Conformation of the core sequence in melanocortin peptides directs selectivity for the melanocortin MC3 and MC4 receptors.

Melanocortin peptides regulate a variety of physiological processes. Five melanocortin receptors (MC-R) have been cloned and the MC3R and MC4R are the main brain MC receptors. The aim of this study was to identify structural requirements in both ligand and receptor that determine gamma-melanocyte-stimulating hormone (MSH) selectivity for the MC3R versus the MC4R. Substitution of Asp10 in [Nle4]Lys-gamma2-MSH for Gly10 from [Nle4]alpha-MSH, increased both activity and affinity for the MC4R while the MC3R remained unaffected. Analysis of chimeric MC3R/MC4Rs and mutant MC4Rs showed that Tyr268 of the MC4R mainly determined the low affinity for [Nle4]Lys-gamma2-MSH. The data demonstrate that Asp10 determines selectivity for the MC3R, however, not through direct side chain interactions, but probably by influencing how the melanocortin core sequence is presented to the receptor-binding pocket. This is supported by mutagenesis of Tyr268 to Ile in the MC4R which increased affinity and activity for [Nle4]Lys-gamma2-MSH, but decreased affinity for two peptides with constrained cyclic structure of the melanocortin core sequence, MT-II and [D-Tyr4]MT-II, that also displayed lower affinity for the MC3R. This study provides a general concept for peptide receptor selectivity, in which the major determinant for a selective receptor interaction is the conformational presentation of the core sequence in related peptides to the receptor-binding pocket.     

Metal ion-mediated agonism and agonist enhancement in melanocortin MC1 and MC4 receptors

An endogenous metal-ion site in the melanocortin MC1 and MC4 receptors was characterized mainly in transiently transfected COS-7 cells. ZnCl(2) alone stimulated signaling through the Gs pathway with a potency of 11 and 13 microm and an efficacy of 50 and 20% of that of alpha-melanocortin stimulating hormone (alpha-MSH) in the MC1 and MC4 receptors, respectively. In the presence of peptide agonist, Zn(II) acted as an enhancer on both receptors, because it shifted the dose-response curves to the left: most pronounced was a 6-fold increase in alpha-MSH potency on the MC1 receptor. The effect of the metal ion appeared to be additive, because the maximal cAMP response for alpha-MSH in the presence of Zn(II) was 60% above the maximal response for the peptide alone. The affinity of Zn(II) could be increased through binding of the metal ion in complex with small hydrophobic chelators. The binding affinities and profiles were similar for a number of the 2,2'-bipyridine and 1,10-phenanthroline analogs in complex with Zn(II) in the MC1 and MC4 receptors. However, the potencies and efficacies of the metal-ion complexes were very different in the two receptors, and close to full agonism was obtained in the MC1 receptor. Metal ion-chelator complexes having antagonistic properties were also found. An initial attempt to map the metal-ion binding site in the MC1 receptor indicated that Cys(271) in extracellular loop 3 and possibly Asp(119) at the extracellular end of TM-III, which are both conserved among all MC receptors, are parts of the site. It is concluded that the function of the MC1 and MC4 receptors can be positively modulated by metal ions acting both as partial agonists and as potentiators for other agonists, including the endogenous peptide ligand alpha-MSH at Zn(II) concentrations that could be physiological. Furthermore, the metal ion-chelator complexes may serve as leads in the development of novel melanocortin receptor modulators.     

Functional characterization of two melanocortin (MC) receptors in lamprey showing orthology to the MC1 and MC4 receptor subtypes

Background  

The melanocortin (MC) receptors have a key role in regulating body weight and pigmentation. They belong to the rhodopsin family of G protein-coupled receptors (GPCRs). The purpose of this study was to identify ancestral MC receptors in agnathan, river lamprey.     

Mapping of the ACTH,MSH, and neural (MC3 and MC4) melanocortin receptors in the mouse and human

The melanocortin peptides regulate a wide variety of physiological processes, including pigmentation and glucocorticoid production, and also have several activities in the central and peripheral nervous systems. The melanocortin receptor family includes the melanocytestimulating hormone receptor (MSH-R), adrenocorticotropic hormone receptor (ACTH-R), and two neural receptors, MC3-R and MC4-R. In the human these receptors map to 16q24 (MSH-R), 18p11.2 (ACTH-R), 20q13.2 (MC3-R), and 18q22 (MC4-R). The corresponding locations in the mouse are 8, 18, and 2; a variant for mapping MC4-R has not yet been identified. The data reported here also show that the neural MC3 receptor maps close to a disease locus for benign neonatal epilepsy in human and near the El-2 epilepsy susceptibility locus in the mouse.     

Predicting the coupling specificity of GPCRs to G-proteins by support vector machines

G-protein coupled receptors （GPCRs） represent one of the most important classes of drug targets for pharmaceutical industry and play important roles in cellular signal transduction. Predicting the coupling specificity of GPCRs to G-proteins is vital for further understanding the mechanism of signal transduction and the function of the receptors within a cell, which can provide new clues for pharmaceutical research and development. In this study, the features of amino acid compositions and physiochemical properties of the full-length GPCR sequences have been analyzed and extracted. Based on these features, classifiers have been developed to predict the coupling specificity of GPCRs to G-protelns using support vector machines. The testing results show that this method could obtain better prediction accuracy.     

A method for the prediction of GPCRs coupling specificity to G-proteins using refined profile Hidden Markov Models

Background  

G- Protein coupled receptors (GPCRs) comprise the largest group of eukaryotic cell surface receptors with great pharmacological interest. A broad range of native ligands interact and activate GPCRs, leading to signal transduction within cells. Most of these responses are mediated through the interaction of GPCRs with heterotrimeric GTP-binding proteins (G-proteins). Due to the information explosion in biological sequence databases, the development of software algorithms that could predict properties of GPCRs is important. Experimental data reported in the literature suggest that heterotrimeric G-proteins interact with parts of the activated receptor at the transmembrane helix-intracellular loop interface. Utilizing this information and membrane topology information, we have developed an intensive exploratory approach to generate a refined library of statistical models (Hidden Markov Models) that predict the coupling preference of GPCRs to heterotrimeric G-proteins. The method predicts the coupling preferences of GPCRs to G_s, G_i/o and G_q/11, but not G_12/13 subfamilies.     

Rat cerebral cortex corticotropin-releasing hormone receptors: evidence for receptor coupling to multiple G-proteins

The wide distribution of corticotrophin-releasing hormone (CRH) receptors in brain and periphery appear to be important in integrating the responses of the brain, endocrine and immune systems to physiological, psychological and immunological stimuli. The type 1 receptors are highly expressed throughout the cerebral cortex, a region involved in cognitive function and modulation of stress responses, where they are coupled to the adenylyl cyclase system. Using techniques that analyse receptor-mediated guanine-nucleotide binding protein (G-proteins) activation, we recently demonstrated that expressed type 1alpha CRH receptors are capable of activating multiple G-proteins, which suggests that CRH can regulate multiple signalling pathways. In an effort to characterize the intracellular signals generated by CRH in the rat cerebral cortex we sought to identify G-proteins activated by CRH in a physiological membrane environment. Rat cerebral cortical membrane suspensions were analysed for the ability of CRH to stimulate incorporation of [alpha-32P]-GTP-gamma-azidoanilide to various G-protein alpha-chains. Our results show that CRH receptors are coupled to and activate at least five different G-proteins (Gs, Gi, Gq/11, Go and Gz) with subsequent stimulation of at least two intracellular signalling cascades. In addition, the photoaffinity experiments indicated that the CRH receptors preferentially activate the 45 kDa form of the Gs alpha-protein. This data may help elucidate the intracellular signalling pathways mediating the multiple actions of CRH especially under different physiological conditions.     

A naive Bayes model to predict coupling between seven transmembrane domain receptors and G-proteins

MOTIVATION: An understanding of the coupling between a G-protein coupled receptor (GPCR) and a specific class of heterotrimeric GTP-binding proteins (G-proteins) is vital for further comprehending the function of the receptor within a cell. However, predicting G-protein coupling based on the amino acid sequence of a receptor has been a daunting task. While experimental data for G-protein coupling exist, published models that rely on sequence based prediction are few. In this study, we have developed a Naive Bayes model to successfully predict G-protein coupling specificity by training over 80 GPCRs with known coupling. Each intracellular domain of GPCRs was treated as a discrete random variable, conditionally independent of one another. In order to determine the conditional probability distributions of these variables, ClustalW-generated phylogenetic trees were used as an approximation for the clustering of the intracellular domain sequences. The sampling of an intracellular domain sequence was achieved by identifying the cluster containing the homologue with the highest sequence similarity. RESULTS: Out of 55 GPCRs validated, the model yielded a correct classification rate of 72%. Our model also predicted multiple G-protein coupling for most of the GPCRs in the validation set. The Bayesian approach in this work offers an alternative to the experimental approach in order to answer the biological problem of GPCR/G-protein coupling selectivity. AVAILABILITY: Academic users should send their request for the perl program for calculating likelihood probabilities at jack.cao@astrazeneca.com. SUPPLEMENTARY INFORMATION: The materials can be viewed at http://www.astrazeneca-montreal.com/AZRDM_info/supporting_info.pdf.     

Further evidence for ancient role of ACTH peptides at melanocortin (MC) receptors; pharmacology of dogfish and lamprey peptides at dogfish MC receptors

The cloning of melanocortin (MC) receptors in distant species has provided us tools to get insight in how the ligand-receptors interactions in the MC system have evolved. We have however lacked studies on pharmacology of native ancient melanocortin peptides at the ancient MC receptors. In this paper we synthesized melanocortin peptides from both the sea lamprey (Petromyzon marinus) and spiny dogfish (Squalus acanthias) and tested them on the MC3 and MC4 receptors from spiny dogfish. The results show that both the dogfish and lamprey ACTH peptides have similar or higher affinity than the dogfish alpha-, beta- and gamma-MSH peptides to the dogfish MC3 and MC4 receptors. Moreover, both the dogfish and lamprey ACTH peptides have more than 10-fold higher affinity than alpha-MSH to the dogfish MC4 receptor. We also show that dogfish delta-MSH is able to bind to MC receptors and its potency is higher than of dogfish beta-MSH, which is considered to be its precursor. Our results provide the first evidence that native ACTH ligands from dogfish and lamprey have a preference above native MSH peptides to ancient version of the MC3 and MC4 receptors. This further strengthens the hypotheses that the ligand contributing to the first version of the melanocortin ligand-receptor system resembled ACTH.     

Pharmacological comparison of rat and human melanocortin 3 and 4 receptors in vitro

The melanocortin 3 and 4 receptors are G-protein-coupled receptors found in the hypothalamus with important role in regulation of the energy balance. In this study, we performed pharmacological comparison of the rat and human melancortin (MC) 3 and MC4 receptors. We transiently expressed the genes for these receptors individually in a mammalian cell line and determined the binding affinities to several MSH peptides. The results showed no major difference between the rat and human MC3 receptors while the rat MC4 receptor had higher affinity to several peptides compared with the human MC4 receptor. NDP-, alpha-, beta-, gamma-MSH, ACTH(1-24), HS014 and MTII had from 5- to 34-fold higher affinity for the rat MC4 receptor, while SHU9119, HS024 and HS028 had similar affinity for both the MC4 receptors. Pharmacological species difference have earlier been reported for the MC1 and MC5 receptors but this is the first report showing important differences between the rat and human MC4 receptors.     

Molecular mechanisms of G-proteins coupling with membrane receptors and second messenger systems

G-proteins transmit the signals from hormone receptors onto intracellular effector systems which take part in production of the second messengers such as cAMP, IP3, DAG and Ca2+. Molecular mechanisms of G-protein participation in the coupling of the seven-domain receptors to adenylate cyclase, phospholipase C and channels for Ca2+ and K+ ions are discussed in this paper. G-protein is a heterotrimers built of alpha-, beta- and gamma-subunits, which dissociate onto alpha- and beta gamma-subunits during interaction with hormone-receptor complex. alpha-subunit as well as beta gamma-dimmer may interact with effector system that leads to acceleration or slowing down of second messengers formation. Molecular mechanisms of such regulatory signal diversification are described. Seven-domain receptors possess very high recognition specificity of G-proteins. It is defined by combination of both alpha- and beta gamma-subunits in the G-protein structure. There is well-defined interaction specificity of G-protein alpha-subunit with effector systems. Combinations of different beta- and gamma-subunits involved in complex formation define interaction specificity of G-protein beta gamma-complex with effector systems. The highest interaction specificity of receptors with G-proteins and G-proteins with effector systems is found during triple complex formations: receptor--G-protein--effector. Such specificity is stronger in living cells than in membrane preparations. It can be an evidence of intracellular factors influence on the processes of interaction of the proteins involved in transmembrane regulatory signal transduction.     

Prediction of the coupling specificity of GPCRs to four families of G-proteins using hidden Markov models and artificial neural networks

MOTIVATION: G-protein coupled receptors are a major class of eukaryotic cell-surface receptors. A very important aspect of their function is the specific interaction (coupling) with members of four G-protein families. A single GPCR may interact with members of more than one G-protein families (promiscuous coupling). To date all published methods that predict the coupling specificity of GPCRs are restricted to three main coupling groups G(i/o), G(q/11) and G(s), not including G(12/13)-coupled or other promiscuous receptors. RESULTS: We present a method that combines hidden Markov models and a feed-forward artificial neural network to overcome these limitations, while producing the most accurate predictions currently available. Using an up-to-date curated dataset, our method yields a 94% correct classification rate in a 5-fold cross-validation test. The method predicts also promiscuous coupling preferences, including coupling to G(12/13), whereas unlike other methods avoids overpredictions (false positives) when non-GPCR sequences are encountered. AVAILABILITY: A webserver for academic users is available at http://bioinformatics.biol.uoa.gr/PRED-COUPLE2     

Fluorescence anisotropy assay for pharmacological characterization of ligand binding dynamics to melanocortin 4 receptors

Fluorescence anisotropy assay was implemented for characterization of ligand binding dynamics to melanocortin 4 (MC₄) receptors. This approach enables on-line monitoring of reactions that is essential for estimation of more correct binding parameters, understanding of ligand binding and its regulation mechanisms, and design of new drugs with desirable properties. Two different red-shifted fluorophore-labeled peptide ligands, Cy3B-NDP-α-MSH and TAMRA-NDP-α-MSH, were used and compared in assays that monitored their binding to MC₄ receptors in membranes of Sf9 insect cells. The Cy3B dye-labeled ligand exhibited improved performance in assays when compared with the TAMRA-labeled ligand, having higher photostability, insensitivity to buffer properties, and better signal/noise ratio. The binding of both ligands to membranes of Sf9 cells expressing MC₄ receptors was saturable and with high affinity. All studied MC₄ receptor-specific nonlabeled ligands displaced fluoroligands’ binding in a concentration-dependent manner with potencies in agreement with their pharmacological activities. On-line monitoring of the reactions revealed that equilibrium of peptide binding was not reached even after 3 h. Real-time monitoring of ligand binding dynamics enabled us to find optimal experimental conditions for each particular ligand and an improved estimate of their binding parameters.     

Identification of the dual specificity and the functional domains of the cardiac-specific protein kinase TNNI3K

Molecular cloning of cardiac troponin I-interacting kinase (TNNI3K), a novel cardiac-specific protein kinase containing seven N-terminal ankyrin (ANK) repeats followed by a protein kinase domain and a C-terminal Ser-rich domain, has previously been reported. In the present study, we show that the C-terminal functional region of TNNI3K negatively regulates the kinase activity, and the N-terminal ANK domain is necessary for autophosphorylation. An in vitro kinase assay shows that TNNI3K exhibits dual-specific kinase activity and forms dimers or oligomers that may be necessary for its activation.     

Rescue of melanocortin 4 receptor (MC4R) nonsense mutations by aminoglycoside-mediated read-through

Aminoglycoside-mediated read-through of stop codons was recently demonstrated for a variety of diseases in vitro and in vivo. About 30 percent of human genetic diseases are the consequence of nonsense mutations. Nonsense mutations in obesity-associated genes like the melanocortin 4 receptor (MC4R), expressed in the hypothalamus, show the impact of premature stop codons on energy homeostasis. Therefore, the MC4R could be a potential pharmaceutical target for obesity treatment and targeting MC4R stop mutations could serve as proof of principle for nonsense mutations in genes expressed in the brain. We investigated four naturally occurring nonsense mutations in the MC4R (W16X, Y35X, E61X, Q307X) located at different positions in the receptor for aminoglycoside-mediated functional rescue in vitro. We determined localization and amount of full-length protein before and after aminoglycoside treatment by fluorescence microscopy, cell surface and total enzyme linked immunosorbent assay (ELISA). Signal transduction properties were analyzed by cyclic adenosine monophosphate (cAMP) assays after transient transfection of MC4R wild type and mutant receptors into COS-7 cells. Functional rescue of stop mutations in the MC4R is dependent on: (i) triplet sequence of the stop codon, (ii) surrounding sequence, (iii) location within the receptor, (iv) applied aminoglycoside and ligand. Functional rescue was possible for W16X, Y35X (N-terminus), less successful for Q307X (C-terminus) and barely feasible for E61X (first transmembrane domain). Restoration of full-length proteins by PTC124 could not be confirmed. Future pharmaceutical applications must consider the potency of aminoglycosides to restore receptor function as well as the ability to pass the blood-brain barrier.     

Synthesis and biological evaluation of imidazole-based small molecule antagonists of the melanocortin 4 receptor (MC4-R)

A novel series of imidazole-based small molecule antagonists of the melanocortin 4 receptor (MC4-R) is reported. Members of this series have been identified, which exhibit sub-micromolar binding affinity for the MC4-R, functional potency <100nM, and good oral exposure in rat. Antagonists of the MC4-R are potentially useful in the therapeutic treatment of involuntary weight loss due to advanced age or disease (e.g. cancer or AIDS), an area of large, unmet medical need.