Molecular determinants of ivermectin sensitivity at the glycine receptor chloride channel

Ivermectin is an anthelmintic drug that works by activating glutamate-gated chloride channel receptors (GluClRs) in nematode parasites. GluClRs belong to the Cys-loop receptor family that also includes glycine receptor (GlyR) chloride channels. GluClRs and A288G mutant GlyRs are both activated by low nanomolar ivermectin concentrations. The crystal structure of the Caenorhabditis elegans α GluClR complexed with ivermectin has recently been published. Here, we probed ivermectin sensitivity determinants on the α1 GlyR using site-directed mutagenesis and electrophysiology. Based on a mutagenesis screen of transmembrane residues, we identified Ala288 and Pro230 as crucial sensitivity determinants. A comparison of the actions of selamectin and ivermectin suggested the benzofuran C05-OH was required for high efficacy. When taken together with docking simulations, these results supported a GlyR ivermectin binding orientation similar to that seen in the GluClR crystal structure. However, whereas the crystal structure shows that ivermectin interacts with the α GluClR via H-bonds with Leu218, Ser260, and Thr285 (α GluClR numbering), our data indicate that H-bonds with residues homologous to Ser260 and Thr285 are not important for high ivermectin sensitivity or direct agonist efficacy in A288G α1 GlyRs or three other GluClRs. Our data also suggest that van der Waals interactions between the ivermectin disaccharide and GlyR M2-M3 loop residues are unimportant for high ivermectin sensitivity. Thus, although our results corroborate the ivermectin binding orientation as revealed by the crystal structure, they demonstrate that some of the binding interactions revealed by this structure do not pertain to other highly ivermectin-sensitive Cys-loop receptors.     

Incompatibility between a pair of residues from the pre-M1 linker and Cys-loop blocks surface expression of the glycine receptor

Regulation of cell membrane excitability can be achieved either by modulating the functional properties of cell membrane-expressed single channels or by varying the number of expressed channels. Whereas the structural basis underlying single channel properties has been intensively studied, the structural basis contributing to surface expression is less well characterized. Here we demonstrate that homologous substitution of the pre-M1 linker from the β subunit prevents surface expression of the α1 glycine receptor chloride channel. By investigating a series of chimeras comprising α1 and β subunits, we hypothesized that this effect was due to incompatibility between a pair of positively charged residues, which lie in close proximity to each other in the tertiary structure, from the pre-M1 linker and Cys-loop. Abolishing either positive charge restored surface expression. We propose that incompatibility (electrostatic repulsion) between this pair of residues misfolds the glycine receptor, and in consequence, the protein is retained in the cytoplasm and prevented from surface expression by the quality control machinery. This hypothesis suggests a novel mechanism, i.e. residue incompatibility, for explaining the mutation-induced reduction in channel surface expression, often present in the cases of hereditary hyperekplexia.     

Analysis of Hyperekplexia Mutations Identifies Transmembrane Domain Rearrangements That Mediate Glycine Receptor Activation

Pentameric ligand-gated ion channels (pLGICs) mediate numerous physiological processes and are therapeutic targets for a wide range of clinical indications. Elucidating the structural differences between their closed and open states may help in designing improved drugs that bias receptors toward the desired conformational state. We recently showed that two new hyperekplexia mutations, Q226E and V280M, induced spontaneous activity in α1 glycine receptors. Gln-226, located near the top of transmembrane (TM) 1, is closely apposed to Arg-271 at the top of TM2 in the neighboring subunit. Using mutant cycle analysis, we inferred that Q226E induces activation via an enhanced electrostatic attraction to Arg-271. This would tilt the top of TM2 toward TM1 and hence away from the pore axis to open the channel. We also concluded that the increased side chain volume of V280M, in the TM2-TM3 loop, exerts a steric repulsion against Ile-225 at the top of TM1 in the neighboring subunit. We infer that this steric repulsion would tilt the top of TM3 radially outwards against the stationary TM1 and thus provide space for TM2 to relax away from the pore axis to create an open channel. Because the transmembrane domain movements inferred from this functional analysis are consistent with the structural differences evident in the x-ray atomic structures of closed and open state bacterial pLGICs, we propyose that the model of pLGIC activation as outlined here may be broadly applicable across the eukaryotic pLGIC receptor family.     

Desensitization of alpha7 nicotinic receptor is governed by coupling strength relative to gate tightness

Binding of a neurotransmitter to its membrane receptor opens an integral ion conducting pore. However, prolonged exposure to the neurotransmitter drives the receptor to a refractory state termed desensitization, which plays an important role in shaping synaptic transmission. Despite intensive research in the past, the structural mechanism of desensitization is still elusive. Using mutagenesis and voltage clamp in an oocyte expression system, we provide several lines of evidence supporting a novel hypothesis that uncoupling between binding and gating machinery is the underlying mechanism for α7 nicotinic receptor (nAChR) desensitization. First, the decrease in gate tightness was highly correlated to the reduced desensitization. Second, nonfunctional mutants in three important coupling loops (loop 2, loop 7, and the M2-M3 linker) could be rescued by a gating mutant. Furthermore, the decrease in coupling strength in these rescued coupling loop mutants reversed the gating effect on desensitization. Finally, coupling between M1 and hinge region of the M2-M3 linker also influenced the receptor desensitization. Thus, the uncoupling between N-terminal domain and transmembrane domain, governed by the balance of coupling strength and gate tightness, underlies the mechanism of desensitization for the α7 nAChR.     

Contributions of conserved residues at the gating interface of glycine receptors

Glycine receptors (GlyRs) are chloride channels that mediate fast inhibitory neurotransmission and are members of the pentameric ligand-gated ion channel (pLGIC) family. The interface between the ligand binding domain and the transmembrane domain of pLGICs has been proposed to be crucial for channel gating and is lined by a number of charged and aromatic side chains that are highly conserved among different pLGICs. However, little is known about specific interactions between these residues that are likely to be important for gating in α1 GlyRs. Here we use the introduction of cysteine pairs and the in vivo nonsense suppression method to incorporate unnatural amino acids to probe the electrostatic and hydrophobic contributions of five highly conserved side chains near the interface, Glu-53, Phe-145, Asp-148, Phe-187, and Arg-218. Our results suggest a salt bridge between Asp-148 in loop 7 and Arg-218 in the pre-M1 domain that is crucial for channel gating. We further propose that Phe-145 and Phe-187 play important roles in stabilizing this interaction by providing a hydrophobic environment. In contrast to the equivalent residues in loop 2 of other pLGICs, the negative charge at Glu-53 α1 GlyRs is not crucial for normal channel function. These findings help decipher the GlyR gating pathway and show that distinct residue interaction patterns exist in different pLGICs. Furthermore, a salt bridge between Asp-148 and Arg-218 would provide a possible mechanistic explanation for the pathophysiologically relevant hyperekplexia, or startle disease, mutant Arg-218 → Gln.     

The Minimum M3-M4 Loop Length of Neurotransmitter-activated Pentameric Receptors Is Critical for the Structural Integrity of Cytoplasmic Portals

The 5-HT₃A receptor homology model, based on the partial structure of the nicotinic acetylcholine receptor from Torpedo marmorata, reveals an asymmetric ion channel with five portals framed by adjacent helical amphipathic (HA) stretches within the 114-residue loop between the M3 and M4 membrane-spanning domains. The positive charge of Arg-436, located within the HA stretch, is a rate-limiting determinant of single channel conductance (γ). Further analysis reveals that positive charge and volume of residue 436 are determinants of 5-HT₃A receptor inward rectification, exposing an additional role for portals. A structurally unresolved stretch of 85 residues constitutes the bulk of the M3-M4 loop, leaving a >45-Å gap in the model between M3 and the HA stretch. There are no additional structural data for this loop, which is vestigial in bacterial pentameric ligand-gated ion channels and was largely removed for crystallization of the Caenorhabditis elegans glutamate-activated pentameric ligand-gated ion channels. We created 5-HT3A subunit loop truncation mutants, in which sequences framing the putative portals were retained, to determine the minimum number of residues required to maintain their functional integrity. Truncation to between 90 and 75 amino acids produced 5-HT₃A receptors with unaltered rectification. Truncation to 70 residues abolished rectification and increased γ. These findings reveal a critical M3-M4 loop length required for functions attributable to cytoplasmic portals. Examination of all 44 subunits of the human neurotransmitter-activated Cys-loop receptors reveals that, despite considerable variability in their sequences and lengths, all M3-M4 loops exceed 70 residues, suggesting a fundamental requirement for portal integrity.     

Distinct conformational changes in activated agonist-bound and agonist-free glycine receptor subunits

Ligand binding to Cys-loop receptors produces either global conformational changes that lead to activation or local conformational changes that do not. We found that the fluorescence of a fluorophore tethered to R271C in the extracellular M2 region of the α1 glycine receptor increases during glycine activation but not during ivermectin activation. This prompted the hypothesis that this signal reports a glycine-mediated conformational change not essential for activation. We tested this by investigating whether the fluorescence signal depended on whether the fluorophore was attached to a glycine-free or a glycine-bound subunit. Agonist-free subunits were created by incorporating T204A and R65K mutations, which disrupted glycine binding to both (+) and (−) subunit interfaces. In heteromeric receptors comprising wild-type and R65K,T204A,R271C triple-mutant subunits, the fluorescence response exhibited a drastically reduced glycine sensitivity relative to the current response. Two conclusions can be drawn from this. First, because the labeled glycine-free subunits were activated by glycine binding to neighboring wild-type subunits, our results provide evidence for a cooperative activation mechanism. However, because the fluorescent label on glycine-free subunits does not reflect movements at the channel gate, we conclude that glycine binding also produces a local non-concerted conformational change that is not essential for receptor activation.     

Structural basis of M3 muscarinic receptor dimer/oligomer formation

Class A G protein-coupled receptors (GPCRs) are known to form dimers and/or oligomeric arrays in vitro and in vivo. These complexes are thought to play important roles in modulating class A GPCR function. Many studies suggest that residues located on the "outer" (lipid-facing) surface of the transmembrane (TM) receptor core are critically involved in the formation of class A receptor dimers (oligomers). However, no clear consensus has emerged regarding the identity of the TM helices or TM subsegments involved in this process. To shed light on this issue, we have used the M(3) muscarinic acetylcholine receptor (M3R), a prototypic class A GPCR, as a model system. Using a comprehensive and unbiased approach, we subjected all outward-facing residues (70 amino acids total) of the TM helical bundle (TM1-7) of the M3R to systematic alanine substitution mutagenesis. We then characterized the resulting mutant receptors in radioligand binding and functional studies and determined their ability to form dimers (oligomers) in bioluminescence resonance energy transfer saturation assays. We found that M3R/M3R interactions are not dependent on the presence of one specific structural motif but involve the outer surfaces of multiple TM subsegments (TM1-5 and -7) located within the central and endofacial portions of the TM receptor core. Moreover, we demonstrated that the outward-facing surfaces of most TM helices play critical roles in proper receptor folding and/or function. Guided by the bioluminescence resonance energy transfer data, molecular modeling studies suggested the existence of multiple dimeric/oligomeric M3R arrangements, which may exist in a dynamic equilibrium. Given the high structural homology found among all class A GPCRs, our results should be of considerable general relevance.     

Conformational changes at the agonist binding domain of the N-methyl-D-aspartic acid receptor

The conformational changes in the agonist binding domain of the glycine-binding GluN1 and glutamate-binding GluN2A subunits of the N-methyl D-aspartic acid receptor upon binding agonists of varying efficacy have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances indicate a cleft closure conformational change at the GluN1 subunit upon binding agonists; however, no significant changes in the cleft closure are observed between partial and full agonists. This is consistent with the previously reported crystal structures for the isolated agonist binding domain of this receptor. Additionally, the LRET-based distances show that the agonist binding domain of the glutamate-binding GluN2A subunit exhibits a graded cleft closure with the extent of cleft closure being proportional to the extent of activation, indicating that the mechanism of activation in this subunit is similar to that of the glutamate binding α-amino-5-methyl-3-hydroxy-4-isoxazole propionate and kainate subtypes of the ionotropic glutamate receptors.     

Identification of interaction sites for dimerization and adapter recruitment in Toll/interleukin-1 receptor (TIR) domain of Toll-like receptor 4

Toll-like receptor signaling requires interactions of the Toll/IL-1 receptor (TIR) domains of the receptor and adapter proteins. Using the mammalian protein-protein interaction trap strategy, homology modeling, and site-directed mutagenesis, we identify the interaction surfaces in the TLR4 TIR domain for the TLR4-TLR4, TLR4-MyD88 adapter-like (MAL), and TLR4-TRIF-related adapter molecule (TRAM) interaction. Two binding sites are equally important for TLR4 dimerization and adapter recruitment. In a model based on the crystal structure of the dimeric TLR10 TIR domain, the first binding site mediates TLR4-TLR4 TIR-TIR interaction. Upon dimerization, two identical second binding sites of the TLR4 TIR domain are juxtaposed and form an extended binding platform for both MAL and TRAM. In our mammalian protein-protein interaction trap assay, MAL and TRAM compete for binding to this platform. Our data suggest that adapter binding can stabilize the TLR4 TIR dimerization.     

Delineation of the structural determinants of the N-methyl-D-aspartate receptor glycine binding site

In this study, we have further delineated the domains of the N-methyl-D-aspartate receptor NR1 subunit that contribute to the glycine co-agonist binding site. Taking an iterative approach, we have constructed truncation mutants of the NR1 subunit, transiently expressed them in HEK-293 cells, and determined the binding of the glycine site antagonist [3H]L-689,560. Amino acids 380-811 were sufficient to form a glycine binding site with affinities for [3H]L-689,560 and glycine that were not significantly different from wild-type NR1. More extensive deletions, from either the amino- or the carboxy-terminal end, resulted in loss of ligand binding. Additional constructs were made starting from amino acids 380-843 of NR1, replacing the transmembrane (TMI-TMIII) domain with intervening linker sequences while retaining the TMIV domain so as to anchor the polypeptide to the membrane. Although robust amounts of polypeptides were synthesised by transfected cells, only low levels of [3H]L-689,560 binding sites could be detected. This suggests that only a small proportion of the synthesised polypeptide folds in the appropriate manner so as to form a ligand binding site. These data indicate that although it is possible to reduce the glycine binding site to minimal so-called S1 and S2 domains, efficient folding of the polypeptide so as to form a ligand binding site may require sequences within the TMI-TMIII domain.     

T cell receptors are structures capable of initiating signaling in the absence of large conformational rearrangements

Native and non-native ligands of the T cell receptor (TCR), including antibodies, have been proposed to induce signaling in T cells via intra- or intersubunit conformational rearrangements within the extracellular regions of TCR complexes. We have investigated whether any signatures can be found for such postulated structural changes during TCR triggering induced by antibodies, using crystallographic and mutagenesis-based approaches. The crystal structure of murine CD3ε complexed with the mitogenic anti-CD3ε antibody 2C11 enabled the first direct structural comparisons of antibody-liganded and unliganded forms of CD3ε from a single species, which revealed that antibody binding does not induce any substantial rearrangements within CD3ε. Saturation mutagenesis of surface-exposed CD3ε residues, coupled with assays of antibody-induced signaling by the mutated complexes, suggests a new configuration for the complex within which CD3ε is highly exposed and reveals that no large new CD3ε interfaces are required to form during antibody-induced signaling. The TCR complex therefore appears to be a structure that is capable of initiating intracellular signaling in T cells without substantial structural rearrangements within or between the component subunits. Our findings raise the possibility that signaling by native ligands might also be initiated in the absence of large structural rearrangements in the receptor.     

Opportunities at the interface of chemistry and biology

The combination of the tools and principles of chemistry, together with the tools of modern molecular biology, allow us to create complex synthetic and natural molecules, and processes with novel biological, chemical and physical properties. This article illustrates the tremendous opportunity that lies at this interface of chemistry and biology by describing a number of examples, ranging from efforts to expand the genetic code of living organisms to the use of combinatorial methods to generate biologically active synthetic molecules.     

N-glycosylation determines ionic permeability and desensitization of the TRPV1 capsaicin receptor

The balance of glycosylation and deglycosylation of ion channels can markedly influence their function and regulation. However, the functional importance of glycosylation of the TRPV1 receptor, a key sensor of pain-sensing nerves, is not well understood, and whether TRPV1 is glycosylated in neurons is unclear. We report that TRPV1 is N-glycosylated and that N-glycosylation is a major determinant of capsaicin-evoked desensitization and ionic permeability. Both N-glycosylated and unglycosylated TRPV1 was detected in extracts of peripheral sensory nerves by Western blotting. TRPV1 expressed in HEK-293 cells exhibited various degrees of glycosylation. A mutant of asparagine 604 (N604T) was not glycosylated but did not alter plasma membrane expression of TRPV1. Capsaicin-evoked increases in intracellular calcium ([Ca(2+)](i)) were sustained in wild-type TRPV1 HEK-293 cells but were rapidly desensitized in N604T TRPV1 cells. There was marked cell-to-cell variability in capsaicin responses and desensitization between individual cells expressing wild-type TRPV1 but highly uniform responses in cells expressing N604T TRPV1, consistent with variable levels of glycosylation of the wild-type channel. These differences were also apparent when wild-type or N604T TRPV1-GFP fusion proteins were expressed in neurons from trpv1(-/-) mice. Capsaicin evoked a marked, concentration-dependent increase in uptake of the large cationic dye YO-PRO-1 in cells expressing wild-type TRPV1, indicative of loss of ion selectivity, that was completely absent in cells expressing N604T TRPV1. Thus, TRPV1 is variably N-glycosylated and glycosylation is a key determinant of capsaicin regulation of TRPV1 desensitization and permeability. Our findings suggest that physiological or pathological alterations in TRPV1 glycosylation would affect TRPV1 function and pain transmission.     

Agonist- and Ca2+-dependent desensitization of TRPV1 channel targets the receptor to lysosomes for degradation

TRPV1 receptor agonists such as the vanilloid capsaicin and the potent analog resiniferatoxin are well known potent analgesics. Depending on the vanilloid, dose, and administration site, nociceptor refractoriness may last from minutes up to months, suggesting the contribution of different cellular mechanisms ranging from channel receptor desensitization to Ca(2+) cytotoxicity of TRPV1-expressing neurons. The molecular mechanisms underlying agonist-induced TRPV1 desensitization and/or tachyphylaxis are still incompletely understood. Here, we report that prolonged exposure of TRPV1 to agonists induces rapid receptor endocytosis and lysosomal degradation in both sensory neurons and recombinant systems. Agonist-induced receptor internalization followed a clathrin- and dynamin-independent endocytic route, triggered by TRPV1 channel activation and Ca(2+) influx through the receptor. This process appears strongly modulated by PKA-dependent phosphorylation. Taken together, these findings indicate that TRPV1 agonists induce long-term receptor down-regulation by modulating the expression level of the channel through a mechanism that promotes receptor endocytosis and degradation and lend support to the notion that cAMP signaling sensitizes nociceptors through several mechanisms.     

The interaction between two extracellular linker regions controls sustained opening of acid-sensing ion channel 1

Activation of acid-sensing ion channels (ASICs) contributes to neuronal death during stroke, to axonal degeneration during neuroinflammation, and to pain during inflammation. Although understanding ASIC gating may help to modulate ASIC activity during these pathologic situations, at present it is poorly understood. The ligand, H(+), probably binds to several sites, among them amino acids within the large extracellular domain. The extracellular domain is linked to the two transmembrane domains by the wrist region that is connected to two anti-parallel β-strands, β1 and β12. Thus, the wrist region together with those β-strands may have a crucial role in transmitting ligand binding to pore opening and closing. Here we show that amino acids in the β1-β2 linker determine constitutive opening of ASIC1b from shark. The most crucial residue within the β1-β2 linker (Asp(110)), when mutated from aspartate to cysteine, can be altered by cysteine-modifying reagents much more readily when channels are closed than when they are desensitized. Finally, engineering of a cysteine at position 110 and at an adjacent position in the β11-β12 linker leads to spontaneous formation of a disulfide bond that traps the channel in the desensitized conformation. Collectively, our results suggest that the β1-β2 and β11-β12 linkers are dynamic during gating and tightly appose to each other during desensitization gating. Hindrance of this tight apposition leads to reopening of the channel. It follows that the β1-β2 and β11-β12 linkers modulate gating movements of ASIC1 and may thus be drug targets to modulate ASIC activity.     

N-formyl peptide receptor 3 (FPR3) departs from the homologous FPR2/ALX receptor with regard to the major processes governing chemoattractant receptor regulation, expression at the cell surface, and phosphorylation

Among human N-formyl peptide chemoattractant receptors, FPR2/ALX and FPR3 share the highest degree of amino acid identity (83%), and trigger similar cell responses upon ligand binding. Although FPR2/ALX is a promiscuous receptor, FPR3 has only one specific high affinity ligand, F2L, and a more restricted tissue/cell distribution. In this study, we showed that FPR2/ALX behaved as the prototypical receptor FPR1. The agonist-dependent phosphorylation used a hierarchical mechanism with a prominent role of Ser(329), Thr(332), and Thr(335). Phosphorylation of FPR2/ALX was essential for its desensitization but the lack of phosphorylation did not result in enhanced or sustained responses. In contrast, resting FPR3 displayed a marked level of phosphorylation, which was only slightly increased upon agonist stimulation. Another noticeable difference between the two receptors was their subcellular distribution in unstimulated cells. Although FPR2/ALX was evenly distributed at the plasma membrane FPR3 was localized in small intracellular vesicles. By swapping domains between FPR2/ALX and FPR3, we uncovered the determinants involved in the basal phosphorylation of FPR3. Experiments aimed at monitoring receptor-bound antibody uptake showed that the intracellular distribution of FPR3 resulted from a constitutive internalization that was independent of C terminus phosphorylation. Unexpectedly, exchanging residues 1 to 53, which encompass the N-terminal extracellular region and the first transmembrane domain, between FPR2/ALX and FPR3 switched localization of the receptors from the plasma membrane to intracellular vesicles and vice versa. A clathrin-independent, possibly caveolae-dependent, mechanism was involved in FPR3 constitutive internalization. The peculiar behavior of FPR3 most probably serves distinct physiological functions that remain largely unknown.     

Molecular Determinants of Allosteric Modulation at the M1 Muscarinic Acetylcholine Receptor

Benzylquinolone carboxylic acid (BQCA) is an unprecedented example of a selective positive allosteric modulator of acetylcholine at the M₁ muscarinic acetylcholine receptor (mAChR). To probe the structural basis underlying its selectivity, we utilized site-directed mutagenesis, analytical modeling, and molecular dynamics to delineate regions of the M₁ mAChR that govern modulator binding and transmission of cooperativity. We identified Tyr-85^2.64 in transmembrane domain 2 (TMII), Tyr-179 and Phe-182 in the second extracellular loop (ECL2), and Glu-397^7.32 and Trp-400^7.35 in TMVII as residues that contribute to the BQCA binding pocket at the M₁ mAChR, as well as to the transmission of cooperativity with the orthosteric agonist carbachol. As such, the BQCA binding pocket partially overlaps with the previously described “common” allosteric site in the extracellular vestibule of the M₁ mAChR, suggesting that its high subtype selectivity derives from either additional contacts outside this region or through a subtype-specific cooperativity mechanism. Mutation of amino acid residues that form the orthosteric binding pocket caused a loss of carbachol response that could be rescued by BQCA. Two of these residues (Leu-102^3.29 and Asp-105^3.32) were also identified as indirect contributors to the binding affinity of the modulator. This new insight into the structural basis of binding and function of BQCA can guide the design of new allosteric ligands with tailored pharmacological properties.     

A comparison of glycine- and ivermectin-mediated conformational changes in the glycine receptor ligand-binding domain

Glycine receptor chloride channels are Cys-loop receptor proteins that isomerize between a low affinity closed state and a high affinity ion-conducting state. There is currently much interest in understanding the mechanisms that link affinity changes with conductance changes. This essentially involves an agonist binding in the glycine receptor ligand-binding site initiating local conformational changes that propagate in a wave towards the channel gate. However, it has proved difficult to convincingly distinguish those agonist-induced domain movements that are critical for triggering activation from those that are simply local deformations to accommodate ligands in the site. We employed voltage-clamp fluorometry to compare conformational changes in the ligand-binding site in response to activation by glycine, which binds locally, and ivermectin, which binds in the transmembrane domain. We reasoned that ivermectin-mediated activation should initiate a conformational wave that propagates from the pore-lining domain towards the ligand-binding domain, eliciting conformational changes in those extracellular domains that are allosterically linked to the gate. We found that ivermectin indeed elicited conformational changes in ligand-binding domain loops C, D and F. This implies that conformational changes in these domains are important for activation. This result also provides a mechanism to explain how ivermectin potentiates glycine-induced channel activation.     

Role of the transmembrane domain 4/extracellular loop 2 junction of the human gonadotropin-releasing hormone receptor in ligand binding and receptor conformational selection

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.