Modeling and docking of the three-dimensional structure of the human melanocortin 4 receptor

A three-dimensional structure of the human melanocortin 4 receptor (hMC4R) is constructed in this study using a computer-aided molecular modeling approach. Human melanocortin 4 receptor is a G Protein-Coupled Receptor (GPCR). We structurally aligned transmembrane helices with bovine rhodopsin transmembrane domains, simulated both intracellular and extracellular loop domains on homologous loop regions in other proteins of known 3D structure and modeled the C terminus on the corresponding part of bovine rhodopsin. Then tandem minimization and dynamics calculations were run to refine the crude structure. The simulative model was tested by docking with a triplet peptide (RFF) ligand. It was found that the ligand is located among transmembrane regions TM3, TM4, TM5, and TM6 of hMC4R. In consistence with mutational and biochemical data, binding site is mainly formed as a hydrophobic and negatively charged pocket. The model constructed here might provide a structural framework for making rational predictions in relevant fields.     

Molecular modeling of human MT2 melatonin receptor: the role of Val204, Leu272 and Tyr298 in ligand binding

A model of the helical part of the human MT2 melatonin (hMT2) receptor, a member of the G protein-coupled receptors superfamily has been generated, based on the structure of bovine rhodopsin. Modeling has been combined with site-directed mutagenesis to investigate the role of the specific amino acid residues within the transmembrane domains (TM) numbers V, VI and VII of hMT2 receptor in the interaction with 2-iodomelatonin. Saturation binding assays with 2-iodomelatonin demonstrated that the substitution V204A (TMV) resulted in total loss of binding while the mutation V205A had no effect. The replacement of F209 with alanine led to a significant decrease in the Bmax value of receptor binding while mutations V205A and F209A also within TM V did not significantly change binding properties of the hMT2 receptor. In the case of TM VI, the substitution G271T caused substantial decrease in 2-iodomelatonin binding to the hMT2 receptor. The change L272A (TM VI) as well as mutation Y298A within TM VII completely abolished ligand binding to the receptor. These data suggest that several new amino acid residues within TM V, VI and VII are involved in ligand-MT2 receptor interaction.     

The non-competitive antagonists 2-methyl-6-(phenylethynyl)pyridine and 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester interact with overlapping binding pockets in the transmembrane region of group I metabotropic glutamate receptors

We have investigated the mechanism of inhibition and site of action of the novel human metabotropic glutamate receptor 5 (hmGluR5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), which is structurally unrelated to classical metabotropic glutamate receptor (mGluR) ligands. Schild analysis indicated that MPEP acts in a non-competitive manner. MPEP also inhibited to a large extent constitutive receptor activity in cells transiently overexpressing rat mGluR5, suggesting that MPEP acts as an inverse agonist. To investigate the molecular determinants that govern selective ligand binding, a mutagenesis study was performed using chimeras and single amino acid substitutions of hmGluR1 and hmGluR5. The mutants were tested for binding of the novel mGluR5 radioligand [(3)H]2-methyl-6-(3-methoxyphenyl)ethynyl pyridine (M-MPEP), a close analog of MPEP. Replacement of Ala-810 in transmembrane (TM) VII or Pro-655 and Ser-658 in TMIII with the homologous residues of hmGluR1 abolished radioligand binding. In contrast, the reciprocal hmGluR1 mutant bearing these three residues of hmGluR5 showed high affinity for [(3)H]M-MPEP. Radioligand binding to these mutants was also inhibited by 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt), a structurally unrelated non-competitive mGluR1 antagonist previously shown to interact with residues Thr-815 and Ala-818 in TMVII of hmGluR1. These results indicate that MPEP and CPCCOEt bind to overlapping binding pockets in the TM region of group I mGluRs but interact with different non-conserved residues.     

Mutational analysis and molecular modeling of the allosteric binding site of a novel,selective, noncompetitive antagonist of the metabotropic glutamate 1 receptor

A model of the rmGlu1 seven-transmembrane domain complexed with a negative allosteric modulator, 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)- 1,6-dihydro-pyrimidine-5-carbonitrile (EM-TBPC) was constructed. Although the mGlu receptors belong to the family 3 G-protein-coupled receptors with a low primary sequence similarity to rhodopsin-like receptors, the high resolution crystal structure of rhodopsin was successfully applied as a template in this model and used to select residues for site-directed mutagenesis. Three mutations, F801(6.51)A, Y805(6.55)A, and T815(7.39)M caused complete loss of the [(3)H]EM-TBPC binding and blocked the EM-TBPC-mediated inhibition of glutamate-evoked G-protein-coupled inwardly rectifying K(+) channel current and [Ca(2+)](i) response. The mutation W798(6.48)F increased the binding affinity of antagonist by 10-fold and also resulted in a marked decrease in the IC(50) value (4 versus 128 nm) compared with wild type. The V757(5.47)L mutation led to a dramatic reduction in binding affinity by 13-fold and a large increase in the IC(50) value (1160 versus 128 nm). Two mutations, N7474(5.51)A and N7504(5.54)A, increased the efficacy of the EM-TBPC block of the glutamate-evoked [Ca(2+)](i) response. We observed a striking conservation in the position of critical residues. The residues Val-757(5.47), Trp-798(6.48), Phe-801(6.51), Tyr-805(6.55), and Thr-815(7.39) are critical determinants of the EM-TBPC-binding pocket of the mGlu1 receptor, validating the rhodopsin crystal structure as a template for the family 3 G-protein-coupled receptors. In our model, the aromatic ring of EM-TBPC might interact with the cluster of aromatic residues formed from Trp-798(6.48), Phe-801(6.51), and Tyr-805(6.55), thereby blocking the movement of the TM6 helix, which is crucial for receptor activation.     

Defining the roles of Asn-128, Glu-129 and Phe-130 in loop A of the 5-HT3 receptor

The ligand binding pocket of Cys-loop receptors consists of a number of binding loops termed A-F. Here we examine the 5-HT3 receptor loop A residues Asn-128, Glu-129 and Phe-130 using modelling, mutagenesis, radioligand binding and functional studies on HEK 293 cells. Replacement of Asn-128 results in receptors that have wild type [3H]granisetron binding characteristics but large changes (ranging from a five-fold decrease to a 1500-fold increase) in the 5-HT EC50 when compared to wild type receptors. Phe-130 mutant receptors show both increases and decreases in Kd and EC50 values, depending on the amino acid substituted. The most critical of these residues appears to be Glu-129; its replacement with a range of other amino acids results in non-binding and non-functional receptors. Lack of binding and function in some, but not all, of these receptors is due to poor membrane expression. These data suggest that Glu-129 is important primarily for receptor expression, although it may also play a role in ligand binding; Phe-130 is important for both ligand binding and receptor function, and Asn-128 plays a larger role in receptor function than ligand binding. In light of these results, we have created two new homology models of the 5-HT3 receptor, with alternative positions of loop A. In our preferred model Glu-129 and Phe-130 contribute to the binding site, while the location of Asn-128 immediately behind the binding pocket could contribute to the conformation changes that result in receptor gating. This study provides a new model of the 5-HT3 receptor binding pocket, and also highlights the importance of experimental data to support modelling studies.     

Phe-308 and Phe-312 in transmembrane domain 7 are major sites of alpha 1-adrenergic receptor antagonist binding. Imidazoline agonists bind like antagonists

Although agonist binding in adrenergic receptors is fairly well understood and involves residues located in transmembrane domains 3 through 6, there are few residues reported that are involved in antagonist binding. In fact, a major docking site for antagonists has never been reported in any G-protein coupled receptor. It has been speculated that antagonist binding is quite diverse depending upon the chemical structure of the antagonist, which can be quite different from agonists. We now report the identification of two phenylalanine residues in transmembrane domain 7 of the alpha(1a)-adrenergic receptor (Phe-312 and Phe-308) that are a major site of antagonist affinity. Mutation of either Phe-308 or Phe-312 resulted in significant losses of affinity (4-1200-fold) for the antagonists prazosin, WB4101, BMY7378, (+) niguldipine, and 5-methylurapidil, with no changes in affinity for phenethylamine-type agonists such as epinephrine, methoxamine, or phenylephrine. Interestingly, both residues are involved in the binding of all imidazoline-type agonists such as oxymetazoline, cirazoline, and clonidine, confirming previous evidence that this class of ligand binds differently than phenethylamine-type agonists and may be more antagonist-like, which may explain their partial agonist properties. In modeling these interactions with previous mutagenesis studies and using the current backbone structure of rhodopsin, we conclude that antagonist binding is docked higher in the pocket closer to the extracellular surface than agonist binding and appears skewed toward transmembrane domain 7.     

Structural basis for ligand binding and specificity in adrenergic receptors: implications for GPCR-targeted drug discovery

Crystal structures of engineered human beta 2-adrenergic receptors (ARs) in complex with an inverse agonist ligand, carazolol, provide three-dimensional snapshots of the disposition of seven transmembrane helices and the ligand-binding site of an important G protein-coupled receptor (GPCR). As expected, beta 2-AR shares substantial structural similarities with rhodopsin, the dim-light photoreceptor of the rod cell. However, although carazolol and the 11- cis-retinylidene moiety of rhodopsin are situated in the same general binding pocket, the second extracellular (E2) loop structures are quite distinct. E2 in rhodopsin shows beta-sheet structure and forms part of the chromophore-binding site. In the beta 2-AR, E2 is alpha-helical and seems to be distinct from the receptor's active site, allowing a potential entry pathway for diffusible ligands. The structures, together with extensive structure-activity relationship (SAR) data from earlier studies, provide insight about possible structural determinants of ligand specificity and how the binding of agonist ligands might alter receptor conformation. We review key features of the new beta 2-AR structures in the context of recent complementary work on the conformational dynamics of GPCRs. We also report 600 ns molecular dynamics simulations that quantified beta 2-AR receptor mobility in a membrane bilayer environment and show how the binding of an agonist ligand, adrenaline (epinephrine), causes conformational changes to the ligand-binding pocket and neighboring helices.     

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.     

Defining the roles of Asn-128, Glu-129 and Phe-130 in loop A of the 5-HT3 receptor

The ligand binding pocket of Cys-loop receptors consists of a number of binding loops termed A–F. Here we examine the 5-HT₃ receptor loop A residues Asn-128, Glu-129 and Phe-130 using modelling, mutagenesis, radioligand binding and functional studies on HEK 293 cells. Replacement of Asn-128 results in receptors that have wild type [³H]granisetron binding characteristics but large changes (ranging from a five-fold decrease to a 1500-fold increase) in the 5-HT EC₅₀ when compared to wild type receptors. Phe-130 mutant receptors show both increases and decreases in K_d and EC₅₀ values, depending on the amino acid substituted. The most critical of these residues appears to be Glu-129; its replacement with a range of other amino acids results in non-binding and non-functional receptors. Lack of binding and function in some, but not all, of these receptors is due to poor membrane expression. These data suggest that Glu-129 is important primarily for receptor expression, although it may also play a role in ligand binding; Phe-130 is important for both ligand binding and receptor function, and Asn-128 plays a larger role in receptor function than ligand binding. In light of these results, we have created two new homology models of the 5-HT₃ receptor, with alternative positions of loop A. In our preferred model Glu-129 and Phe-130 contribute to the binding site, while the location of Asn-128 immediately behind the binding pocket could contribute to the conformation changes that result in receptor gating. This study provides a new model of the 5-HT₃ receptor binding pocket, and also highlights the importance of experimental data to support modelling studies.     

Phenoxybenzamine binding reveals the helical orientation of the third transmembrane domain of adrenergic receptors

Phenoxybenzamine (PB), a classical alpha-adrenergic antagonist, binds irreversibly to the alpha-adrenergic receptors (ARs). Amino acid sequence alignments and the predicted helical arrangement of the seven transmembrane (TM) domains suggested an accessible cysteine residue in transmembrane 3 of the alpha(2)-ARs, in position C(3.36) (in subtypes A, B, and C corresponding to amino acid residue numbers 117/96/135, respectively), as a possible site for the PB interaction. Irreversible binding of PB to recombinant human alpha(2)-ARs (90 nm, 30 min) reduced the ligand binding capacity of alpha(2A)-, alpha(2B)-, and alpha(2C)-AR by 81, 96, and 77%. When the TM3 cysteine, Cys(117), of alpha(2A)-AR was mutated to valine (alpha(2A)-C117V), the receptor became resistant to PB (inactivation, 10%). The beta(2)-AR contains a valine in this position (V(3.36); position number 117) and a cysteine in the preceding position (Cys(116)) and was not inactivated by PB (10 microm, 30 min) (inactivation 26%). The helical orientation of TM3 was tested by exchanging the amino acids at positions 116 and 117 of the alpha(2A)-AR and beta(2)-AR. The alpha(2A)-F116C/C117V mutant was resistant to PB (inactivation, 7%), whereas beta(2)-V117C was irreversibly inactivated (inactivation, 93%), confirming that position 3.36 is exposed to receptor ligands, and position 3.35 is not exposed in the binding pocket.     

Site-directed mutagenesis of a single residue changes the binding properties of the serotonin 5-HT2 receptor from a human to a rat pharmacology.

Mesulergine displays approximately 50-fold higher affinity for the rat 5-HT2 receptor than for the human receptor. Comparison of the deduced amino acid sequences of cDNA clones encoding the human and rat 5-HT2 receptors reveals only 3 amino acid differences in their transmembrane domains. Only one of these differences (Ser----Ala at position 242 of TM5) is near to regions implicated in ligand binding by G protein-coupled receptors. We investigated the effect of mutating Ser242 of the human 5-HT2 receptor to an Ala residue as is found in the rat clone. Both [3H]mesulergine binding and mesulergine competition of [3H]ketanserin binding showed high affinity for rat membranes and the mutant human clone but low affinity for the native human clone, in agreement with previous studies of human postmortem tissue. These studies suggest that a single naturally occurring amino acid change between the human and the rat 5-HT2 receptors makes a major contribution to their pharmacological differences.     

Positive and negative allosteric modulators of the Ca2+-sensing receptor interact within overlapping but not identical binding sites in the transmembrane domain

A three-dimensional model of the human extracellular Ca(2+)-sensing receptor (CaSR) has been used to identify specific residues implicated in the recognition of two negative allosteric CaSR modulators of different chemical structure, NPS 2143 and Calhex 231. To demonstrate the involvement of these residues, we have analyzed dose-inhibition response curves for the effect of these calcilytics on Ca(2+)-induced [(3)H]inositol phosphate accumulation for the selected CaSR mutants transiently expressed in HEK293 cells. These mutants were further used for investigating the binding pocket of two chemically unrelated positive allosteric CaSR modulators, NPS R-568 and (R)-2-[1-(1-naphthyl)ethylaminomethyl]-1H-indole (Calindol), a novel potent calcimimetic that stimulates (EC(50) = 0.31 microM) increases in [(3)H]inositol phosphate levels elicited by activating the wild-type CaSR by 2 mM Ca(2+). Our data validate the involvement of Trp-818(6.48), Phe-821(6.51), Glu-837(7.39), and Ile-841(7.43) located in transmembranes (TM) 6 and TM7, in the binding pocket for both calcimimetics and calcilytics, despite important differences observed between each family of compounds. The TMs involved in the recognition of both calcilytics include residues located in TM3 (Arg-680(3.28), Phe-684(3.32), and Phe-688(3.36)). However, our study indicates subtle differences between the binding of these two compounds. Importantly, the observation that some mutations that have no effect on calcimimetics recognition but which affect the binding of calcilytics in TM3 and TM5, suggests that the binding pocket of positive and negative allosteric modulators is partially overlapping but not identical. Our CaSR model should facilitate the development of novel drugs of this important therapeutic target and the identification of the molecular determinants involved in the binding of allosteric modulators of class 3 G-protein-coupled receptors.     

Clusters of transmembrane residues are critical for human prostacyclin receptor activation

Relaxation of vascular smooth muscle and prevention of blood coagulation are mediated by ligand-induced activation of the human prostacyclin (hIP) receptor, a seven-transmembrane-domain G-protein-coupled receptor (GPCR). In this study, we elucidate the molecular requirements for receptor activation within the region of the ligand-binding pocket, identifying transmembrane residues affecting potency. Eleven of 30 mutated residues in the region of the ligand-binding domain exhibited defective activation (decreased potency). These critical residues localized to four distinct clusters (analysis via a rhodopsin-based human prostacyclin receptor homology model). Residues Y75(2.65) (TMII), F95(3.28) (TMIII), and R279(7.40) (TMVII) comprised the immediate binding-pocket cluster and were shown to be essential for proper receptor activation, compared to equivalent expression levels of the wild-type hIP (WT EC(50) = 1.2 +/- 0.1 nM; Y75(2.65)A EC(50) = 347.3 +/- 62.8 nM, p < 0.001; F95(3.28)A EC(50) = 8.0 +/- 0.6 nM, p < 0.001; R279(7.40)A EC(50) = 130 +/- 63.0 nM, p < 0.001). Residues S20(1.39) (TMI), F24(1.43) (TMI), and F72(2.62) (TMII) were localized to a cluster involving P17(1.36), a critical residue thought to facilitate transmembrane movement during changes in activation conformation. A third cluster formed around amino acid D60(2.50) (TMII), containing the highly conserved (100% of prostanoid receptors) D288(7.49)/P289(7.50) motif located in TMVII. Last, a large hydrophobic cluster composed of aromatic residues F146(4.52) (TMIV), F150(4.56) (TMIV), F184(5.40) (TMV), and Y188(5.44) (TMV) was observed away from the ligand-binding pocket, but still necessary for hIP activation. These results assist in delineating the potential molecular requirements for agonist-induced signaling through the transmembrane domain. Such observations may be generally applicable, as many of these clusters are highly conserved among the prostanoid receptors as well as other class A GPCRs.     

FoldGPCR: Structure prediction protocol for the transmembrane domain of G protein‐coupled receptors from class A

Building reliable structural models of G protein‐coupled receptors (GPCRs) is a difficult task because of the paucity of suitable templates, low sequence identity, and the wide variety of ligand specificities within the superfamily. Template‐based modeling is known to be the most successful method for protein structure prediction. However, refinement of homology models within 1–3 Å Cα RMSD of the native structure remains a major challenge. Here, we address this problem by developing a novel protocol (foldGPCR) for modeling the transmembrane (TM) region of GPCRs in complex with a ligand, aimed to accurately model the structural divergence between the template and target in the TM helices. The protocol is based on predicted conserved inter‐residue contacts between the template and target, and exploits an all‐atom implicit membrane force field. The placement of the ligand in the binding pocket is guided by biochemical data. The foldGPCR protocol is implemented by a stepwise hierarchical approach, in which the TM helical bundle and the ligand are assembled by simulated annealing trials in the first step, and the receptor‐ligand complex is refined with replica exchange sampling in the second step. The protocol is applied to model the human β₂‐adrenergic receptor (β₂AR) bound to carazolol, using contacts derived from the template structure of bovine rhodopsin. Comparison with the X‐ray crystal structure of the β₂AR shows that our protocol is particularly successful in accurately capturing helix backbone irregularities and helix‐helix packing interactions that distinguish rhodopsin from β₂AR. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Sulfation of tyrosine 174 in the human C3a receptor is essential for binding of C3a anaphylatoxin

The complement anaphylatoxin C3a and its cellular seven-transmembrane segment receptor, C3aR, are implicated in a variety of pathological inflammatory processes. C3aR is a G-protein-coupled receptor with an exceptionally large second extracellular loop of 172 amino acids. Previously reported deletion studies have shown that at least part of this region plays a critical role in binding C3a. Our data now demonstrate that five tyrosines in the second extracellular loop of the C3aR are posttranslationally modified by the addition of sulfate. Blocking sulfation by mutation of tyrosine to phenylalanine at positions 184, 188, 317, and/or 318 does not affect ligand binding or signal transduction. However, when tyrosine 174 is mutated to phenylalanine, binding of native C3a is completely blocked. This variant efficiently mobilizes calcium in response to synthetic C3a agonist peptides, but not to native C3a. This finding is consistent with a two-site model of ligand association typical of many peptide ligand-receptor interactions and identifies sulfotyrosine 174 as the critical C3a docking site. Tyrosine sulfation in the amino-terminal extracellular domain has been shown to be important in several other seven-transmembrane segment receptors. Our data now demonstrate that tyrosine sulfate in other extracellular domains can function for ligand interactions as well.     

A chemogenomic analysis of the transmembrane binding cavity of human G-protein-coupled receptors

The amino acid sequences of 369 human nonolfactory G-protein-coupled receptors (GPCRs) have been aligned at the seven transmembrane domain (TM) and used to extract the nature of 30 critical residues supposed--from the X-ray structure of bovine rhodopsin bound to retinal--to line the TM binding cavity of ground-state receptors. Interestingly, the clustering of human GPCRs from these 30 residues mirrors the recently described phylogenetic tree of full-sequence human GPCRs (Fredriksson et al., Mol Pharmacol 2003;63:1256-1272) with few exceptions. A TM cavity could be found for all investigated GPCRs with physicochemical properties matching that of their cognate ligands. The current approach allows a very fast comparison of most human GPCRs from the focused perspective of the predicted TM cavity and permits to easily detect key residues that drive ligand selectivity or promiscuity.     

Helix 8 of the viral chemokine receptor ORF74 directs chemokine binding

The constitutively active G-protein-coupled receptor and viral oncogene ORF74, encoded by Kaposi sarcoma-associated herpesvirus (human herpesvirus 8), binds a broad range of chemokines, including CXCL1 (agonist), CXCL8 (neutral ligand), and CXCL10 (inverse agonist). Although chemokines interact with the extracellular N terminus and loops of the receptor, we demonstrate that helix 8 (Hx8) in the intracellular carboxyl tail (C-tail) of ORF74 directs chemokine binding. Partial deletion of the C-tail resulted in a phenotype with reduced constitutive activity but intact regulation by ligands. Complete deletion of the C-tail, including Hx8, resulted in an inactive phenotype that lacks CXCL8 binding sites and has an increased number of binding sites for CXCL10. Similar effects were obtained with the single R7.61(322)W or Q7.62(323)P mutations in Hx8. We propose that the conserved charged or polar side chain at position 7.61 has a specific role in stabilizing the end of transmembrane domain 7 (TM7). Disruption of Hx8 by deletion or mutation distorts an H-bonding network, involving highly conserved amino acids within TM2, TM7, and Hx8, that is crucial for positioning of the TM domains, coupling to Galphaq, and CXCL8 binding. Thus, Hx8 appears to exert a key role in receptor stabilization through the conserved residue R7.61, directing the ligand binding profile of ORF74 and likely also that of other class A G-protein-coupled receptors.     

Molecular identification of the human melanocortin-2 receptor responsible for ligand binding and signaling

The melanocortin-2 receptor (MC2R), also known as the adrenocorticotropic hormone (ACTH) receptor, plays an important role in regulating and maintaining adrenocortical function, specifically steroidogenesis. Mutations of the human MC2R (hMC2R) gene have also been identified in humans with familial glucocorticoid deficiency; however, the molecular basis responsible for hMC2R ligand binding and signaling remains unclear. In this study, both truncated ACTH peptides and site-directed mutagenesis studies were used to determine molecular mechanisms of hMC2R binding ACTH and signaling. Our results indicate that ACTH1-16 is the minimal peptide required for hMC2R binding and signaling. Mutations of common melanocortin receptor family amino acid residues E80 in transmembrane domain 2 (TM2), D107 in TM3, F178 in TM4, F235 and H238 in TM6, and F258 in TM7 significantly reduced ACTH-binding affinity and signaling. Furthermore, mutations of unique amino acids D104 and F108 in TM3 and F168 and F178 in TM4 significantly decreased ACTH binding and signaling. In conclusion, our results suggest that the residues in TM2, TM3, and TM6 of hMC2R share similar binding sites with other MCRs but the residues identified in TM4 and TM7 of hMC2R are unique and required for ACTH selectivity. Our study suggests that hMC2R may have a broad binding pocket in which both conserved and unique amino acid residues are required, which may be the reason why alpha-MSH was not able to bind hMC2R.     

Role of the extracellular loops of G protein-coupled receptors in ligand recognition: a molecular modeling study of the human P2Y1 receptor

The P2Y1 receptor is a G protein-coupled receptor (GPCR) and is stimulated by extracellular ADP and ATP. Site-directed mutagenesis of the three extracellular loops (ELs) of the human P2Y1 receptor indicates the existence of two essential disulfide bridges (Cys124 in EL1 and Cys202 in EL2; Cys42 in the N-terminal segment and Cys296 in EL3) and several specific ionic and H-bonding interactions (involving Glu209 and Arg287). Through molecular modeling and molecular dynamics simulations, an energetically sound conformational hypothesis for the receptor has been calculated that includes transmembrane (TM) domains (using the electron density map of rhodopsin as a template), extracellular loops, and a truncated N-terminal region. ATP may be docked in the receptor, both within the previously defined TM cleft and within two other regions of the receptor, termed meta-binding sites, defined by the extracellular loops. The first meta-binding site is located outside of the TM bundle, between EL2 and EL3, and the second higher energy site is positioned immediately underneath EL2. Binding at both the principal TM binding site and the lower energy meta-binding sites potentially affects the observed ligand potency. In meta-binding site I, the side chain of Glu209 (EL2) is within hydrogen-bonding distance (2.8 A) of the ribose O3', and Arg287 (EL3) coordinates both alpha- and beta-phosphates of the triphosphate chain, consistent with the insensitivity in potency of the 5'-monophosphate agonist, HT-AMP, to mutation of Arg287 to Lys. Moreover, the selective reduction in potency of 3'NH2-ATP in activating the E209R mutant receptor is consistent with the hypothesis of direct contact between EL2 and nucleotide ligands. Our findings support ATP binding to at least two distinct domains of the P2Y1 receptor, both outside and within the TM core. The two disulfide bridges present in the human P2Y1 receptor play a major role in the structure and stability of the receptor, to constrain the loops within the receptor, specifically stretching the EL2 over the opening of the TM cleft and thus defining the path of access to the binding site.     

GPC receptors and not ligands decide the binding mode in neuropeptide Y multireceptor/multiligand system

Many G protein-coupled receptors belong to families of different receptor subtypes, which are recognized by a variety of distinct ligands. To study such a multireceptor/multiligand system, we investigated the Y-receptor family. This family consists of four G protein-coupled Y receptors in humans (hY 1R, hY 2R, hY 4R, and hY 5R) and is activated by the so-called NPY hormone family, which itself consists of three native peptide ligands named neuropeptide Y (NPY), pancreatic polypeptide (PP), and peptide YY (PYY). The hY 5R shows high affinity for all ligands, although for PP binding, the affinity is slightly decreased. As a rational explanation, we suggest that Tyr (27) is lost as a contact point between PP and the hY 5R in contrast to NPY or PYY. Furthermore, several important residues for ligand binding were identified by the first extensive mutagenesis study of the hY 5R. Using a complementary mutagenesis approach, we were able to discover a novel interaction point between hY 5R and NPY. The interaction between NPY(Arg (25)) and hY 5R(Asp (2.68)) as well as between NPY(Arg (33)) and hY 5R(Asp (6.59)) is maintained in the binding of PYY and PP to hY 5R but different to the PP-hY 4R and NPY-hY 1R contact points. Therefore, we provide evidence that the receptor subtype and not the pre-orientated conformation of the ligand at the membrane decides the binding mode. Furthermore, the first hY 5R model was set up on the basis of the crystal structure of bovine rhodopsin. We can show that most of the residues identified to be critical for ligand binding are located within the now postulated binding pocket.