Substituted NDP-MSH peptides paired with mutant melanocortin-4 receptors demonstrate the role of transmembrane 6 in receptor activation

The melanocortin-4 receptor (MC4R) is involved in regulating energy homeostasis and is a potential therapeutic target for obesity and cachexia. Molecular interactions between peptide ligands and MC4R have been studied in detail. Less is known regarding the role of these interactions in the mechanism of MC4R activation. The aim of this study was to investigate the molecular mechanism of human MC4R activation by [Nle4, d-Phe7]alpha-melanocyte-stimulating hormone (NDP-MSH), by first defining the role of the His6-d-Phe7-Arg8-Trp9 residues in receptor activation (Emax for stimulation of cAMP accumulation) using modified peptides, then understanding how their interaction with the receptor modulates activation using site-directed mutagenesis and a molecular model of NDP-MSH bound to the active state of the receptor. Alanine substitution indicated that the d-Phe7, Arg8, and Trp9 side chains contribute binding energy but are not essential for the receptor activation event. Conversely, His6 to Ala6 substitution reduced receptor activation but did not affect affinity. Chlorine substitutions on the d-Phe7 side chain also inhibited receptor activation. F261(6.51)A and F284(7.35)A receptor mutations acted as gain-of-function mutations, restoring efficacy to the His6 and d-Phe7 substituted peptides that had lost efficacy at the wild-type receptor. Based on a model of NDP-MSH and MC4R interaction, the antagonist behavior of these peptides is consistent with the prevention of transmembrane 6 (TM6) rotation. This data supports the hypothesis that increasing the size of d-Phe7 directly interferes with TM6 rotation, preventing receptor activation. We further propose that removing the interaction with the His6 side chain reorients the peptide within the binding pocket, indirectly impeding TM6 rotation by strengthening peptide interaction with F261(6.51) and F284(7.35). These findings refine the molecular basis for the mechanism of ligand-stimulated hMC4R activation and will be useful for the development of hMC4R agonists and antagonists.     

Molecular determinants of ligand binding to the human melanocortin-4 receptor

To elucidate the molecular basis for the interaction of ligands with the human melanocortin-4 receptor (hMC4R), agonist structure-activity studies and receptor point mutagenesis were performed. Structure-activity studies of [Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) identified D-Phe7-Arg8-Trp9 as the minimal NDP-MSH fragment that possesses full agonist efficacy at the hMC4R. In an effort to identify receptor residues that might interact with amino acids in this tripeptide sequence 24 hMC4R transmembrane (TM) residues were mutated (the rationale for choosing specific receptor residues for mutation is outlined in the Results section). Mutation of TM3 residues D122 and D126 and TM6 residues F261 and H264 decreased the binding affinity of NDP-MSH 5-fold or greater, thereby identifying these receptor residues as sites potentially involved in the sought after ligand-receptor interactions. By examination of the binding affinities and potencies of substituted NDP-MSH peptides at receptor mutants, evidence was found that core melanocortin peptide residue Arg8 interacts at a molecular level with hMC4R TM3 residue D122. TM3 mutations were also observed to decrease the binding of hMC4R antagonists. Notably, mutation of TM3 residue D126 to alanine decreased the binding affinity of AGRP (87-132), a C-terminal derivative of the endogenous melanocortin antagonist, 8-fold, and simultaneous mutations D122A/D126A completely abolished AGRP (87-132) binding. In addition, mutation of TM3 residue D122 or D126 decreased the binding affinity of hMC4R antagonist SHU 9119. These results provide further insight into the molecular determinants of hMC4R ligand binding.     

Contribution of the conserved amino acids of the melanocortin-4 receptor in [corrected] [Nle4,D-Phe7]-alpha-melanocyte-stimulating [corrected] hormone binding and signaling

Melanocortin 4 receptor (MC4R) plays an important role in the regulation of food intake and body weight. To determine the molecular basis of human MC4R (hMC4R) responsible for alpha-melanocortin-stimulating hormone (alpha-MSH) binding, in this study, we utilized both receptor domain exchange and site-directed mutagenesis studies to investigate the molecular determinants of hMC4R responsible for alpha-MSH binding and signaling. alpha-MSH is a potent agonist at hMC4R but not at hMC2R. Cassette substitutions of the second, third, fourth, fifth, and sixth transmembrane regions (TM) of the hMC4R with the homologous regions of hMC2R were performed and alpha-MSH binding and signaling were examined. Our results indicate that each chimeric receptor was expressed at the cell surface and the expression levels remain similar to that of the wild-type receptor. The cassette substitutions of the second, fourth, fifth, and sixth TMs of the hMC4R with homologous regions of the hMC2R did not significantly alter alpha-MSH binding affinity and potency except substitution of the TM3 of the hMC4R, suggesting that the conserved residues in TMs of the hMC4R are crucial for alpha-MSH binding and signaling. Further mutagenesis studies indicate that conserved residues Glu(100) in TM2, Asp(122), Asp(126) in TM3 and Trp(258), Phe(261), His(264) in TM6 are involved in alpha-MSH binding and signaling. In conclusion, our results suggest that the conserved residues in the TM2, TM3, and TM6 of the hMC4R are responsible for alpha-MSH binding and signaling.     

Residues Leu261, Trp264, and Phe265 account for apolipoprotein E-induced dyslipidemia and affect the formation of apolipoprotein E-containing high-density lipoprotein

Overexpression of apolipoprotein E (apoE) induces hypertriglyceridemia in apoE-deficient mice, which is abrogated by deletion of the carboxy-terminal segment of residues 260-299. We have used adenovirus-mediated gene transfer in apoE-/- and apoA-I-/- mice to test the effect of three sets of apoE mutations within the region of residues 261-265 on the induction of hypertriglyceridemia, the esterification of cholesterol of very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL), and the formation of spherical or discoidal apoE-containing HDL. A single-amino acid substitution (apoE4[Phe265Ala]) induced hypertriglyceridemia in apoE-/- or apoA-I-/- mice, promoted the accumulation of free cholesterol in the very low-density lipoprotein (VLDL) and HDL region, and decreased HDL cholesterol levels. A double substitution (apoE4[Leu261Ala/Trp264Ala]) induced milder hypertriglyceridemia and increased HDL cholesterol levels. A triple substitution (apoE4[Leu261Ala/Trp264Ala/Phe265Ala] or apoE2[Leu261Ala/Trp264Ala/Phe265Ala]) did not induce hypertriglyceridemia and increased greatly the HDL cholesterol levels. Electron microscopy (EM) analysis of the HDL fractions showed that apoE4[Leu261Ala/Trp264Ala/Phe265Ala] and apoE2[Leu261Ala/Trp264Ala/Phe265Ala] contained spherical HDL, apoE4[Leu261Ala/Trp264Ala] contained mostly spherical and few discoidal HDL particles, and apoE4[Phe265Ala] contained discoidal HDL. We conclude that residues Leu261, Trp264, and Phe265 play an important role in apoE-induced hypertriglyceridemia, the accumulation of free cholesterol in VLDL and HDL, and the formation of discoidal HDL. Substitution of these residues with Ala improves the apoE functions by preventing hypertriglyceridemia and promoting formation of spherical apoE-containing HDL.     

Interactions of the Melanocortin-4 Receptor with the Peptide Agonist NDP-MSH

Melanocortin-4 receptor (MC4R) has an important regulatory role in energy homeostasis and food intake. Peptide agonists of the MC4R are characterized by the conserved sequence His₆-Phe₇-Arg₈-Trp₉, which is crucial for their interaction with the receptor. This investigation utilized the covalent attachment approach to identify receptor residues in close proximity to the bound ligand [Nle₄,d-Phe₇]melanocyte-stimulating hormone (NDP-MSH), thereby differentiating between residues directly involved in ligand binding and those mutations that compromise ligand binding by inducing conformational changes in the receptor. Also, recent X-ray structures of G-protein-coupled receptors were utilized to refine a model of human MC4R in the active state (R^?), which was used to generate a better understanding of the binding mode of the ligand NDP-MSH at the atomic level.The mutation of residues in the human MC4R—such as Leu106 of extracellular loop 1, and Asp122, Ile125, and Asp126 of transmembrane (TM) helix 3, His264 (TM6), and Met292 (TM7)—to Cys residues produced definitive indications of proximity to the side chains of residues in the core region of the peptide ligand. Of particular interest was the contact between d-Phe₇ on the ligand and Ile125 of TM3 on the MC4R. Additionally, Met292 (TM7) equivalent to Lys(7.45) (Ballesteros numbering scheme) involved in covalently attaching retinal in rhodopsin is shown to be in close proximity to Trp₉.For the first time, the interactions between the terminal regions of NDP-MSH and the receptor are described. The amino-terminus appears to be adjacent to a series of hydrophilic residues with novel interactions at Cys196 (TM5) and Asp189 (extracellular loop 2). These interactions are reminiscent of sequential ligand binding exhibited by the β₂-adrenergic receptor, with the former interaction being equivalent to the known interaction involving Ser204 of the β₂-adrenergic receptor.     

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.     

Effect of pH, urea, peptide length, and neighboring amino acids on alanine alpha-proton random coil chemical shifts

Accurate random coil alpha-proton chemical shift values are essential for precise protein structure analysis using chemical shift index (CSI) calculations. The current study determines the chemical shift effects of pH, urea, peptide length and neighboring amino acids on the alpha-proton of Ala using model peptides of the general sequence GnXaaAYaaGn, where Xaa and Yaa are Leu, Val, Phe, Tyr, His, Trp or Pro, and n = 1-3. Changes in pH (2-6), urea (0-1M), and peptide length (n = 1-3) had no effect on Ala alpha-proton chemical shifts. Denaturing concentrations of urea (8M) caused significant downfield shifts (0.10 +/- 0.01 ppm) relative to an external DSS reference. Neighboring aliphatic residues (Leu, Val) had no effect, whereas aromatic amino acids (Phe, Tyr, His and Trp) and Pro caused significant shifts in the alanine alpha-proton, with the extent of the shifts dependent on the nature and position of the amino acid. Smaller aromatic residues (Phe, Tyr, His) caused larger shift effects when present in the C-terminal position (approximately 0.10 vs. 0.05 ppm N-terminal), and the larger aromatic tryptophan caused greater effects in the N-terminal position (0.15 ppm vs. 0.10 C-terminal). Proline affected both significant upfield (0.06 ppm, N-terminal) and downfield (0.25 ppm, C-terminal) chemical shifts. These new Ala correction factors detail the magnitude and range of variation in environmental chemical shift effects, in addition to providing insight into the molecular level interactions that govern protein folding.     

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.     

Complex of an active mu-opioid receptor with a cyclic peptide agonist modeled from experimental constraints

Site-directed mutagenesis and design of Zn(2+)-binding centers have been used to determine a set of specific tertiary interactions between the mu-opioid receptor, a rhodopsin-like G protein-coupled receptor (GPCR), and its cyclic peptide agonist ligand, Tyr(1)-c(S-Et-S)[d-Cys(2)-Phe(3)-d-Pen(4)]NH(2) (JOM6). The binding affinity of the tetrapeptide is strongly dependent on the nature of its first and third residues and on substitutions at positions 213, 216, 237, 300, 315, and 318 of the mu-opioid receptor. His(1) and His(3) analogues of the ligand were able to form metal-binding complexes with the V300C and G213C/T315C receptor mutants, respectively. Direct contact of the Phe(3) residue of JOM6 with Gly(213), Asp(216), Thr(315), and Trp(318) of the receptor was suggested by the binding affinities of His(3)-, Nle(3)-, Leu(3)-, Aci(3)-, Delta(E)Phe(3)-, and Delta(Z)Phe(3)-substituted peptides with the G213C/T315C, D216V, T315C, and W318L mutants. The improved binding affinity of the free carboxylate analogue of JOM6 for binding to the E229D mutant revealed an interaction between the C-terminal group of the peptide and Glu(229) of the receptor. The experimental constraints that were obtained were applied for distance geometry modeling of the mu-receptor in complex with the tetrapeptide agonist ligand, JOM6. The active conformation of the opioid receptor was calculated using the crystal structure of "inactive" rhodopsin and published engineered and intrinsic metal-binding sites and disulfide bonds that allow or facilitate activation of GPCRs. Interhelical H-bonds existing in the mu-receptor were applied as additional distance constraints. The calculated model of the receptor-ligand complex can serve as a prototype of the active state for all rhodopsin-like GPCRs. It displays a strongly shifted transmembrane helix 6 (TM6) and reorientation of the conserved Trp(293) residue in TM6 upon its interaction with the agonist. Importantly, the binding pockets of the active and inactive states are not identical, which implies distinct interaction modes of agonists and antagonists. In the active state, the binding pocket of the mu-receptor is complementary to the previously proposed receptor-bound conformation of JOM6.     

Molecular modelling and site-directed mutagenesis of human GALR1 galanin receptor defines determinants of receptor subtype specificity

Human galanin is a 30 amino acid neuropeptide that elicits a range of biological activities by interaction with G protein-coupled receptors. We have generated a model of the human GALR1 galanin receptor subtype (hGALR1) based on the alpha carbon maps of frog rhodopsin and investigated the significance of potential contact residues suggested by the model using site-directed mutagenesis. Mutation of Phe186 within the second extracellular loop to Ala resulted in a 6-fold decrease in affinity for galanin, representing a change in free energy consistent with hydrophobic interaction. Our model suggests interaction between Phe186 of hGALR1 and Ala7 or Leu11 of galanin. Receptor subtype specificity was investigated by replacement of residues in hGALR1 with the corresponding residues in hGALR2 and use of the hGALR2-specific ligands hGalanin(2-30) and [D-Trp2]hGalanin(1-30). The His267Ile mutant receptor exhibited a pharmacological profile corresponding to that of hGALR1, suggesting that His267 is not involved in a receptor-ligand interaction. The mutation Phe115Ala resulted in a decreased binding affinity for hGalanin and for hGALR2-specific analogues, indicating Phe115 to be of structural importance to the ligand binding pocket of hGALR1 but not involved in direct ligand interaction. Analysis of Glu271Trp suggested that Glu271 of hGALR1 interacts with the N-terminus of galanin and that the Trp residue in the corresponding position in hGALR2 is involved in receptor subtype specificity of binding. Our model supports previous reports of Phe282 of hGALR1 interacting with Trp2 of galanin and His264 of hGALR1 interacting with Tyr9 of galanin.     

Peptide and small molecules rescue the functional activity and agonist potency of dysfunctional human melanocortin-4 receptor polymorphisms

The melanocortin pathway, specifically the melanocortin-4 receptor and the cognate endogenous agonist and antagonist ligands, have been strongly implicated in the regulation of energy homeostasis and satiety. Genetic studies of morbidly obese human patients and normal weight control patients have resulted in the discovery of over 70 human melanocortin-4 receptor (MC4R) polymorphisms observed as both heterozygous and homozygous forms. A number of laboratories have been studying these hMC4R polymorphisms attempting to understand the molecular mechanism(s) that might explain the obese human phenotype. Herein, we have studied 13 polymorphic hMC4Rs that have been identified to possess statistically significant decreased endogenous agonist potency with synthetic peptides and small molecules attempting to identify ligands that can pharmacologically rescue the hMC4R polymorphic agonist response. The ligands examined in this study include NDP-MSH, MTII, Ac-His-DPhe-Arg-Trp-NH2 (JRH887-9), Ac-Anc-DPhe-Arg-Trp-NH2 (amino-2-naphtylcarboxylic acid, Anc, JRH420-12), Ac-His-(pI)DPhe-Arg-Trp-NH2 (JRH322-18), chimeric AGRP-melanocortin based ligands (Tyr-c[Cys-His-DPhe-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH2, AMW3-130 and Ac-mini-(His-DPhe-Arg-Trp)-hAGRP-NH2, AMW3-106), and the small molecules JB25 and THIQ. The hMC4R polymorphisms included in this study are S58C, N97D, I102S, L106P, S127L, T150I, R165Q, R165W, L250Q, G252S, C271Y, Y287Stop, and I301T. These studies resulted in the NDP-MSH, MTII, AMW3-130, THIQ, and AMW3-106 ligands possessing nanomolar to subnanomolar agonist potency at the hMC4R polymorphisms examined in this study. Thus, these ligands could generically rescue the potency and stimulatory response of the abnormally functioning hMC4Rs studied and may provide tools to further clarify the molecular mechanism(s) involving these receptor modifications.     

Structure activity studies of the melanocortin-4 receptor by in vitro mutagenesis: identification of agouti-related protein (AGRP), melanocortin agonist and synthetic peptide antagonist interaction determinants

In vitro mutagenesis of the mouse melanocortin-4 receptor (mMC4R) has been performed, based upon homology molecular modeling and previous melanocortin receptor mutagenesis studies that identified putative ligand-receptor interactions. Twenty-three mMC4 receptor mutants were generated and pharmacologically characterized using several melanocortin-based ligands [alpha-MSH, NDP-MSH, MTII, DNal (1')(7)-MTII, Nal(2')(7)-MTII, SHU9119, and SHU9005]. Selected mutant receptors possessing significant differences in the melanocortin-based peptide agonist and/or antagonist pharmacology were further evaluated using the endogenous antagonist agouti-related protein fragment hAGRP(83-132) and hAGRP(109-118) molecules. These studies of the mouse MC4R provide further experimental data suggesting that the conserved melanocortin receptor residues Glu92 (TM2), Asp114 (TM3), and Asp118 (TM3) (mouse MC4R numbering) are important for melanocortin-based peptide molecular recognition. Additionally, the Glu92 and Asp118 mMC4R residues are important for molecular recognition and binding of AGRP(83-132). We have identified the Phe176 (TM4), Tyr179 (TM4), Phe254 (TM6), and Phe259 (TM6) receptor residues as putatively interacting with the melanocortin-based ligand Phe(7) by differences between alpha-MSH and NDP-MSH agonist potencies. The Glu92, Asp118, and Phe253 mMC4R receptor residues appear to be critical for hAGRP(83-132) molecular recognition and binding while Phe176 appears to be important for functional antagonism of AGRP(83-132) and AGRP(109-118) but not molecular recognition. The Phe253 mMC4R residue appears to be important for AGRP(83-132) molecular recognition and general mMC4 receptor stimulation. The Phe254 and Phe259 mMC4R amino acids may participate in the differentiation of agonist versus antagonist activity of the melanocortin-based peptide antagonists SHU9119 and SHU9005, but not AGRP(83-132) or AGRP(109-118). The Met192 side chain when mutated to a Phe results in a constitutively active mMC4R that does not effect agonist ligand binding or potency. Melanocortin-based peptides modified at the 7 position of MTII with DPhe, DNal(1'), Nal(2'), and DNal(2') have been pharmacologically characterized at these mutant mouse MC4Rs. These data suggest a revised hypothesis for the mechanism of SHU9119 antagonism at the MC4R which may be attributed to the presence of a "bulky" naphthyl moiety at the 7 position (original hypothesis), and additionally that both the stereochemistry and naphthyl ring position (2' versus 1') are important for positioning of the ligand Arg(8) residue with the corresponding mMC4R amino acids.     

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.     

Importance of the C-terminal phenylalanine of gastrin for binding to the human CCK(2) receptor.

The importance of the C-terminal Phe of gastrin and structural requirements at position 17 for binding to the human CCK2 receptor were assessed using analogs of [Leu15]G(11-17). The following peptides were synthesized, Ac[Leu15]G(11-17), Ac[Leu15]G(11-16)NH2, [Leu15]G(11-17), [Leu15,Ala17]G(11-17), [Leu15,Abu17]G(11-17), [Leu15,Val17]G(11-17), [Leu15,Leu17]G(11-17), [Leu15,Cha17]G(11-17), [Leu15,Trp17]G(11-17), [Leu15,Tic17]G(11-17), [Leu15, d-Phe17]G(11-17) and [Leu15,p-X-Phe17]G(11-17), where X = F, Cl, Br, I, OH, CH3, NH2 and NO2. Competition binding experiments with [3H]CCK-8 were performed using human CCK2 receptors stably expressed in CHO cells. Phe17 was shown to be important for binding. A hydrophobic side-chain larger than Leu is required at position 17 but aromaticity does not appear to be essential. Constraint of the aromatic side-chain either in the g+ or g- conformation, as in the case of Tic, results in a significant decrease in affinity. In addition, the peptide conformation induced by incorporation of d-Phe decreases binding. The size and electron withdrawing/donating properties of the para substituent are not important for interaction with the receptor. The current study shows that the use of des-Phe analogs of gastrin is not a viable strategy for development of antagonists for the human CCK2 receptor.     

Cyclic analogs of alpha-melanocyte-stimulating hormone (alphaMSH) with high agonist potency and selectivity at human melanocortin receptor 1b

Alpha-melanotropin (alphaMSH), Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9-Gly10-Lys11-Pro12-Val13-NH2,(1) has been long recognized as an important physiological regulator of skin and hair pigmentation in mammals. Binding of this peptide to the melanocortin receptor 1 (MC1R) leads to activation of tyrosinase, the key enzyme of the melanin biosynthesis pathway. In this study, interactions of the human MC1bR (an isoform of the receptor 1a) with the synthetic cyclic analogs of alphaMSH were studied. These ligands were analogs of MTII, Ac-Nle4-cyclo-(Asp5-His6-D-Phe7-Arg8-Trp9-Lys10)-NH2, a potent pan-agonist at the human melanocortin receptors (hMC1,3-5R). In the structure of MTII, the His6-D-Phe7-Arg8-Trp9 segment has been recognized as "essential" for molecular recognition at the human melanocortin receptors (hMC1,3-5R). Herein, the role of the Trp9 in the ligand interactions with the hMC1b,3-5R has been reevaluated. Analogs with various amino acids in place of Trp9 were synthesized and tested in vitro in receptor affinity binding and cAMP functional assays at human melanocortin receptors 1b, 3, 4 and 5 (hMC1b,3-5R). Several of the new peptides were high potency agonists (partial) at hMC1bR (EC50 from 0.5 to 20 nM) and largely inactive at hMC3-5R. The bulky aromatic side chain in position 9, such as that in Trp, was found not to be essential to agonism (partial) of the studied peptides at hMC1bR.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Identification of the critical residues of bradykinin receptor B1 for interaction with the kinins guided by site-directed mutagenesis and molecular modeling

We report the critical residues for the interaction of the kinins with human bradykinin receptor 1 (B1) using site-directed mutagenesis in conjunction with molecular modeling of the binding modes of the kinins in the homology model of the B1 receptor. Mutation of Lys118 in transmembrane (TM) helix 3, Ala270 in TM6, and Leu294 in TM7 causes a significant decrease in the affinity for the peptide agonists des-Arg10kallidin (KD) and des-Arg9BK but not the peptide antagonist des-Arg10Leu9KD. In contrast, mutations in TM2, TM3, TM6, and TM7 cause a significant decrease in the affinity for both the peptide agonists and the antagonist. These data indicate that the B1 bradykinin binding pocket for agonists and antagonists is similar, but the manners in which they interact with the receptor do not completely overlap. Therefore, there is a potential to influence the receptor's ligand selectivity.     

Molecular basis of melanocortin-4 receptor for AGRP inverse agonism

We have investigated receptor structural components of the melanocortin-4 receptor (MC4R) responsible for ligand-dependent inverse agonism. We utilized agouti-related protein (AGRP), an inverse agonist which reduces MC4R basal cAMP production, as a tool to determine the molecular mechanism. We tested a series of chimeric receptors and utilized MC4R and MC1R as templates, in which AGRP is an inverse agonist for MC4R but not for MC1R. Our results indicate that replacements of the extracellular loops 1, 2 and 3 of MC4R with the corresponding regions of MC1R did not affect AGRP inverse agonist activity. However, replacement of the N terminus of MC4R with the same region of MC1R decreases AGRP inverse agonism. Replacement of transmembrane domains 3, 4, 5 and 6 of MC4R with the corresponding regions of MC1R did not affect AGRP inverse agonist activity but mutation of D90A in transmembrane 2 (TM2) and D298A in TM7 abolished AGRP inverse activity. Deletion of the distal MC4R C terminus fails to maintain AGRP mediated reduction in basal cAMP production although it maintains NDP-MSH mediated cAMP production. In conclusion, our results indicate that the N terminus and the distal C terminus of MC4R do appear to play important roles in AGRP inverse agonism but not NDP-MSH mediated receptor activation. Our results also indicate that the residues D90 in TM2 and D298 in TM7 of hMC4R are involved in not only NDP-MSH mediated receptor activation but also AGRP mediated inverse agonism.     

Control of conformational equilibria in the human B2 bradykinin receptor. Modeling of nonpeptidic ligand action and comparison to the rhodopsin structure

A prototypic study of the molecular mechanisms of activation or inactivation of peptide hormone G protein-coupled receptors was carried out on the human B2 bradykinin receptor. A detailed pharmacological analysis of receptor mutants possessing either increased constitutive activity or impaired activation or ligand recognition allowed us to propose key residues participating in intramolecular interaction networks stabilizing receptor inactive or active conformations: Asn(113) and Tyr(115) (TM III), Trp(256) and Phe(259) (TM VI), Tyr(295) (TM VII) which are homologous of the rhodopsin residues Gly(120), Glu(122), Trp(265), Tyr(268), and Lys(296), respectively. An essential experimental finding was the spatial proximity between Asn(113), which is the cornerstone of inactive conformations, and Trp(256) which plays a subtle role in controlling the balance between active and inactive conformations. Molecular modeling and mutagenesis data showed that Trp(256) and Tyr(295) constitute, together with Gln(288), receptor contact points with original nonpeptidic ligands. It provided an explanation for the ligand inverse agonist behavior on the WT receptor, with underlying restricted motions of TMs III, VI, and VII, and its agonist behavior on the Ala(113) and Phe(256) constitutively activated mutants. These data on the B2 receptor emphasize that conformational equilibria are controlled in a coordinated fashion by key residues which are located at strategic positions for several G protein-coupled receptors. They are discussed in comparison with the recently determined rhodopsin crystallographic structure.