of allosteric modulation by GTP was observed (10). In that study, 10-fold decreases in the affinity of this mutant receptor for several agonists and antagonists was observed, although no change was found in the affinities for iodo-LSD or spiperone. In our present study, we find no significant change in the affinity of any of the ligands studied for this mutant. This discrepancy may reflect differences in the effect of this mutation in the rat and human receptors or differences in the radioligand binding assay utilized in the two studies.

The exploration of the structural and pharmacological properties of the 5-HT receptor by mutagenesis and computational studies has demonstrated a functional and spatial relationship of the conserved TMH-2 Asp and TMH-7 Asn found in most GPCRs. The results for the functional double revertant mutant implicate both residues in a common hydrogen-bonding network that is involved in the conformational changes produced in the receptor as a consequence of agonist binding. This approach provides insight into the arrangement of the transmembrane helix domains of GPCRs, and computational simulations with the three-dimensional molecular model suggest conformational changes that occur during receptor activation. The causes for differences in the effects of single substitutions at the two positions in the 5-HT receptor and in the GnRH receptor should be addressable in a similar manner through combined experimental explorations and computational modeling.

  
Table: Analysis of saturation binding and PI hydrolysis data for 5-HT receptor constructs

Binding affinities (K values) were obtained from saturation binding with [H]Ketanserin. EC values were obtained from concentration-response curves for 5-HT. The E values are expressed as a percent of the maximal stimulation above basal levels obtained with activation of the wild-type receptor, which, in these experiments, was 4.2 ± 0.7-fold above basal levels. Values represent means ± S.E. of two experiments for the binding data and three experiments for the PI assays.


  
Table: Affinity for [H]Ketanserin-labeled sites in 5-HTreceptor constructs

The K values were derived from two competition experiments. A representative experiment for two of the competing ligands is shown in Fig. 2B. Competition with unlabeled 5-HT was also performed for the Asp Ala, Asn Asp, and Asn Ala receptor mutants, and K values of 1638 ± 553, 249 ± 28, and 549 ± 15, respectively, were obtained.



FOOTNOTES

*
This work was supported by National Institutes of Health Grants DA09088, DA09083, and KO5 DA00060. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Fishberg Center for Neurobiology Research, P. O. Box 1065, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029. Tel.: 212-241-7075; Fax: 212-996-9785.

The abbreviations used are: GPCR, G-protein-coupled receptor; TMH, transmembrane helix; GnRH, gonadotropin-releasing hormone; 5-HT, 5-hydroxytryptamine; PI, phosphatidylinositol.


ACKNOWLEDGEMENTS

Dr. Alan G. Saltzman is gratefully acknowledged for providing the human 5-HT receptor cDNA. We thank Dr. Saul Maayani for many helpful suggestions.


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