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.     

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.     

Synthesis and characterization of new radioligands for the mammalian melanin-concentrating hormone (MCH) receptor

Melanin-concentrating hormone (MCH) is a neuropeptide present in the brain of all vertebrates. For the characterization of MCH receptors, a monoiodinated [Phe13, Tyr19]-MCH radioligand analogue was developed. The high susceptibility of [125I]-[Phe13, Tyr19]-MCH to oxidative damage and its very lipophilic nature made it necessary to develop new MCH radioligands. To increase the stability, native methionines were replaced by non-sulphur containing amino acid residues. In one analogue, the L-enantiomer of the phenylalanine residue at position 13 was substituted by the D-enantiomer, which increased the relative affinity of the ensuing [125I]-[D-Phe13, Tyr19]-MCH about 7-fold. The different analogues were iodinated by an enzymatic reaction and used for binding studies with mouse melanoma cells. [125I]-[Met(O)4,8, Phe13, Tyr19]-MCH and [125I]-[Hse4,8, Phe13, Tyr19]-MCH showed only about 19% of total binding and [125I]-[Ser4,8, Phe13, Tyr19]-MCH displayed about 44% of total binding when compared with [125I]-[Phe13, Tyr19]-MCH. Non-specific binding for all tracers was below 11% of total binding of [125I]-[Phe13, Tyr19]-MCH binding. [125I]-[D-Phe13, Tyr19]-MCH was used for saturation binding studies and revealed a KD of 122.7 +/- 15.3 pmol/l. This radioligand was further characterized by association and dissociation binding studies.     

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.     

Expression and intracellular localisation of odorant receptors in mammalian cell lines using Semliki Forest virus vectors

Odorant receptors are members of the G protein-coupled receptor superfamily. They are expressed on the surface of cilia of olfactory neurons, where they bind ligand (odorant). Studies of the molecular mechanisms of olfaction are complicated by the extremely large number of receptor genes, and difficulties in pairing a particular mammalian receptor to a specific odorant ligand in vivo. Here we report expression and localisation studies of two rat odorant receptor genes (17 and OR5), and C. elegans odr-10, using the Semliki Forest virus (SFV) system. All receptors were epitope-tagged at the N- or C-terminus in order to facilitate their detection in infected cells, and determine the localisation and membrane-orientation of recombinant proteins. The immortalised mouse olfactory neuronal cell line OLF 442, rat cortical and striatal primary neuron cultures, and the baby hamster kidney (BHK) cells, were infected and tested. Immunofluorescence and confocal microscopy studies performed on permeabilised, non-permeabilised and native cells revealed that in BHK cells the rat receptors 17 and OR5 were not targeted to the plasma membrane and remained in the endoplasmic reticulum. In contrast, in the mouse olfactory cell line OLF 442 both rat receptors were correctly inserted into the plasma membrane. Similar results were obtained using primary neurons, indicating that like mature neurons, the immortalised OLF 442 cells are capable of providing for correct odorant receptor processing and targeting.     

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.     

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.     

Effect of salmon melanin-concentrating hormone and mammalian gonadotrophin-releasing hormone on somatolactin release in pituitary culture of Cichlasoma dimerus

We detected a close morphological association between melanin-concentrating hormone (MCH)-immunoreactive (ir) fibers and somatolactin (SL)-ir cells in the pars intermedia of the cichlid fish Cichlasoma dimerus by double-label immunofluorescence. Male pituitaries obtained from adult C. dimerus were incubated with 0.1-10 muM salmon MCH, and the amount of SL released into the culture medium was semi-quantified by Western blot. This assay showed an increase of SL release in a dose-dependent manner (linear regression: P < 0.05). A close association of GnRH-ir fibers with SL-ir cells was also detected at the pars intermedia level. Male pituitaries were also incubated with 0.1-10 muM of mammalian GnRH, and SL release was semi-quantified by Western blot, showing an increase of released SL levels in a dose-dependent manner (linear regression: P < 0.05). In contrast, SL release was unaffected from female pituitaries incubated with salmon MCH; however, an increasing tendency was observed when mammalian GnRH was used. Hypothalamic close association of MCH-ir perikarya and GnRH-ir fibers was found by double-label immunofluorescence indicating a possible relationship between them. These results suggest that SL, like other pituitary hormones, is under hypothalamic control and is involved in diverse physiological processes including background adaptation and reproduction. This study has also shown that the in vitro culture of a single C. dimerus pituitary is a feasible method for studying the control of SL release and other pituitary hormones.     

Expression and regulation of phospholipase D isoforms in mammalian cell lines

Phospholipase D (PLD) is activated in mammalian cells in response to diverse stimuli that include growth factors, activators of protein kinase C, and agonists binding to G-protein-coupled receptors. Two forms of mammalian PLD, PLD1 and PLD2, have been identified. Expression of mRNA and protein for PLD1 and PLD2 was analyzed in the following cell lines: A7r5 (rat vascular smooth muscle); EL4 (mouse thymoma); HL-60 (human myeloid leukemia); Jurkat (human leukemia); PC-3 (human prostate adenocarcinoma); PC-12K (rat phaeochromocytoma); and Rat-1 HIR (rat fibroblast). All, with the exception of EL4, express agonist-activated PLD activity. PLD1 is expressed in A7r5, HL-60, PC-3, and Rat-1, while PLD2 is expressed in A7r5, Jurkat, PC12K, PC-3, and Rat-1. Neither isoform is expressed in EL4. Guanine nucleotide-independent PLD activity is present in membranes from all cells expressing PLD2. In PC12K cells, which express only PLD2, treatment with nerve growth factor causes neurite outgrowth and increases expression of PLD2 mRNA and protein within 6-12 h. A corresponding increase is observed in membrane PLD activity and in phorbol-12-myristate-13-acetate (PMA)-stimulated PLD activity in intact cells. These results show that PLD2 can be regulated both pretranslationally and posttranslationally by agonists.     

Expression of interleukin 2 receptors on interleukin 3-dependent cell lines

Several mouse IL 3-dependent cell lines, IC2, LT4, FDC-P2, and PB-3C, derived from spleen or bone marrow cells were shown to express low affinity receptors for IL 2 (Kd; 0.5 to 8 X 10(-8) M). High affinity receptors for IL 2 were not detected on the IL 3-dependent cells within the experimental limitation of this study. The clones did not respond to IL 2 at all at the concentration as high as 25 micrograms/ml. The number of the receptors expressed on those clones was estimated to be 0.2 to 2 X 10(5)/cell, which is comparable with the number of those on IL 2-dependent T cell clones. Expression of IL 2 receptor was confirmed in mRNA levels for both IC2 and LT4 cells. A relatively low level expression of one (4.5 Kb) of four IL 2 receptor mRNA species was observed with those IL 3-dependent clones compared with IL 2-dependent T cells. It seems that these low affinity receptors may be expressed on IL 3-dependent cells that undergo differentiation or maturation in mast cell and some myeloid cell lineages.     

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.     

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-R(1) 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-R(1) or MCH-R(2) genes in HEK293, Chinese hamster ovary, COS-7, or 3T3-L1 cells, or amplified MCH-R(1) 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 Ca(2+) 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.     

Expression of recombinant sea urchin cellulase SnEG54 using mammalian cell lines

We previously identified the cellulase SnEG54 from Japanese purple sea urchin Strongylocentrotus nudus, the molecular mass of which is about 54 kDa on SDS-PAGE. It is difficult to express and purify a recombinant cellulase protein using bacteria such as Escherichia coli or yeast. In this study, we generated mammalian expression vectors encoding SnEG54 to transiently express SnEG54 in mammalian cells. Both SnEG54 expressed in mammalian cells and SnEG54 released into the culture supernatant showed hydrolytic activity toward carboxymethyl cellulose. By using a retroviral expression system, we also established a mammalian cell line that constitutively produces SnEG54. Unexpectedly, SnEG54 released into the culture medium was not stable, and the peak time showing the highest concentration was approximately 1-2 days after seeding into fresh culture media. These findings suggest that non-mammalian sea urchin cellulase can be generated in human cell lines but that recombinant SnEG54 is unstable in culture medium due to an unidentified mechanism.     

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.