Cellular models for the study of the pharmacology and signaling of melanin-concentrating hormone receptors

Cellular models for the study of the neuropeptide melanin-concentrating hormone (MCH) have become indispensable tools for pharmacological profiling and signaling analysis of MCH and its synthetic analogues. Although expression of MCH receptors is most abundant in the brain, MCH-R₁ is also found in different peripheral tissues. Therefore, not only cell lines derived from nervous tissue but also from peripheral tissues that naturally express MCH receptors have been used to study receptor signaling and regulation. For screening of novel compounds, however, heterologous expression of MCH-R₁ or MCH-R₂ genes in HEK293, Chinese hamster ovary, COS-7, or 3T3-L1 cells, or amplified MCH-R₁ expression/signaling in IRM23 cells transfected with the G_q protein gene are the preferred tools because of more distinct pharmacological effects induced by MCH, which include inhibition of cAMP formation, stimulation of inositol triphosphate production, increase in intracellular free Ca²⁺ and/or activation of mitogen-activated protein kinases. Most of the published data originate from this type of model system, whereas data based on studies with cell lines endogenously expressing MCH receptors are more limited. This review presents an update on the different cellular models currently used for the analysis of MCH receptor interaction and signaling.     

Europium-labeled melanin-concentrating hormone analogues: ligands for measuring binding to melanin-concentrating hormone receptors 1 and 2

We investigated the use of Eu3+ chelate-labeled analogues of melanin-concentrating hormone (MCH) as ligands for both human MCH receptors (MCHR1 and MCHR2). The analogues employed were Ala17 MCH, S36057 (Y-ADO-RC*MLGRVFRPC*W, where ADO=8-amino-3,6-dioxyoctanoyl and *=disulfide bond), and R2P (RC*MLGRVFRPC*Y-NH2). The peptides were readily labeled on the alpha-amino residue with the Eu3+ chelate of N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-tetraacetic acid and then purified by reverse-phase fast-performance liquid chromatography at neutral pH to maintain Eu3+ chelation. Both labeled Ala17 MCH and S36057 had high affinity for MCHR1 ( Kd = 0.37 and 0.059nM, respectively) while Eu3+ -labeled S36057 and R2P had high affinity for MCHR2 ( Kd = 0.16 and 0.10nM, respectively). Labeled Ala17 MCH had little demonstrable binding affinity for MCHR2. Eu3+ -labeled S36057 and R2P were full agonists at MCHR1 when assessed by measurement of agonist-stimulated GTPgamma(35)S binding. Competition binding experiments with both MCHR isoforms, a series of previously characterized alanine scan MCH analogues, and a recently identified nonpeptide MCHR1-selective antagonist T-226296 confirmed the expected receptor selectivity. These studies further extend the utility of Eu3+ chelate time-resolved fluorescence for the development of high-sensitivity, nonradioactive receptor binding assays and demonstrate the need to select the optimal ligand for labeling.     

Melanin-concentrating hormone and melanin-concentrating hormone receptors in mammalian skin physiopathology

To date, there is a dearth of evidence to support functions for melanin-concentrating hormone (MCH) and melanin-concentrating hormone receptors (MCH-R) in mammalian skin physiology including pigmentation, inflammation and immune responses and skin cell proliferation. Much research is therefore still needed to define the roles of the hormone and its receptors in mammalian skin. This will be a crucial step to identifying pathogenic mechanisms that may involve the MCH/MCH-R system in the context of inflammatory and autoimmune skin diseases as well as skin cancers. The following review summarizes the studies which have been carried out to examine the expression and function of MCH and MCH-R in mammalian skin. Recent findings with regard to humoral immune responses to the MCH-R1 in patients with the skin depigmenting disease vitiligo are also discussed.     

Short segment of human melanin-concentrating hormone that is sufficient for full activation of human melanin-concentrating hormone receptors 1 and 2

Human melanin-concentrating hormone (hMCH) is a potent but nonselective agonist at human melanin-concentrating hormone receptors 1 and 2 (hMCH-1R and hMCH-2R, respectively). To determine the structural features of this neuropeptide which are necessary for efficient binding to and activation of the receptors, Ala-substituted, open-chain, and truncated analogues were synthesized and tested in the binding assays in CHO cells expressing hMCH-1R and hMCH-2R, and in functional assays measuring the level of intracellular calcium mobilization in human HEK-293 cells expressing these receptors. A compound consisting merely of the cyclic core of hMCH with the Arg attached to the N-terminus of the disulfide ring was found to activate both hMCH-1R and hMCH-2R about as effectively as full-length hMCH. Thus, the sequence Arg-cyclo(S-S)(Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys) appears to constitute the "active core" that is necessary for agonist potency at hMCH-1R and hMCH-2R. A potent and approximately 4-fold more selective agonist at hMCH-1R than at hMCH-2R is also reported.     

Signalling pathway of goldfish melanin-concentrating hormone receptors 1 and 2

Melanin-concentrating hormone (MCH) is the natural ligand for the MCH-1 receptor (MCHR1) and MCH-2 receptor (MCHR2). The MCH-MCHR1 system plays a central role in energy metabolism in rodents. Recently, we identified MCHR1 and MCHR2 orthologues in goldfish, designated gfMCHR1 and gfMCHR2. In a mammalian cell-based assay, calcium mobilization was evoked by gfMCHR2 via both Gαi/o and Gαq, while the gfMCHR1-mediated response was exclusively dependent on Gαq. This coupling capacity to G proteins is in contrast to human MCHR1 and MCHR2. Here, we extended our previous characterization of the two gfMCHRs by examining their different signalling pathway. We found that MCH caused activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) via both gfMCHR1 and gfMCHR2 in dose-dependent manners. Unlike the case for gfMCHR2, gfMCHR1 signalling was not sensitive to pertussis toxin, suggesting Gαq coupling of gfMCHR1 in the ERK1/2 pathway as well as a calcium mobilization system. Cyclic AMP assays revealed that gfMCHR2 was efficiently coupled to Gαi/o, while gfMCHR1 was weakly coupled to Gαs. Finally, we investigated the transduction features stimulated by two mammalian MCH analogues. As expected, Compound 15, which is a full agonist of human MCHR1, was a potent gfMCHR1 agonist in multiple signalling pathways. On the other hand, Compound 30, which is a human MCHR1-selective antagonist with negligible agonist potency, unexpectedly acted as a selective agonist of gfMCHR1. These results are the first to demonstrate that gfMCHR1 and gfMCHR2 have quite different signalling properties from human MCHRs.     

Expression and characterization of melanin-concentrating hormone receptors on mammalian cell lines

The neuropeptide melanin-concentrating hormone (MCH) is expressed in central and peripheral tissues where it participates in the complex network regulating energy homeostasis as well as in other physiologically important functions. Two MCH receptor subtypes, MCH-R1 and MCH-R2, have been cloned which signal through activation of Gi/o/q proteins and hence regulate different intracellular signals, such as inhibition of cAMP formation, stimulation of IP3 production, increase in intracellular free Ca2+ and/or activation of MAP kinases. Most of the data were obtained with cell systems heterologously expressing either of the MCH receptors. Fewer reports exist on studies with cell lines which endogenously express MCH receptors. Here, we describe human and other mammalian cell lines with which MCH receptor activation can be studied under "natural" conditions and we summarize the characteristics and signaling pathways of the MCH receptors in the different cell systems.     

Cellular signaling in eclosion hormone action

Eclosion hormone (EH) is a 62 amino acid neuropeptide that plays an integral role in triggering ecdysis behavior at the end of each molt. At least three populations of cells are thought to be targets for EH, each of which show an EH-stimulated increase in the intracellular messenger guanosine 3', 5' cyclic monophosphate (cGMP). These EH target cells are believed to include two pairs of neurons in each of the ganglia of the ventral nerve cord (VNC) that contain the neuropeptide crustacean cardioactive peptide (CCAP), the Inka cells of the peripheral epitracheal glands and intrinsic non-neuronal cells in the abdominal transverse nerves. This review describes likely signaling cascades that result in the EH-stimulated cGMP increase. Several lines of evidence suggest the involvement of a novel nitric oxide insensitive soluble guanylyl cyclase (GC). A novel GC with these properties has recently been identified and we also present evidence to suggest that it is activated by EH and describe possible pathways for its activation. In addition, we review our current knowledge on the cellular and molecular events that take place downstream of the increase in cGMP.     

Expression of receptors for melanin-concentrating hormone (MCH) in different tissues and cell lines

Melanin-concentrating hormone (MCH) is a potent orexigenic neuropeptide and a physiological antagonist of alpha-melanocyte-stimulating hormone (alpha-MSH) in the brain as well as at peripheral sites, including the pigmentary systems of specific vertebrates. Two receptor subtypes for MCH, MCH-R1 and MCH-R2, have been cloned, but other receptor subtypes are likely to exist. Based on our own data and the current literature, we have compared the expression of different receptors for MCH in various mammalian cell lines and tissues. Summarizing all data currently available, we conclude that the two cloned MCH receptors, MCH-R1 and MCH-R2, exhibit differences in their expression pattern, although MCH-R1 is generally colocalized in all tissues where MCH-R2 expression is found. It appears that MCH-R1 is more abundant and has a wider distribution pattern than MCH-R2. Other hypothetical MCH-R subtypes may be expressed in specific tissues, e.g., in the pigment cell system.     

Cellular signaling by fibroblast growth factor receptors

The 22 members of the fibroblast growth factor (FGF) family of growth factors mediate their cellular responses by binding to and activating the different isoforms encoded by the four receptor tyrosine kinases (RTKs) designated FGFR1, FGFR2, FGFR3 and FGFR4. Unlike other growth factors, FGFs act in concert with heparin or heparan sulfate proteoglycan (HSPG) to activate FGFRs and to induce the pleiotropic responses that lead to the variety of cellular responses induced by this large family of growth factors. A variety of human skeletal dysplasias have been linked to specific point mutations in FGFR1, FGFR2 and FGFR3 leading to severe impairment in cranial, digital and skeletal development. Gain of function mutations in FGFRs were also identified in a variety of human cancers such as myeloproliferative syndromes, lymphomas, prostate and breast cancers as well as other malignant diseases. The binding of FGF and HSPG to the extracellular ligand domain of FGFR induces receptor dimerization, activation and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of the receptor molecule. A variety of signaling proteins are phosphorylated in response to FGF stimulation including Shc, phospholipase-Cgamma, STAT1, Gab1 and FRS2alpha leading to stimulation of intracellular signaling pathways that control cell proliferation, cell differentiation, cell migration, cell survival and cell shape. The docking proteins FRS2alpha and FRS2beta are major mediators of the Ras/MAPK and PI-3 kinase/Akt signaling pathways as well as negative feedback mechanisms that fine-tune the signal that is initiated at the cell surface following FGFR stimulation.     

Cardiovascular effects of melanin-concentrating hormone

Melanin-concentrating hormone (MCH) is a cyclic 19-amino acid neuropeptide exclusively synthesized in the lateral hypothalamic area (LHA) and the zona incerta (ZI) that has been implicated in the regulation of energy balance. Despite what is known about the orexigenic effect of MCH, whether MCH has distinct cardiovascular and metabolic effects has yet to be determined. Thus, our goal here was to characterize the concurrent cardiovascular, metabolic, and behavioral responses of male rats to chronic intracerebroventricular (icv) infusion of MCH. Male Long-Evans rats were instrumented with telemetry transmitters for measurement of heart rate (HR) and housed in room calorimeters for assessment of food intake and oxygen consumption (VO(2)) at standard lab ambient temperature (23 degrees C) in order to examine physiological responses to chronic infusion of MCH (8 microg/d and 16 microg/d). Our findings provide the first evidence that chronic administration of MCH induces bradycardia and reduced mean arterial pressure, while it did not affect VO(2). A second experiment was performed in which the physiological responses to an acute icv infusion of MCH were observed. The results of experiment 2 indicate that MCH leads to a low HR that is maintained during the first 2 h post-infusion, the time period during which MCH acutely stimulated feeding. Collectively, these findings confirm that MCH may be an important modulator of sympathetic nervous system activity and thus may play a critical role in coordinating normal responses to negative energy balance.     

Structure-activity relationships of melanin-concentrating hormone

Melanin-concentrating hormone (MCH) is a cyclic heptadecapeptide (H-Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-O H) that induces aggregation of melanin granules within the melanophores of teleost fishes. Chemical and enzymatic modifications of MCH were conducted in order to deduce the structure-activity relationship using an in vitro bioassay with fish scales, and a radioimmunoassay using a specific antiserum to synthetic MCH. Micro-modification of MCH was employed with the natural peptide, and the modified form was purified by reverse-phase HPLC. MCH1-14 and NPS-Trp15-MCH were equipotent to MCH. Reduction and carboxamidomethylation of MCH caused complete loss of biological activity. Modification of the Tyr residue with tetranitromethane and Arg residues with 1,2-cyclohexadione significantly reduced activity, while oxidation with hydrogen peroxide caused only partial loss (10%) of activity. These results suggest that the configuration of the S-S loop is essential for activity, and Arg and Tyr may play an important role in the biological activity. In the radioimmunoassay, MCH1-14, MCH5-14 and CAM-Cys5,14-MCH showed no cross-reactivity, whereas MCH5-17 and other derivatives gave inhibition slopes parallel to the MCH standard, suggesting that the antigenic determinant of the antiserum is located in the carboxy-terminal.     

Binding sites for melanin-concentrating hormone in the human brain

Binding sites for melanin-concentrating hormone (MCH) in human brain were investigated and characterized by radioligand binding. Specific binding sites for MCH were present in every region of human brain (cerebral cortex, cerebellum, thalamus, hypothalamus, pons, and medulla oblongata) obtained at autopsy. alpha-Melanocyte stimulating hormone or ACTH was a poor inhibitor of (125)I-MCH binding (IC(50) 1 microM) compared with MCH (IC(50) = 0.3 +/- 0.07 nM, mean +/- SEM, n = 3). Scatchard plots of (125)I-MCH binding in human brain (thalamus) gave a dissociation constant of 0.2 +/- 0.06 nM and maximal binding of 5.8 +/- 0.3 fmol/mg protein (n = 3). These findings suggest that specific MCH binding sites that differ from the melanocortin receptors exist in human brain.     

Structural features of the inactive and active states of the melanin-concentrating hormone receptors: insights from molecular simulations

Comparative molecular dynamics simulations of both subtypes 1 and 2 of the melanin-concentrating hormone receptor (MCHR1 and MCHR2, respectively) in their free and hormone-bound forms have been carried out. The hormone has been used in its full-length and truncated forms, as well as in 16 mutated forms. Moreover, MCHR1 has been simulated in complex with T-226296, a novel orally active and selective antagonist. The comparative analysis of an extended number of receptor configurations suggests that the differences between inactive (i.e., free and antagonist-bound) and active (i.e., agonist-bound) states of MCHRs involve the receptor portions close to the E/DRY and NPxxY motifs, with prominence to the cytosolic extensions of helices 2, 3, 6, and 7. In fact, the active forms of these receptors share the release of selected intramolecular interactions found in the inactive forms, such as that between R3.50 of the E/DRY motif and D2.40, and that between Y7.53 of the NPxxY motif and F7.60. Another feature of the active forms of both MCHRs is the approach of "helix 8" to the cytosolic extension of helix 3. These features of the active forms are concurrent with the opening of a cleft at the cytosolic end of the helix bundle. For both MCHRs, the agonist-induced chemical information transfer from the extracellular to the cytosolic domains is mediated by a cluster of aromatic amino acids in helix 6, following the ligand interaction with selected amino acids in the extracellular half of the receptor.     

Regulation of melanin-concentrating hormone receptor 1 signaling by RGS8 with the receptor third intracellular loop

Melanin-concentrating hormone (MCH) receptor 1 (MCH1R) belongs to the class A G protein-coupled receptors (GPCRs). The MCH-MCH1R system plays a central role in energy metabolism, and thus the regulation of signaling pathways activated by this receptor is of particular interest. Regulator of G protein signaling (RGS) proteins work by increasing the GTPase activity of G protein alpha subunits and attenuate cellular responses coupled with G proteins. Recent evidence has shown that RGS proteins are not simple G protein regulators but equally inhibit the signaling from various GPCRs. Here, we demonstrate that RGS8, which is highly expressed in the brain, functions as a negative modulator of MCH1R signaling. By using biochemical approaches, RGS8 was found to selectively and directly bind to the third intracellular (i3) loop of MCH1R in vitro. When expressed in HEK293T cells, RGS8 and MCH1R colocalized to the plasma membrane and RGS8 potently inhibited the calcium mobilization induced by MCH. The N-terminal 9 amino acids of RGS8 were required for the optimal capacity to downregulate the receptor signaling. Furthermore, Arg(253) and Arg(256) at the distal end of the i3 loop were found to comprise a structurally important site for the functional interaction with RGS8, since coexpression of RGS8 with R253Q/R256Q mutant receptors resulted in a loss of induction of MCH-stimulated calcium mobilization. This functional association suggests that RGS8 may represent a new therapeutic target for the development of novel pharmaceutical agents.     

Functions of melanin-concentrating hormone in fish

Melanin-concentrating hormone (MCH) was originally discovered in fish, in which it causes aggregation or concentration of melanin granules in melanophores, thus regulating body color. MCH is a cyclic neuropeptide synthesized as a preprohormone in the hypothalamus of all vertebrates. Mammalian MCH plays an important role as a neurotransmitter or neuromodulator in regulating food intake and energy homeostasis. MCH signaling system may involve in regulating food intake also in fish. This neuropeptide binds to G-protein-coupled seven transmembrane receptor[s] to mediate its functions. This article reviews MCH and MCH receptor signaling systems in body color change and food intake in fish.     

Importance of melanin-concentrating hormone receptor for the acute effects of ghrelin

The hypothalamic peptide melanin-concentrating hormone (MCH) and the gastric hormone ghrelin take part in the regulation of energy homeostasis and stimulate food intake. In the present study, ghrelin was administered centrally to MCH-receptor knockout (MCHr KO) mice. MCHr KO mice and wild type (WT) controls both consumed more food when treated with ghrelin. After ghrelin administration, the serum levels of insulin increased only in WT mice whereas the serum levels of corticosterone increased both in WT and MCHr KO mice. The level of growth hormone (GH) mRNA in the pituitary gland was markedly increased in response to ghrelin injection in the WT mice but was unaffected in the MCHr KO mice. The different ghrelin responses could not be explained by a difference in growth hormone secretagogue receptor expression between MCHr KO and WT mice in the pituitary or hypothalamus. In summary, the MCHr is not required for ghrelin induced feeding. However, the MCHr does play a role for the effect of ghrelin on GH expression in the pituitary and serum insulin levels.     

Membrane organization of luteinizing hormone receptors differs between actively signaling and desensitized receptors

Signaling by the luteinizing hormone/choriogonadotropin receptor (LHR) is of considerable interest because of its requirement for successful reproduction. Time-resolved phosphorescence anisotropy and fluorescence resonance energy transfer were used to investigate the organization of endogenous LHRs in porcine follicular membranes in two distinct signaling states, active and desensitized. Desensitized LHRs exhibited approximately 3-fold slower rotational correlation times compared with active LHRs (59 +/- 4 and 21 +/- 9 mus, respectively), suggesting that with agonist-dependent desensitization the receptors are organized into larger protein complexes. Incubation of membranes with inhibitors of LHR desensitization, such as neutralizing anti-arrestin antibodies, a synthetic peptide corresponding to the third intracellular loop of the LHR but not the corresponding scrambled peptide, or catalytically inactive ARNO, resulted in faster rotational diffusion times equivalent to those of actively signaling LHRs. Furthermore, desensitized LHRs exhibited a 2.4-fold increase in fluorescence resonance energy transfer between LHRs suggesting that the larger protein aggregates formed during desensitization contain more self-associated LHRs. These results indicate that agonist-dependent LHR desensitization precedes organization of LHRs at the cells surface into larger protein aggregates.     

Role of the melanin-concentrating hormone neuropeptide in sleep regulation

Melanin-concentrating hormone (MCH), a neuropeptide secreted by a limited number of neurons within the tuberal hypothalamus, has been drawn in the field of sleep only fairly recently in 2003. Since then, growing experimental evidence indicates that MCH may play a crucial role in the homeostatic regulation of paradoxical sleep (PS). MCH-expressing neurons fire specifically during PS. When injected icv MCH induces a 200% increase in PS quantities in rats and the lack of MCH induces a decrease in sleep quantities in transgenic mice. Here, we review recent studies suggesting a role for MCH in the regulation of the sleep–wake cycle, in particular PS, including insights on (1) the specific activity of MCH neurons during PS; (2) how they might be controlled across the sleep–wake cycle; (3) how they might modulate PS; (4) and finally whether MCH might take part in the expression of some symptoms observed in primary sleep disorders.