Identification and pharmacological characterization of a novel human melanin-concentrating hormone receptor, mch-r2.

Melanin-concentrating hormone (MCH) is a neuropeptide highly expressed in the brain that regulates several physiological functions mediated by receptors in the G protein-coupled receptor family. Recently an orphan receptor, SLC-1, has been identified as an MCH receptor (MCH-R1). Herein we identify and characterize a novel receptor for human MCH (MCH-R2). The receptor is composed of 340 amino acids encoded by a 1023-base pair cDNA and is 35% homologous to SLC-1. (125)I-MCH specifically bound to Chinese hamster ovary cells stably expressing MCH-R2. MCH stimulated dose-dependent increases in intracellular free Ca(2+) and inositol phosphate production in these cells but did not affect cAMP production. The pharmacological profile for mammalian MCH, [Phe(13),Tyr(19)]MCH, and salmon MCH at MCH-R2 differed compared with MCH-R1 as assessed by intracellular signaling and radioligand binding assays. The EC(50) in signaling assays and the IC(50) in radioligand binding assays of salmon MCH was an order of magnitude higher than mammalian MCH at MCH-R2. By comparison, the EC(50) and IC(50) values of salmon MCH and mammalian MCH at MCH-R1 were relatively similar. Blot hybridization revealed exclusive expression of MCH-R2 mRNA in several distinct brain regions, particularly in the cortical area, suggesting the involvement of MCH-R2 in the central regulation of MCH-mediated functions.     

The receptor for the orexigenic peptide melanin-concentrating hormone is a G-protein-coupled receptor

Gene-knockout studies of melanin-concentrating hormone (MCH) and its effect on feeding and energy balance have firmly established MCH as an orexigenic (appetite-stimulating) peptide hormone. Here we identify MCH as the ligand for the orphan receptor SLC-1. The rat SLC-1 is activated by nanomolar concentrations of MCH and is coupled to the G protein G alpha i/o. The pattern of SLC-1 messenger RNA expression coincides with the distribution of MCH-containing nerve terminals and is consistent with the known central effects of MCH. Our identification of an MCH receptor could have implications for the development of new anti-obesity therapies.     

Endogenous melanin-concentrating hormone receptor SLC-1 in human melanoma SK-MEL-37 cells.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that regulates several physiological functions. The orphan G protein-coupled receptors SLC-1 and MCHR2 were recently found to bind MCH with high affinity. We show here that the human melanoma cell line SK-MEL-37 expresses SLC-1 mRNA but not MCHR2 by RT-PCR analysis and immunofluorescence studies. Using Chinese hamster ovary cells and 293 cells overexpressing SLC-1 by cDNA transfection, it was shown that SLC-1 coupled to both G alpha(i)/G alpha(o) and G alpha(q) proteins. In SK-MEL-37 cells, MCH inhibited forskolin-stimulated cyclic AMP accumulation and induced mitogen-activated protein kinase (MAPK) in a pertussis toxin-(PTX)-sensitive manner. The MAPK activity leads to the production of phosphorylated forms of p42/p44 MAPK. However, an increase in the intracellular free Ca(2+) concentration was not elicited by MCH in SK-MEL-37 cells. These results show that SLC-1 is coupled only to PTX-sensitive G alpha(i)/G alpha(o) in SK-MEL-37 cells. This study provides for the first time a skin-derived cellular model to analyze the molecular mechanism of the MCH signaling pathway.     

Molecular cloning and sequencing of a cDNA encoding a human alpha 1A adrenergic receptor.

DNA from a rat hippocampus cDNA library and sets of highly degenerate oligonucleotide primers directed toward conserved regions of previously cloned G-protein receptors were used in the polymerase chain reaction to selectively amplify and clone new members of this gene family. A human hippocampus cDNA library was screened with a 610 base pair fragment generated by PCR and a cDNA clone, H318/3, was isolated. The deduced amino acid sequence of this clone encoded a protein of 501 amino acids that showed strong sequence homology to previously cloned G-protein receptors. Nucleotide sequence analysis revealed clone H318/3 was 78% homologous to a rat alpha 1A adrenergic receptor with homology being 95% when comparisons were made in the region that lies between the first to the seventh transmembrane domains. Based on this high degree of sequence homology, we conclude that clone H318/3 represents a cDNA for a human alpha 1A adrenergic receptor.     

Discovery of a receptor related to the galanin receptors

We report the isolation of a cDNA clone named GPR54, which encodes a novel G protein-coupled receptor (GPCR). A PCR search of rat brain cDNA retrieved a clone partially encoding a GPCR. In a library screening this clone was used to isolate a cDNA with an open reading frame (ORF) encoding a receptor of 396 amino acids long which shared significant identities in the transmembrane regions with rat galanin receptors GalR1 (45%), GalR3 (45%) and GalR2 (44%). Northern blot and in situ hybridization analyses revealed that GPR54 is expressed in brain regions (pons, midbrain, thalamus, hypothalamus, hippocampus, amygdala, cortex, frontal cortex, and striatum) as well as peripheral regions (liver and intestine). In COS cell expression of GPR54 no specific binding was observed for 125I-galanin. A recent BLAST search with the rat GPR54 ORF nucleotide sequence recovered the human orthologue of GPR54 in a 3.5 Mb contig localized to chromosome 19p13.3.     

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.     

Identification of melanin concentrating hormone (MCH) as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1).

To identify possible ligands of the orphan somatostatin-like receptor 1 (SLC-1), rat brain extracts were analyzed by using the functional expression system of Xenopus oocytes injected with cRNAs encoding SLC-1 and G protein-gated inwardly rectifying potassium channels (GIRK). A strong inward current was observed with crude rat brain extracts which upon further purification by cation exchange chromatography and high performance liquid chromatography (HPLC) yielded two peptides with a high agonist activity. Mass spectrometry and partial peptide sequencing revealed that one peptide is identical with the neuropeptide melanin concentrating hormone (MCH), the other represents a truncated version of MCH lacking the three N-terminal amino acid residues. Xenopus oocytes expressing the MCH receptor responded to nM concentrations of synthetic MCH not only by the activation of GIRK-mediated currents but also by the induction of Ca(2+) dependent chloride currents mediated by phospholipase C. This indicates that the MCH receptor can couple either to the G(i)- or G(q)-mediated signal transduction pathway, suggesting that MCH may serve for a number of distinct brain functions including food uptake behavior.     

Identification of a histamine H4 receptor on human eosinophils--role in eosinophil chemotaxis

Eosinophils are recruited to sites of inflammation via the action of a number of chemical mediators, including PAF, leukotrienes, eotaxins, ECF-A and histamine. Although many of the cell-surface receptors for these mediators have been identified, histamine-driven chemotaxis has not been conclusively attributed to any of the three known histamine receptor subtypes, suggesting the possibility of a 4th histamine-responsive receptor on eosinophils. We have identified and cloned a novel G protein-coupled receptor (GPCR), termed Pfi-013, from an IL-5 stimulated eosinophil cDNA library which is homologous to the human histamine H3 receptor, both at the sequence and gene structure level. Expression data indicates that Pfi-013 is predominantly expressed in peripheral blood leukocytes, with lower expression levels in spleen, testis and colon. Ligand-binding studies using Pfi-013 expressed in HEK-293Galpha15 cells, demonstrates specific binding to histamine with a Kd of 3.28 +/- 0.76 nM and possesses a unique rank order of potency against known histaminergic compounds in a competitive ligand-binding assay (histamine > clobenpropit > iodophenpropit > thioperamide > R-alpha-methylhistamine > cimetidine > pyrilamine). We have therefore termed this receptor human histamine H4. Chemotaxis studies on isolated human eosinophils have confirmed that histamine is chemotactic and that agonists of the known histamine receptors (H1, H2, and H3) do not induce such a response. Furthermore, studies employing histamine-receptor antagonists have shown an inhibition of chemotaxis only by the H3 antagonists clobenpropit and thioperamide. Since these compounds are also antagonists of hH4 we postulate that the receptor mediating histaminergic chemotaxis is this novel histamine H4 receptor.     

Cloning and expression of a novel mouse somatostatin receptor (SSTR2B).

A mouse somatostatin (SS) receptor cDNA was cloned from neuroblastoma x glioma (NG108-15) cells. The sequence is almost identical to that of the mouse SSTR2 receptor [(1992) Proc. Natl. Acad. Sci. USA 89, 251)] but lacks about 300 nucleotides between transmembrane domain VII and the C-terminus. This spliced variant of SSTR2 (designated SSTR2B) encodes a protein which is 23 residues shorter than that predicted from the SSTR2 sequence, and differs in 15 amino acids at the C-terminus. mRNA corresponding to SSTR2B occurs in mouse tissues in higher abundance than that of SSTR2. SSTR2B binds SS peptides with high affinity when expressed in mammalian cells.     

Cloning and characterization of Rhesus monkey neuropeptide Y receptor subtypes

Neuropeptide Y (NPY) is a 36 amino acid peptide that is abundant in the brain and peripheral nervous system. NPY has a variety of effects when administered into the brain including a pronounced feeding effect, anxiolysis, regulation of neuroendocrine axes and inhibition of neurotransmitter release. These effects are mediated by up to 6 G protein coupled receptors designated Y1, Y2, Y3, Y4, Y5 and y6. To better understand the phylogeny and pharmacology of NPY in non-human primates, we have cloned and expressed the NPY Y1, Y2 and Y5 receptor subtypes from the Rhesus monkey. No cDNA sequence encoding a Y4 receptor was found suggesting substantial sequence differences when compared to the human sequence. Comparison of these sequences with those from human indicated strong sequence conservation of Y1, Y2 and Y5 between the two species. The displacement of (125)I-PYY binding to the Rhesus monkey and human receptors by various peptides was compared to evaluate the pharmacology of the two species. Similar pharmacologies were noted across the species at the various receptor subtypes. These results indicate the Rhesus monkey and human NPY receptor subtypes have a close amino acid sequence conservation and that the peptide recognition domains are conserved as well.     

Structure-activity relationship studies of melanin-concentrating hormone (MCH)-related peptide ligands at SLC-1, the human MCH receptor

Melanin-concentrating hormone (MCH) is a cyclic nonadecapeptide involved in the regulation of feeding behavior, which acts through a G protein-coupled receptor (SLC-1) inhibiting adenylcyclase activity. In this study, 57 analogues of MCH were investigated on the recently cloned human MCH receptor stably expressed in HEK293 cells, on both the inhibition of forskolin-stimulated cAMP production and guanosine-5'-O-(3-[(35)S]thiotriphosphate ([(35)S]- GTPgammaS) binding. The dodecapeptide MCH-(6-17) (MCH ring between Cys(7) and Cys(16), with a single extra amino acid at the N terminus (Arg(6)) and at the C terminus (Trp(17))) was found to be the minimal sequence required for a full and potent agonistic response on cAMP formation and [(35)S]- GTPgammaS binding. We Ala-scanned this dodecapeptide and found that only 3 of 8 amino acids of the ring, namely Met(8), Arg(11), and Tyr(13), were essential to elicit full and potent responses in both tests. Deletions inside the ring led either to inactivity or to poor antagonists with potencies in the micromolar range. Cys(7) and Cys(16) were substituted by Asp and Lys or one of their analogues, in an attempt to replace the disulfide bridge by an amide bond. However, those modifications were deleterious for agonistic activity. In [(35)S]- GTPgammaS binding, these compounds behaved as weak antagonists (K(B) 1-4 microm). Finally, substitution in MCH-(6-17) of 6 out of 12 amino acids by non-natural residues and concomitant replacement of the disulfide bond by an amide bond led to three compounds with potent antagonistic properties (K(B) = 0.1-0.2 microm). Exploitation of these structure-activity relationships should open the way to the design of short and stable MCH peptide antagonists.     

Expression of melanin-concentrating hormone receptors in insulin-producing cells: MCH stimulates insulin release in RINm5F and CRI-G1 cell-lines

Melanin-concentrating hormone (MCH) is a hypothalamic orexigenic peptide. Recently, an orphan G-protein-coupled receptor (SLC-1) was identified that binds MCH with high affinity. Here, we demonstrate the mRNA expression of this receptor in insulin-producing cells including CRI-G1 and RINm5F cells, and in rat islets of Langerhans. Immunofluorescence studies in CRI-G1 and RINm5F cell-lines demonstrated cell-surface expression of the receptor. Rat MCH significantly stimulated insulin secretion in both cell-lines. The potency and the efficacy of MCH were significantly increased in the simultaneous presence of forskolin, suggesting that MCH may amplify the insulinotropic effect of cyclic AMP elevating stimuli. Salmon MCH, which differs from rat/human MCH by six amino acids, was less efficacious than rat/human MCH in stimulating insulin release. The data provide evidence for the expression of MCH receptors in insulin producing cells. The insulinotropic effect of MCH may contribute to the regulation of metabolism and energy balance by this peptide.     

EXPRESSION OF RECEPTORS FOR MELANIN-CONCENTRATING HORMONE (MCH) IN DIFFERENT TISSUES AND CELL LINES

ABSTRACT

Melanin-concentrating hormone (MCH) is a potent orexigenic neuropeptide and a physiological antagonist of α-melanocyte-stimulating hormone (α-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.     

Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide.

Vasoactive intestinal polypeptide (VIP), a 28 amino acid peptide hormone, plays many physiological roles in the peripheral and central nerve systems. A functional cDNA clone of the VIP receptor was isolated from a rat lung cDNA library by cross-hybridization with the secretin receptor cDNA. VIP bound the cloned VIP receptor expressed in mouse COP cells and stimulated adenylate cyclase through the cloned receptor. The rat VIP receptor consists of 459 amino acids with a calculated Mr of 52,054 and contains seven transmembrane segments. It is structurally related to the secretin, calcitonin, and parathyroid hormone receptors, suggesting that they constitute a new subfamily of the Gs protein-coupled receptors. VIP receptor mRNA was detected in various rat tissues including liver, lung, intestines, and brain. In situ hybridization revealed that VIP receptor mRNA is widely distributed in neuronal cells of the adult rat brain, with a relatively high expression in the cerebral cortex and hippocampus.     

Molecular cloning of the human transmembrane secretory component (poly-Ig receptor) and its mRNA expression in human tissues

A 2.5 kilobase (kb) cDNA clone containing 92% of the coding region for human transmembrane secretory component (SC) or poly-Ig receptor, was isolated from a mammary gland cDNA library. The cDNA clone encoded a protein of 693 amino acids which showed 99% homology with the primary amino acid sequence of human free SC as reported by Eiffert et al. (1), and 54% homology with the deduced amino acid sequence of rabbit transmembrane SC for which cDNA was cloned by Mostov et al. (2). Northern blot analysis showed mRNA expression in various human exocrine tissues in good agreement with our previous immunohistochemical studies of SC.