Automated Patch Clamp Recordings of GPCR-Gated Ion Channels: Targeting the MC4-R/Kir7.1 Potassium Channel Complex.

Automated patch clamp recording is a valuable technique in drug discovery and the study of ion channels. It allows for the precise measurement and manipulation of channel currents, providing insights into their function and modulation by drugs or other compounds. The melanocortin 4 receptor (MC4-R) is a G protein-coupled receptor (GPCR) crucial to appetite regulation, energy balance, and body weight. MC4-R signaling is complex and involves interactions with other receptors and neuropeptides in the appetite-regulating circuitry. MC4-Rs, like other GPCRs, are known to modulate ion channels such as Kir7.1, an inward rectifier potassium channel, in response to ligand binding. This modulation is critical for controlling ion flow across the cell membrane, which can influence membrane potential, excitability, and neurotransmission. The MC4-R is the target for the anti-obesity drug Imcivree. However, this drug is known to lack optimal potency and also has side effects. Using high-throughput techniques for studying the MC4-R/Kir7.1 complex allows researchers to rapidly screen many compounds or conditions, aiding the development of drugs that target this system. Additionally, automated patch clamp recording of this receptor-channel complex and its ligands can provide valuable functional and pharmacological insights supporting the development of novel therapeutic strategies. This approach can be generalized to other GPCR-gated ion channel functional complexes, potentially accelerating the pace of research in different fields with the promise to uncover previously unknown aspects of receptor-ion channel interactions.

Novel Cocrystal Structures of Peptide Antagonists Bound to the Human Melanocortin Receptor 4 Unveil Unexplored Grounds for Structure-Based Drug Design.

Melanocortin 4 receptor (MC4-R) antagonists are actively sought for treating cancer cachexia. We determined the structures of complexes with PG-934 and SBL-MC-31. These peptides differ from SHU9119 by substituting His6 with Pro6 and inserting Gly10 or Arg10. The structures revealed two subpockets at the TM7-TM1-TM2 domains, separated by N2857.36. Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study. Most ligands retained the SHU9119 potency, but several SBL-MC-31-derived peptides significantly enhanced MC4-R selectivity over MC1-R by 60- to 132-fold. We also investigated MC4-R coupling to the K+ channel, Kir7.1. Some peptides activated the channel, whereas others induced channel closure independently of G protein coupling. In cell culture studies, channel activation correlated with increased feeding, while a peptide with Kir7.1 inhibitory activity reduced eating. These results highlight the potential for targeting the MC4-R:Kir7.1 complex for treating positive and restrictive eating disorders.

Inhibition of the melanocortin-3 receptor (MC3R) causes generalized sensitization to anorectic agents.

The melanocortin-3 receptor (MC3R) acts presynaptically to regulate GABA release from agouti-related protein (AgRP) nerve terminals and thus may be a negative regulator of multiple circuits involved in feeding behavior and energy homeostasis. Here, we examined the role of MC3R in regulating the response to various anorexigenic agents. Our findings reveal that genetic deletion or pharmacological inhibition of MC3R improves the dose responsiveness to Glucagon-like peptide 1 (GLP1) agonists, as assayed by inhibition of food intake and weight loss. An enhanced anorectic response to other agents, including the acute satiety factors peptide YY (PYY3-36) and cholecystokinin (CCK) and the long-term adipostatic factor, leptin, demonstrated that increased sensitivity to anorectic agents is a generalized result of MC3R antagonism. Enhanced neuronal activation in multiple nuclei, including ARH, VMH, and DMH, was observed using Fos immunohistochemistry following low-dose liraglutide in MC3R knockout mice (Mc3r-/-), supporting the hypothesis that the MC3R is a negative regulator of circuits regulating multiple aspects of feeding behavior. The enhanced anorectic response in Mc3r -/- mice after administration of GLP1 analogs was also independent of the incretin effects and malaise induced by GLP1R analogs, suggesting that MC3R antagonists may have value in enhancing the dose-response range of obesity therapeutics.

Targeting the central melanocortin system for the treatment of metabolic disorders.

A large body of preclinical and clinical data shows that the central melanocortin system is a promising therapeutic target for treating various metabolic disorders such as obesity and cachexia, as well as anorexia nervosa. Setmelanotide, which functions by engaging the central melanocortin circuitry, was approved by the FDA in 2020 for use in certain forms of syndromic obesity. Furthermore, the FDA approvals in 2019 of two peptide drugs targeting melanocortin receptors for the treatment of generalized hypoactive sexual desire disorder (bremelanotide) and erythropoietic protoporphyria-associated phototoxicity (afamelanotide) demonstrate the safety of this class of peptides. These approvals have also renewed excitement in the development of therapeutics targeting the melanocortin system. Here, we review the anatomy and function of the melanocortin system, discuss progress and challenges in developing melanocortin receptor-based therapeutics, and outline potential metabolic and behavioural disorders that could be addressed using pharmacological agents targeting these receptors.

The unique structural characteristics of the Kir 7.1 inward rectifier potassium channel: a novel player in energy homeostasis control.

The inward rectifier potassium channel Kir7.1, encoded by the KCNJ13 gene, is a tetramer composed of two-transmembrane domain-spanning monomers, closer in homology to Kir channels associated with potassium transport such as Kir1.1, 1.2, and 1.3. Compared with other channels, Kir7.1 exhibits small unitary conductance and low dependence on external potassium. Kir7.1 channels also show a phosphatidylinositol 4,5-bisphosphate (PIP2) dependence for opening. Accordingly, retinopathy-associated Kir7.1 mutations mapped at the binding site for PIP2 resulted in channel gating defects leading to channelopathies such as snowflake vitreoretinal degeneration and Leber congenital amaurosis in blind patients. Lately, this channel's role in energy homeostasis was reported due to the direct interaction with the melanocortin type 4 receptor (MC4R) in the hypothalamus. As this channel seems to play a multipronged role in potassium homeostasis and neuronal excitability, we will discuss what is predicted from a structural viewpoint and its possible implications for hunger control.

Demonstration of a Common DPhe7 to DNal(2')7 Peptide Ligand Antagonist Switch for Melanocortin-3 and Melanocortin-4 Receptors Identifies the Systematic Mischaracterization of the Pharmacological Properties of Melanocortin Peptides.

Melanocortin peptides containing a 3-(2-naphthyl)-d-alanine residue in position 7 (DNal(2')7), reported as melanocortin-3 receptor (MC3R) subtype-specific agonists in two separate publications, were found to lack significant MC3R agonist activity. The cell lines used at the University of Arizona for pharmacological characterization of these peptides, consisting of HEK293 cells stably transfected with human melanocortin receptor subtypes MC1R, MC3R, MC4R, or MC5R, were then obtained and characterized by quantitative polymerase chain reaction (PCR). While the MC1R cell line correctly expressed only hMCR1, the three other cell lines were mischaracterized with regard to receptor subtype expression. The demonstration that a 3-(2-naphthyl)-d-alanine residue in position 7, irrespective of the melanocortin peptide template, results primarily in the antagonism of MC3R and MC4R then allowed us to search the published literature for additional errors. The erroneously characterized DNal(2')7-containing peptides date back to 2003; thus, our analysis suggests that systematic mischaracterization of the pharmacological properties of melanocortin peptides occurred.

The melanocortin-3 receptor is a pharmacological target for the regulation of anorexia.

Ablation of hypothalamic AgRP (Agouti-related protein) neurons is known to lead to fatal anorexia, whereas their activation stimulates voracious feeding and suppresses other motivational states including fear and anxiety. Despite the critical role of AgRP neurons in bidirectionally controlling feeding, there are currently no therapeutics available specifically targeting this circuitry. The melanocortin-3 receptor (MC3R) is expressed in multiple brain regions and exhibits sexual dimorphism of expression in some of those regions in both mice and humans. MC3R deletion produced multiple forms of sexually dimorphic anorexia that resembled aspects of human anorexia nervosa. However, there was no sexual dimorphism in the expression of MC3R in AgRP neurons, 97% of which expressed MC3R. Chemogenetic manipulation of arcuate MC3R neurons and pharmacologic manipulation of MC3R each exerted potent bidirectional regulation over feeding behavior in male and female mice, whereas global ablation of MC3R-expressing cells produced fatal anorexia. Pharmacological effects of MC3R compounds on feeding were dependent on intact AgRP circuitry in the mice. Thus, the dominant effect of MC3R appears to be the regulation of the AgRP circuitry in both male and female mice, with sexually dimorphic sites playing specialized and subordinate roles in feeding behavior. Therefore, MC3R is a potential therapeutic target for disorders characterized by anorexia, as well as a potential target for weight loss therapeutics.

Structure-Based Design of Melanocortin 4 Receptor Ligands Based on the SHU-9119-hMC4R Cocrystal Structure†.

The melanocortin receptors (MC1R-MC5R) belong to class A G-protein-coupled receptors (GPCRs) and are known to have receptor-specific roles in normal and diseased states. Selectivity for MC4R is of particular interest due to its involvement in various metabolic disorders, including obesity, feeding regulation, and sexual dysfunctions. To further improve the potency and selectivity of MC4R (ant)agonist peptide ligands, we designed and synthesized a series of cyclic peptides based on the recent crystal structure of MC4R in complex with the well-characterized antagonist SHU-9119 (Ac-Nle4-c[Asp5-His6-DNal(2')7-Arg8-Trp9-Lys10]-NH2). These analogues were pharmacologically characterized in vitro, giving key insights into exploiting binding site subpockets to deliver more selective ligands. More specifically, the side chains of the Nle4, DNal(2')7, and Trp9 residues in SHU-9119, as well as the amide linkage between the Asp5 and Lys10 side chains, were found to represent structural features engaging a hMC4R/hMC3R selectivity switch.

Membrane orientation and oligomerization of the melanocortin receptor accessory protein 2.

The melanocortin receptor accessory protein 2 (MRAP2) plays a pivotal role in the regulation of several G protein-coupled receptors that are essential for energy balance and food intake. MRAP2 loss-of-function results in obesity in mammals. MRAP2 and its homolog MRAP1 have an unusual membrane topology and are the only known eukaryotic proteins that thread into the membrane in both orientations. In this study, we demonstrate that the conserved polybasic motif that dictates the membrane topology and dimerization of MRAP1 does not control the membrane orientation and dimerization of MRAP2. We also show that MRAP2 dimerizes through its transmembrane domain and can form higher-order oligomers that arrange MRAP2 monomers in a parallel orientation. Investigating the molecular details of MRAP2 structure is essential for understanding the mechanism by which it regulates G protein-coupled receptors and will aid in elucidating the pathways involved in metabolic dysfunction.

Determination of the melanocortin-4 receptor structure identifies Ca2+ as a cofactor for ligand binding.

The melanocortin-4 receptor (MC4R) is involved in energy homeostasis and is an important drug target for syndromic obesity. We report the structure of the antagonist SHU9119-bound human MC4R at 2.8-angstrom resolution. Ca2+ is identified as a cofactor that is complexed with residues from both the receptor and peptide ligand. Extracellular Ca2+ increases the affinity and potency of the endogenous agonist α-melanocyte-stimulating hormone at the MC4R by 37- and 600-fold, respectively. The ability of the MC4R crystallized construct to couple to ion channel Kir7.1, while lacking cyclic adenosine monophosphate stimulation, highlights a heterotrimeric GTP-binding protein (G protein)-independent mechanism for this signaling modality. MC4R is revealed as a structurally divergent G protein-coupled receptor (GPCR), with more similarity to lipidic GPCRs than to the homologous peptidic GPCRs.

The GPCR accessory protein MRAP2 regulates both biased signaling and constitutive activity of the ghrelin receptor GHSR1a.

Ghrelin is a hormone secreted by the stomach during fasting periods and acts through its receptor, the growth hormone secretagogue 1a (GHSR1a), to promote food intake and prevent hypoglycemia. As such, GHSR1a is an important regulator of energy and glucose homeostasis and a target for the treatment of obesity. Here, we showed that the accessory protein MRAP2 altered GHSR1a signaling by inhibiting its constitutive activity, as well as by enhancing its G protein-dependent signaling and blocking the recruitment and signaling of ß-arrestin in response to ghrelin. In addition, the effects of MRAP2 on the Gαq and ß-arrestin pathways were independent and involved distinct regions of MRAP2. These findings may have implications for the regulation of ghrelin function in vivo and the role of MRAP2 in energy homeostasis. They also show that accessory proteins can bias signaling downstream of GPCRs in response to their endogenous agonist.

60 YEARS OF POMC: Regulation of feeding and energy homeostasis by α-MSH.

The melanocortin peptides derived from pro-opiomelanocortin (POMC) were originally understood in terms of the biological actions of α-melanocyte-stimulating hormone (α-MSH) on pigmentation and adrenocorticotrophic hormone on adrenocortical glucocorticoid production. However, the discovery of POMC mRNA and melanocortin peptides in the CNS generated activities directed at understanding the direct biological actions of melanocortins in the brain. Ultimately, discovery of unique melanocortin receptors expressed in the CNS, the melanocortin-3 (MC3R) and melanocortin-4 (MC4R) receptors, led to the development of pharmacological tools and genetic models leading to the demonstration that the central melanocortin system plays a critical role in the regulation of energy homeostasis. Indeed, mutations in MC4R are now known to be the most common cause of early onset syndromic obesity, accounting for 2-5% of all cases. This review discusses the history of these discoveries, as well as the latest work attempting to understand the molecular and cellular basis of regulation of feeding and energy homeostasis by the predominant melanocortin peptide in the CNS, α-MSH.

A G Protein-biased Designer G Protein-coupled Receptor Useful for Studying the Physiological Relevance of Gq/11-dependent Signaling Pathways.

Designerreceptorsexclusivelyactivated by adesignerdrug (DREADDs) are clozapine-N-oxide-sensitive designer G protein-coupled receptors (GPCRs) that have emerged as powerful novel chemogenetic tools to study the physiological relevance of GPCR signaling pathways in specific cell types or tissues. Like endogenous GPCRs, clozapine-N-oxide-activated DREADDs do not only activate heterotrimeric G proteins but can also trigger ß-arrestin-dependent (G protein-independent) signaling. To dissect the relative physiological relevance of G protein-mediatedversusß-arrestin-mediated signaling in different cell types or physiological processes, the availability of G protein- and ß-arrestin-biased DREADDs would be highly desirable. In this study, we report the development of a mutationally modified version of a non-biased DREADD derived from the M3muscarinic receptor that can activate Gq/11with high efficacy but lacks the ability to interact with ß-arrestins. We also demonstrate that this novel DREADD is activein vivoand that cell type-selective expression of this new designer receptor can provide novel insights into the physiological roles of G protein (Gq/11)-dependentversusß-arrestin-dependent signaling in hepatocytes. Thus, this novel Gq/11-biased DREADD represents a powerful new tool to study the physiological relevance of Gq/11-dependent signaling in distinct tissues and cell types, in the absence of ß-arrestin-mediated cellular effects. Such studies should guide the development of novel classes of functionally biased ligands that show high efficacy in various pathophysiological conditions but display a reduced incidence of side effects.

Salmeterol Efficacy and Bias in the Activation and Kinase-Mediated Desensitization of ß2-Adrenergic Receptors.

Salmeterol is a long-acting ß2-adrenergic receptor (ß2AR) agonist that is widely used as a bronchodilator for the treatment of persistent asthma and chronic obstructive pulmonary disease in conjunction with steroids. Previous studies demonstrated that salmeterol showed weak efficacy for activation of adenylyl cyclase; however, its efficacy in the complex desensitization of the ß2AR remains poorly understood. In this work, we provide insights into the roles played by the G protein-coupled receptor kinase/arrestin and protein kinase A in salmeterol-mediated desensitization through bioluminescence resonance energy transfer (BRET) studies of liganded-ß2AR binding to arrestin and through kinetic studies of cAMP turnover. First, BRET demonstrated a much reduced efficacy for salmeterol recruitment of arrestin to ß2AR relative to isoproterenol. The ratio of BRETISO/BRETSALM after 5-minute stimulation was 20 and decreased to 5 after 35 minutes, reflecting a progressive decline in BRETISO and a stable BRETSALM. Second, to assess salmeterol efficacy for functional desensitization, we examined the kinetics of salmeterol-induced cAMP accumulation (0-30 minutes) in human airway smooth muscle cells in the presence and absence of phosphodiesterase inhibition. Analysis of shaping of cAMP turnover for both agonists demonstrated significant salmeterol desensitization, although it was reduced relative to isoproterenol. Using an isoproterenol rescue protocol after either short-term (10 minutes) or long-term (2 and 14 hours) salmeterol pretreatments, we found that salmeterol progressively depressed isoproterenol stimulation but did not prevent subsequent rescue by isoproterenol and additional isoproterenol-mediated desensitization. Our findings reveal a complex efficacy for functional desensitization, demonstrating that although salmeterol shows weak efficacy for adenylyl cyclase activation and G protein-coupled receptor kinase/arrestin-mediated desensitization, it acts as a strong agonist in highly amplified protein kinase A-mediated events.

Peptide modifications differentially alter G protein-coupled receptor internalization and signaling bias.

Although G protein-coupled receptors (GPCRs) are targeted by more clinically used drugs than any other type of protein, their ligand development is particularly challenging. Humans have four neuropeptide Y receptors: hY1R and hY5R are orexigenic, while hY2R and hY4R are anorexigenic, and represent important anti-obesity drug targets. We show for the first time that PEGylation and lipidation, chemical modifications that prolong the plasma half-lives of peptides, confer additional benefits. Both modifications enhance pancreatic polypeptide preference for hY2R/hY4R over hY1R/hY5R. Lipidation biases the ligand towards arrestin recruitment and internalization, whereas PEGylation confers the opposite bias. These effects were independent of the cell system and modified residue. We thus provide novel insights into the mode of action of peptide modifications and open innovative venues for generating peptide agonists with extended therapeutic potential.

Mutations in arrestin-3 differentially affect binding to neuropeptide Y receptor subtypes.

Based on the identification of residues that determine receptor selectivity in arrestins and the phylogenetic analysis of the arrestin (arr) family, we introduced fifteen mutations of receptor-discriminator residues in arr-3, which were identified previously using mutagenesis, in vitro binding, and BRET-based recruitment assay in intact cells. The effects of these mutations were tested using neuropeptide Y receptors Y1R and Y2R. NPY-elicited arr-3 recruitment to Y1R was not affected by these mutations, or even alanine substitution of all ten residues (arr-3-NCA), which prevented arr-3 binding to other receptors tested so far. However, NCA and two other mutations prevented agonist-independent arr-3 pre-docking to Y1R. In contrast, eight out of 15 mutations significantly reduced agonist-dependent arr-3 recruitment to Y2R. NCA eliminated arr-3 binding to active Y2R, whereas Tyr239Thr reduced it ~7-fold. Thus, manipulation of key residues on the receptor-binding surface generates arr-3 with high preference for Y1R over Y2R. Several mutations differentially affect arr-3 pre-docking and agonist-induced recruitment. Thus, arr-3 recruitment to the receptor involves several mechanistically distinct steps. Targeted mutagenesis can fine-tune arrestins directing them to specific receptors and particular activation states of the same receptor.

Arrestin-3 binds the MAP kinase JNK3α2 via multiple sites on both domains.

Although arrestins bind dozens of non-receptor partners, the interaction sites for most signaling proteins remain unknown. Here we report the identification of arrestin-3 elements involved in binding MAP kinase JNK3α2. Using purified JNK3α2 and MBP fusions containing separated arrestin-3 domains and peptides exposed on the non-receptor-binding surface of arrestin-3 we showed that both domains bind JNK3α2 and identified one element on the N-domain and two on the C-domain that directly interact with JNK3α2. Using in vitro competition we confirmed that JNK3α2 engages identified N-domain element and one of the C-domain peptides in the full-length arrestin-3. The 25-amino acid N-domain element has the highest affinity for JNK3α2, suggesting that it is the key site for JNK3α2 docking. The identification of elements involved in protein-protein interactions paves the way to targeted redesign of signaling proteins to modulate cell signaling in desired ways. The tools and methods developed here to elucidate the molecular mechanism of arrestin-3 interactions with JNK3α2 are suitable for mapping of arrestin-3 sites involved in interactions with other partners.

Targeting individual GPCRs with redesigned nonvisual arrestins.

Numerous human diseases are caused by excessive signaling of mutant G protein-coupled receptors (GPCRs) or receptors that are overstimulated due to upstream signaling imbalances. The feasibility of functional compensation by arrestins with enhanced ability to quench receptor signaling was recently tested in the visual system. The results showed that even in this extremely demanding situation of rods that have no ability to phosphorylate rhodopsin, enhanced arrestin improved rod morphology, light sensitivity, survival, and accelerated photoresponse recovery. Structurally distinct enhanced mutants of arrestins that bind phosphorylated and non-phosphorylated active GPCRs with much higher affinity than parental wild-type (WT) proteins have been constructed. These "super-arrestins" are likely to have the power to dampen the signaling by hyperactive GPCRs. However, most cells express 5-20 GPCR subtypes, only one of which would be overactive, while nonvisual arrestins are remarkably promiscuous, binding hundreds of different GPCRs. Thus, to be therapeutically useful, enhanced versions of nonvisual arrestins must be made fairly specific for particular receptors. Recent identification of very few arrestin residues as key receptor discriminators paves the way to the construction of receptor subtype-specific nonvisual arrestins.

A role for an Hsp70 nucleotide exchange factor in the regulation of synaptic vesicle endocytosis.

Neurotransmission requires a continuously available pool of synaptic vesicles (SVs) that can fuse with the plasma membrane and release their neurotransmitter contents upon stimulation. After fusion, SV membranes and membrane proteins are retrieved from the presynaptic plasma membrane by clathrin-mediated endocytosis. After the internalization of a clathrin-coated vesicle, the vesicle must uncoat to replenish the pool of SVs. Clathrin-coated vesicle uncoating requires ATP and is mediated by the ubiquitous molecular chaperone Hsc70. In vitro, depolymerized clathrin forms a stable complex with Hsc70*ADP. This complex can be dissociated by nucleotide exchange factors (NEFs) that release ADP from Hsc70, allowing ATP to bind and induce disruption of the clathrin:Hsc70 association. Whether NEFs generally play similar roles in vesicle trafficking in vivo and whether they play such roles in SV endocytosis in particular is unknown. To address this question, we used information from recent structural and mechanistic studies of Hsp70:NEF and Hsp70:co-chaperone interactions to design a NEF inhibitor. Using acute perturbations at giant reticulospinal synapses of the sea lamprey (Petromyzon marinus), we found that this NEF inhibitor inhibited SV endocytosis. When this inhibitor was mutated so that it could no longer bind and inhibit Hsp110 (a NEF that we find to be highly abundant in brain cytosol), its ability to inhibit SV endocytosis was eliminated. These observations indicate that the action of a NEF, most likely Hsp110, is normally required during SV trafficking to release clathrin from Hsc70 and make it available for additional rounds of endocytosis.

Modulation of methuselah expression targeted to Drosophila insulin-producing cells extends life and enhances oxidative stress resistance.

Ubiquitously reduced signaling via Methuselah (MTH), a G-protein-coupled receptor (GPCR) required for neurosecretion, has previously been reported to extend life and enhance stress resistance in flies. Whether these effects are due to reduced MTH signalling in specific tissues remains unknown. We determined that reduced expression of mth targeted to the insulin-producing cells (IPCs) of the fly brain was sufficient to extend life and enhance oxidative stress resistance. Paradoxically, we discovered that overexpression of mth targeted to the same cells has similar phenotypic effects to reduced expression due to MTH's interaction with ß-arrestin, which uncouples GPCRs from their G-proteins. We confirmed the functional relationship between MTH and ß-arrestin by finding that IPC-targeted overexpression of ß-arrestin alone mimics the longevity phenotype of reduced MTH signaling. As reduced MTH signaling also inhibits insulin secretion from the IPCs, the most parsimonious mechanistic explanation of its longevity and stress-resistance enhancement might be through reduced insulin/IGF signaling (IIS). However, examination of phenotypic features of long-lived IPC-mth modulated flies as well as several downstream IIS targets implicates enhanced activity of the JNK stress-resistance pathway more directly than insulin signaling in the longevity and stress-resistance phenotypes.