AFFINITY LABELING OF CYSTEINE-MUTANTS EVIDENCES CONTACT RESIDUES IN MODELED RECEPTOR BINDING SITES

ABSTRACT

To investigate the topology of binding sites in two ionotropic receptors, we have initiated a strategy combining affinity labeling with cysteine-scanning mutagenesis. For the GABA_A receptor we have used reactive derivatives of non-competitive blockers (NCBs) to explore interacting positions in its channel. The polypeptide positions of the M₂ segment of the α₁ subunit which we mutated into cysteine were selected for their established accessibility, as determined by the substituted-cysteine accessibility method (SCAM). Using the Xenopus oocyte expression system, we show that receptors containing mutations V257C and S272C are inactivated by several reactive NCBs. These position-selective inactivations lead to an analysis of NCB binding in the channel. For the NMDA receptor glycine-binding site, the prototype antagonist L-701,324 was derivatized at different positions with different reactive groups. The receptor positions to mutate into cysteine were selected after a 3-D homology model. The observed receptor inactivations are mutant- and probe-selective, leading to an unambiguous chemical docking of the antagonist pharmacophore and supporting the model. The site-specificity of the inactivating reactions is assessed by protection experiments and by mutant to wild-type (WT) comparisons. The scope and limitations of the method are briefly discussed.     

Structural model of the N-methyl-D-aspartate receptor glycine site probed by site-directed chemical coupling

The N-methyl-d-aspartate (NMDA) receptor is a ligand-gated ion channel that requires both glutamate and glycine for efficient activation. Here, a strategy combining cysteine scanning mutagenesis and affinity labeling was used to investigate the glycine binding site located on the NR1 subunit. Based on homology modeling to the crystal structure of the glutamate binding site of the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-propionic acid receptor GluR2, cysteines were introduced into the NR1 subunit as chemical sensors for three thiol-reactive derivatives of the competitive antagonist L-701324. After coexpressing the mutant NR1 with wild-type NR2B subunits in Xenopus oocytes, agonist-induced currents were recorded to monitor irreversible receptor inactivation by the reactive antagonists. For each derivative, glycine site-specific inactivations were observed with a distinct subset of cysteine-substituted receptors. Together these inactivating substitutions identified seven NR1 residues (Ile-385, Gln-387, Glu-388, Thr-500, Asn-502, Ala-696, and Val-717) that undergo proximity-induced covalent coupling with specific regions of the bound antagonist and disclose its mode of docking in the glycine binding pocket of the NMDA receptor. Our approach may help to unravel the structural basis of distinct NMDA receptor subtype pharmacologies.     

Mapping the rho1 GABA(C) receptor agonist binding pocket. Constructing a complete model

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. The GABA receptor type C (GABA(C)) is a ligand-gated ion channel with pharmacological properties distinct from the GABA(A) receptor. To date, only three binding domains in the recombinant rho1 GABA(C) receptor have been recognized among six potential regions. In this report, using the substituted cysteine accessibility method, we scanned three potential regions previously unexplored in the rho1 GABA(C) receptor, corresponding to the binding loops A, E, and F in the structural model for ligand-gated ion channels. The cysteine accessibility scanning and agonist/antagonist protection tests have resulted in the identification of residues in loops A and E, but not F, involved in forming the GABA(C) receptor agonist binding pocket. Three of these newly identified residues are in a novel region corresponding to the extended stretch of loop E. In addition, the cysteine accessibility pattern suggests that part of loop A and part of loop E have a beta-strand structure, whereas loop F is a random coil. Finally, when all of the identified ligand binding residues are mapped onto a three-dimensional homology model of the amino-terminal domain of the rho1 GABA(C) receptor, they are facing toward the putative binding pocket. Combined with previous findings, a complete model of the GABA(C) receptor binding pocket was proposed and discussed in comparison with the GABA(A) receptor binding pocket.     

The first extracellular loop of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p undergoes a conformational change upon ligand binding

In this study of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p, we present data indicating that the first extracellular loop (EL1) of the alpha-factor receptor has tertiary structure that limits solvent accessibility and that its conformation changes in a ligand-dependent manner. The substituted cysteine accessibility method was used to probe the solvent exposure of single cysteine residues engineered to replace residues Tyr(101) through Gln(135) of EL1 in the presence and absence of the tridecapeptide alpha-factor and a receptor antagonist. Surprisingly, many residues, especially those at the N-terminal region, were not solvent-accessible, including residues of the binding-competent yet signal transduction-deficient mutants L102C, N105C, S108C, Y111C, and T114C. In striking contrast, two N-terminal residues, Y101C and Y106C, were readily solvent-accessible, but upon incubation with alpha-factor labeling was reduced, suggesting a pheromone-dependent conformational change limiting solvent accessibility had occurred. Labeling in the presence of the antagonist, which binds Ste2p but does not initiate signal transduction, did not significantly alter reactivity with the Y101C and Y106C receptors, suggesting that the alpha-factor-dependent decrease in solvent accessibility was not because of steric hindrance that prevented the labeling reagent access to these residues. Based on these and previous observations, we propose a model in which the N terminus of EL1 is structured such that parts of the loop are buried in a solvent-inaccessible environment interacting with the extracellular part of the transmembrane domain bundle. This study highlights the essential role of an extracellular loop in activation of a G protein-coupled receptor upon ligand binding.     

Reactive affinity probes for the mapping of the glycine-binding site of the NMDA receptor NR1 subunit

The glycine co-agonist binding site of the NMDA receptor is a target for the prevention and treatment of neurotoxic and neurodegenerative conditions. Until now, the interactions taking place at this site, and its structure, have been investigated by ligand structure-activity relationships and by site-directed mutagenesis. On the basis of a structural model which is currently proposed for this site, we have designed and synthesized six affinity markers by substituting electrophilic reactive groups in the 4, the 7 and the 3' positions of L 701,324, a high-affinity glycine site antagonist. These compounds compete with 3H-DCKA binding to rat brain membranes at equilibrium with nanomolar to low-micromolar affinities, and antagonize glycine-evoked currents in oocytes transfected with wild-type NR1-NR2B. However, they do not induce a time-shift in binding equilibria, and do not inactivate irreversibly the glycine evoked currents. Since they react only with cysteine at physiological pH, we conclude that there is no such residue in the site, in agreement with the model. Our affinity markers therefore represent potential topological probes for NMDA receptors with sequence positions related to the glycine-binding site mutated into cysteine.     

Staggering of subunits in NMDAR channels

Functional N-methyl-D-aspartate receptors (NMDARs) are heteromultimers formed by NR1 and NR2 subunits. The M3 segment, as contributed by NR1, forms the core of the extracellular vestibule, including binding sites for channel blockers, and represents a critical molecular link between ligand binding and channel opening. Taking advantage of the substituted cysteine accessibility method along with channel block and multivalent coordination, we studied the contribution of the M3 segment in NR2C to the extracellular vestibule. We find that the M3 segment in NR2C, like that in NR1, contributes to the core of the extracellular vestibule. However, the M3 segments from the two subunits are staggered relative to each other in the vertical axis of the channel. Compared to NR1, homologous positions in NR2C, including those in the highly conserved SYTANLAAF motif, are located about four amino acids more externally. The staggering of subunits may represent a key structural feature underlying the distinct functional properties of NMDARs.     

The GABAA receptor alpha 1 subunit Pro174-Asp191 segment is involved in GABA binding and channel gating

The GABA-binding site undergoes structural rearrangements during the transition from agonist binding to channel opening. To define possible roles of the GABA(A) receptor alpha(1) subunit Pro(174)-Asp(191) segment in these processes, we used the substituted cysteine accessibility method to characterize this region. Each residue was individually mutated to cysteine, expressed with wild-type beta(2) subunits in Xenopus laevis oocytes, and examined using two-electrode voltage clamp. Most mutations did not alter GABA EC(50) values. The D183C mutation produced a 7-fold reduction in GABA sensitivity. There were no significant changes in the K(I) values for the competitive antagonist, SR-95531. N-Biotinylaminoethyl methanethiosulfonate modified P174C-, R176C-, S177C-, V178C-, V180C-, A181C-, D183C-, R186C- and N188C-containing receptors. The pattern of accessibility suggests that this protein segment is aqueous-exposed and adopts a random coil conformation. Both GABA and SR-95531 slowed covalent modification of V178C, V180C, and D183C, indicating that these residues may line the GABA-binding site. Further, pentobarbital-induced channel activation accelerated modification of V180C and A181C and slowed the modification of R186C, suggesting that this region of the alpha(1) subunit may act as a dynamic element during channel-gating transitions.     

Cysteine residues are involved in structure and function of melanocortin 1 receptor: Substitution of a cysteine residue in transmembrane segment two converts an agonist to antagonist

Reduction of disulfide bonds in human melanocortin 1 receptor (hMC1R) with increasing concentrations of DTT (dithiothreitol) resulted in a decrease in the binding of [125I]-ACTH (adrenocorticotropic hormone, L-isomer) in an uniphasic manner and a decrease in [125I]-NDP-MSH ([Nle(4),D-Phe(7)]-alpha-melanocyte stimulating hormone; D-isomer) binding in a biphasic manner. Pretreatment of hMC1R with 10 mM DTT resulted in a 36-fold loss of affinity for alpha-MSH (L-isomer) without affecting the affinity of NDP-MSH (D-isomer). To characterize the role of individual cysteine residues, we employed site-directed mutagenesis to substitute cysteine by glycine at all fourteen positions in hMC1R and analysed wild-type and mutant receptors for ligand binding and cAMP signalling. Single point mutation of four cysteine residues in extracellular loops to glycine (C35G, C267G, C273G, and C275G) resulted in a complete loss of binding for [125I]-NDP-MSH. Moreover, mutants with normal ligand binding, at positions C191G (transmembrane segment 5), C215G (third intracellular loop), and C315G (C-terminal loop) failed to generate cAMP signal in response to both agonists alpha-MSH and NDP-MSH. Mutant at position C78G (with wild-type binding to alpha-MSH as well as NDP-MSH) generated a cAMP signal in response to alpha-MSH (identical to wild-type hMC1R) but interestingly could not be stimulated by NDP-MSH. Moreover, this single amino acid substitution converted NDP-MSH from being an agonist to antagonist at the C78G mutant receptor. These findings demonstrate that (i) alpha-MSH and ACTH (L-isomers) are different from D-isomer NDP-MSH in their sensitivity to DTT for receptor binding, (ii) cysteine residues in N-terminus and extracellular loop three make disulfide bridges and are needed for structural integrity of hMC1R, (iii) cysteine residues in transmembrane segments and intracellular loops are required for receptor-G-protein coupling, (iv) C78 in transmembrane segment two is required for generating a functional response by D-isomer agonist (NDP-MSH) but not by L-isomer agonist (alpha-MSH), and (v) wild-type receptor agonist NDP-MSH is an antagonist at the mutant C78G receptor.     

Structure of the complement factor 5a receptor-ligand complex studied by disulfide trapping and molecular modeling

Complement factor 5a (C5a) is an anaphylatoxin that acts by binding to a G protein-coupled receptor, the C5aR. The relative orientation of this ligand-receptor pair is investigated here using the novel technique of disulfide trapping by random mutagenesis (DTRM) and molecular modeling. In the DTRM technique, an unpaired cysteine is introduced in the ligand, and a library of randomly mutagenized receptors is screened to identify mutants that introduce a cysteine at a position in the receptor that allows functional interactions with the ligand. By repeating this analysis at six positions of C5a, we identify six unique sets of intermolecular interactions for the C5a-C5aR complex, which are then compared with an independently developed computational three-dimensional model of the complex. This analysis reveals that the interface of the receptor N terminus with the cysteine-containing ligand molecules is selected from a variety of possible receptor conformations that exist in dynamic equilibrium. In contrast, DTRM identifies a single position in the second extracellular loop of the receptor that interacts specifically with a cysteine probe placed in the C-terminal tail of the C5a ligand.     

Comparison of the amino acid residues in the sixth transmembrane domains accessible in the binding-site crevices of mu, delta, and kappa opioid receptors.

We have mapped the residues in the sixth transmembrane domains (TMs 6) of the mu, delta, and kappa opioid receptors that are accessible in the binding-site crevices by the substituted cysteine accessibility method (SCAM). We previously showed that ligand binding to the C7.38S mutants of the mu and kappa receptors and the wild-type delta receptor was relatively insensitive to methanethiosulfonate ethylammonium (MTSEA), a positively charged sulfhydryl-specific reagent. These MTSEA-insensitive constructs were used as the templates, and 22 consecutive residues in TM6 (excluding C6.47) of each receptor were mutated to cysteine, 1 at a time. Most mutants retained binding affinities for [3H]diprenorphine, a nonselective opioid antagonist, similar to that of the template receptors. Treatment with MTSEA significantly inhibited [3H]diprenorphine binding to 11 of 22 mutants of the rat mu receptor and 9 of 22 mutants of the human delta receptor and 10 of 22 mutants of the human kappa receptor. Naloxone or diprenorphine protected all sensitive mutants, except the A6.42(287)C mu mutant. Thus, V6.40, F6.44, W6.48, I6.51, Y6.54, V6.55, I6.56, I6.57, K6.58, and A6.59 of the mu receptor; F6.44, I6.51, F6.54, V6.55, I6.56, V6.57, W6.58, T6.59, and L6.60 of the delta receptor; and F6.44, W6.48, I6.51, F6.54, I6.55, L6.56, V6.57, E6.58, A6.59, and L6.60 of the kappa receptor are on the water-accessible surface of the binding-site crevices. The accessibility patterns of residues in the TMs 6 of the mu, delta, and kappa opioid receptors are consistent with the notion that the TMs 6 are in alpha-helical conformations with a narrow strip of accessibility on the intracellular side of 6.54 and a wider area of accessibility on the extracellular side of 6.54, likely due to a proline kink at 6.50 that bends the helix in toward the binding pocket and enables considerable motion in this region. The wider exposure of residues 6.55-6.60 to the binding-site crevice, combined with the divergent amino acid sequences, is consistent with the inferred role of residues in this region in determining ligand binding selectivity. The conservation of the accessibility pattern on the cytoplasmic side of 6.54 suggests that this region may be important for receptor activation. This accessibility pattern is similar to that of the D2 dopamine receptor, the only other GPCR in which TM6 has been mapped by SCAM. That opioid receptors and the remotely related D2 dopamine receptor have similar accessibility patterns in TM6 suggest that these segments of GPCRs in the rhodopsin-like subfamily not only share secondary structure but also are packed similarly into the transmembrane bundle and thus have similar tertiary structure.     

GABA(A) receptor beta 2 Tyr97 and Leu99 line the GABA-binding site. Insights into mechanisms of agonist and antagonist actions.

The identification of residues that line neurotransmitter-binding sites and catalyze allosteric transitions that result in channel gating is crucial for understanding ligand-gated ion channel function. In this study, we used the substituted cysteine accessibility method and two-electrode voltage clamp to identify novel gamma-aminobutyric acid (GABA)-binding site residues and to elucidate the secondary structure of the Trp(92)-Asp(101) region of the beta(2) subunit. Each residue was mutated individually to cysteine and expressed with wild-type alpha(1) subunits in Xenopus oocytes. GABA-gated currents (I(GABA)) were measured before and after exposure to the sulfhydryl reagent, N-biotinylaminoethyl methanethiosulfonate (MTS). V93C, D95C, Y97C, and L99C are accessible to derivatization. This pattern of accessibility is consistent with beta(2)Val(93)-Leu(99) adopting a beta-strand conformation. Both GABA and SR95531 protect Y97C and L99C from modification, indicating that these two residues line the GABA-binding site. In D95C-containing receptors, application of MTS in the presence of SR95531 causes a greater effect on I(GABA) than MTS alone, suggesting that binding of a competitive antagonist can cause movements in the binding site. In addition, we present evidence that beta(2)L99C homomers form spontaneously open channels. Thus, mutation of a binding site residue can alter channel gating, which implies that Leu(99) may be important for coupling agonist binding to channel gating.     

Comparison of taurine- and glycine-induced conformational changes in the M2-M3 domain of the glycine receptor

In the ionotropic glutamate receptor, the global conformational changes induced by partial agonists are smaller than those induced by full agonists. However, in the pentameric ligand-gated ion channel receptor family, the structural basis of partial agonism is not understood. This study investigated whether full and partial agonists induce different conformation changes in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility analysis demonstrated previously that glycine binding induced an increase in surface accessibility of all residues from Arg(271) to Lys(276) in the M2-M3 domain of the homomeric alpha1 GlyR. Here we compare the surface accessibility changes induced by the full agonist, glycine, and the partial agonist, taurine. In GlyRs incorporating the A272C, S273C, L274C, or P275C mutation, the reaction rate of the cysteine-specific compound, methanethiosulfonate ethyltrimethylammonium, depended on how strongly the receptors were activated but was agonist-independent. Reaction rates could not be compared in the R271C and K276C mutant GlyRs because methanethiosulfonate ethyltrimethylammonium did not modify the extremely small currents induced by saturating taurine or equivalent low glycine concentrations. The results indicate that bound taurine and glycine molecules impose identical conformational changes to the M2-M3 domain. We therefore conclude that the higher efficacy of glycine is due to an increased ability to stabilize a common activated configuration.     

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.     

Identification of amino acid residues contributing to the pore of a P2X receptor.

P2X receptors are ion channels opened by extracellular ATP. The seven subunits currently known are encoded by different genes. It is thought that each subunit has two transmembrane domains, a large extracellular loop, and intracellular N- and C-termini, a topology which is fundamentally different from that of other ligand-gated channels such as nicotinic acetylcholine or glutamate receptors. We used the substituted cysteine accessibility method to identify parts of the molecule that form the ionic pore of the P2X2 receptor. Amino acids preceding and throughout the second hydrophobic domain (316-354) were mutated individually to cysteine, and the DNAs were expressed in HEK293 cells. For three of the 38 residues (I328C, N333C, T336C), currents evoked by ATP were inhibited by extracellular application of methanethiosulfonates of either charge (ethyltrimethylammonium, ethylsulfonate) suggesting that they lie in the outer vestibule of the pore. For two further substitutions (L338C, D349C) only the smaller ethylamine derivative inhibited the current. L338C was accessible to cysteine modification whether or not the channel was opened by ATP, but D349C was inhibited only when ATP was concurrently applied. The results indicate that part of the pore of the P2X receptor is formed by the second hydrophobic domain, and that L338 and D349 are on either side of the channel 'gate'.     

A picrotoxin-specific conformational change in the glycine receptor M2-M3 loop

The external loop linking the M2 and M3 transmembrane domains is crucial for coupling agonist binding to channel gating in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility scan previously showed that glycine activation increased the surface accessibility of 6 contiguous residues (Arg271-Lys276) toward the N-terminal end of the homomeric alpha1 GlyR M2-M3 loop. In the present study we used a similar approach to determine whether the allosteric antagonist, picrotoxin, could impose conformational changes to this domain that cannot be induced by varying agonist concentrations alone. Picrotoxin slowed the reaction rate of a sulfhydryl-containing compound (MTSET) with A272C, S273C, and L274C. Before interpreting this as a picrotoxin-specific conformational change, it was necessary to eliminate the possibility of steric competition between picrotoxin and MTSET. Accordingly, we showed that picrotoxin and the structurally unrelated blocker, bilobalide, were both trapped in the R271C GlyR in the closed state and that a point mutation to the pore-lining Thr6' residue abolished inhibition by both compounds. We also demonstrated that the picrotoxin dissociation rate was linearly related to the channel open probability. These observations constitute a strong case for picrotoxin binding in the pore. We thus conclude that the picrotoxin-specific effects on the M2-M3 loop are mediated allosterically. This suggests that the M2-M3 loop responds differently to the occupation of different binding sites.     

On the benzodiazepine binding pocket in GABAA receptors

Benzodiazepines are used for their sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsive effects. They exert their actions through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the gamma-aminobutyric acid, type A (GABA(A)) receptor channel, where they act as positive allosteric modulators. To start to elucidate the relative positioning of benzodiazepine binding site ligands in their binding pocket, GABA(A) receptor residues thought to reside in the site were individually mutated to cysteine and combined with benzodiazepine analogs carrying substituents reactive to cysteine. Direct apposition of such reactive partners is expected to lead to an irreversible site-directed reaction. We describe here the covalent interaction of alpha(1)H101C with a reactive group attached to the C-7 position of diazepam. This interaction was studied at the level of radioactive ligand binding and at the functional level using electrophysiological methods. Covalent reaction occurs concomitantly with occupancy of the binding pocket. It stabilizes the receptor in its allosterically stimulated conformation. Covalent modification is not observed in wild type receptors or when using mutated alpha(1)H101C-containing receptors in combination with the reactive ligand pre-reacted with a sulfhydryl group, and the modification rate is reduced by the binding site ligand Ro15-1788. We present in addition evidence that gamma(2)Ala-79 is probably located in the access pathway of the ligand to its binding pocket.     

Conformational changes at benzodiazepine binding sites of GABA(A) receptors detected with a novel technique

Benzodiazepines are widely used for their anxiolytic, sedative, myorelaxant and anticonvulsant properties. They allosterically modulate GABA(A) receptor function by increasing the apparent affinity of the agonist GABA. We studied conformational changes induced by channel agonists at the benzodiazepine binding site. We used the rate of covalent reaction between a benzodiazepine carrying a cysteine reactive moiety with mutated receptor having a cysteine residue in the benzodiazepine binding pocket, alpha1H101Cbeta2gamma2, as a sensor of its conformation. This reaction rate is sensitive to local conformational changes. Covalent reaction locks the receptor in the conformation stabilized by positive allosteric modulators. By using concatenated subunits we demonstrated that the covalent reaction occurs either exclusively at the alpha/gamma subunit interface, or if it occurs in both alpha1 subunits, exclusively reaction at the alpha/gamma subunit interface can modulate the receptor. We found evidence for an increased rate of reaction of activated receptors, whereas reaction rate with the desensitized state is slowed down. The benzodiazepine antagonist Ro15-1788 efficiently inhibited the covalent reaction in the presence of 100 microm GABA but only partially in its absence or in the presence of 10 microm GABA. It is concluded that Ro15-1788 efficiently protects activated and desensitized states, but not the resting state.     

The role of conserved extracellular cysteine residues in vasopressin V2 receptor function and properties of two naturally occurring mutant receptors with additional extracellular cysteine residues

The G protein-coupled vasopressin V2 receptor (V2 receptor) contains a pair of conserved cysteine residues (C112 and C192) which are thought to form a disulfide bond between the first and second extracellular loops. The conserved cysteine residues were found to be important for the correct formation of the ligand binding domain of some G protein-coupled receptors. Here we have assessed the properties of the V2 receptor after site-directed mutagenesis of its conserved cysteine residues in transiently transfected human embryonic kidney (HEK 293) cells. Mutant receptors (C112S, C112A and C192S, C192A) were non-functional and located mostly in the cell's interior. The conserved cysteine residues of the V2 receptor are thus not only important for the structure of the ligand binding domain but also for efficient intracellular receptor transport. In addition to the functional significance of the conserved cysteine residues, we have also analyzed the defects of two mutant V2 receptors which cause X-linked nephrogenic diabetes insipidus (NDI) by the introduction of additional cysteine residues into the second extracellular loop (mutants G185C, R202C). These mutations are assumed to impair normal disulfide bond formation. Mutant receptor G185C and R202C were efficiently transported to the plasma membrane but were defective in ligand binding. Only in the case of the mutant receptor R202C, the more sensitive adenylyl cyclase activity assay revealed vasopressin-stimulated cAMP formation with a 35-fold increased EC(50) value and with a reduced EC(max), indicating that ligand binding is not completely abolished. Taking the unaffected intracellular transport of both NDI-causing mutant receptors into account, our results indicate that the observed impairment of ligand binding by the additional cysteine residues is not due to the prevention of disulfide bond formation between the conserved cysteine residues.     

Analysis of the third transmembrane domain of the human type 1 angiotensin II receptor by cysteine scanning mutagenesis

Activation of G protein-coupled receptors by agonists involves significant movement of transmembrane domains (TMD) following agonist binding. The underlying structural mechanism by which receptor activation takes place is largely unknown but can be inferred by detecting variability within the environment of the ligand-binding pocket, which is a water-accessible crevice surrounded by the seven TMD helices. Using the substituted-cysteine accessibility method, we identified the residues within the third TMD of the wild-type angiotensin II (AT1) receptor that contribute to the formation of the binding site pocket. Each residue within the Ile103-Tyr127 region was mutated one at a time to a cysteine. Treating the A104C, N111C, and L112C mutant receptors with the charged sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA) strongly inhibited ligand binding, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD3 reporter cysteines engineered in a constitutively active AT1 receptor. Indeed, two additional mutants (S109C and V116C) were found to be sensitive to MTSEA treatment. Our results suggest that constitutive activation of the AT1 receptor causes a minor counterclockwise rotation of TMD3, thereby exposing residues, which are not present in the inactive state, to the binding pocket. This pattern of accessibility of residues in the TMD3 of the AT1 receptor parallels that of homologous residues in rhodopsin. This study identified key elements of TMD3 that contribute to the activation of class A G protein-coupled receptors through structural rearrangements.     

Mutational and Cysteine Scanning Analysis of the Glucagon Receptor N-terminal Domain

The glucagon receptor belongs to the B family of G-protein coupled receptors. Little structural information is available about this receptor and its association with glucagon. We used the substituted cysteine accessibility method and three-dimensional molecular modeling based on the gastrointestinal insulinotropic peptide and glucagon-like peptide 1 receptor structures to study the N-terminal domain of this receptor, a central element for ligand binding and specificity. Our results showed that Asp⁶³, Arg¹¹⁶, and Lys⁹⁸ are essential for the receptor structure and/or ligand binding because mutations of these three residues completely disrupted or markedly impaired the receptor function. In agreement with these data, our models revealed that Asp⁶³ and Arg¹¹⁶ form a salt bridge, whereas Lys⁹⁸ is engaged in cation-π interactions with the conserved tryptophans 68 and 106. The native receptor could not be labeled by hydrophilic cysteine biotinylation reagents, but treatment of intact cells with [2-(trimethylammonium)ethyl]methanethiosulfonate increased the glucagon binding site density. This result suggested that an unidentified protein with at least one free cysteine associated with the receptor prevented glucagon recognition and that [2-(trimethylammonium)ethyl]methanethiosulfonate treatment relieved this inhibition. The substituted cysteine accessibility method was also performed on 15 residues selected using the three-dimensional models. Several receptor mutants, despite a relatively high predicted cysteine accessibility, could not be labeled by specific reagents. The three-dimensional models show that these mutated residues are located on one face of the protein. This could be part of the interface between the receptor and the unidentified inhibitory protein, making these residues inaccessible to biotinylation compounds.