RESUMEN
The melanocortin 1 receptor is a G-protein-coupled receptor, described to be expressed on melanomas and melanocytes. Subsequent RT-PCR studies demonstrated the presence of melanocortin 1 receptor mRNA in other tissues such as pituitary gland and testis. Previously, we have demonstrated that three HLA-A2 binding nonamer peptides derived from melanocortin 1 receptor can elicit peptide-specific CTL which can recognize target cells transfected with the melanocortin 1 receptor gene and MHC class I matched melanoma lines. The potential of targeting melanocortin 1 receptor in therapy and diagnosis will depend on a preferential expression of this receptor in the majority of primary and metastatic melanomas vs normal tissues. We tested a panel of melanomas, carcinomas and other cell lines for the presence of melanocortin 1 receptor, using two monoclonal antibodies. The receptor was detected in 83% of the tested melanoma cell lines but not in other carcinoma lines. Immunohistochemistry revealed a strong expression of melanocortin 1 receptor in all tested primary and metastatic melanomas, but also demonstrated low levels of expression in adrenal medulla, cerebellum, liver and keratinocytes. Flow cytometry studies showed that melanocortin 1 receptor was expressed in in vitro activated monocytes/macrophages and in the THP-1 monocytic leukaemia line at levels of about 1 in 3 to 1 in 5 of that found in melanomas. Peripheral blood-derived dendritic cells, also express melanocortin 1 receptor in vitro. This extensive analysis of melanocortin 1 receptor tissue distribution may be of relevance not only for melanoma immunology, but also for research on the pathogenicity of inflammatory conditions in the skin and neurologic tissues. It remains to be seen if the over-expression of melanocortin 1 receptor in melanomas is sufficiently high to allow a 'therapeutic window' to be exploited in cancer immunotherapy.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Melanoma/metabolismo , Receptores de Corticotropina/metabolismo , Humanos , Monocitos/metabolismo , Receptores de Melanocortina , Distribución Tisular , Células Tumorales CultivadasRESUMEN
Reduction of disulfide bonds in human melanocortin 1 receptor (hMC1R) with increasing concentrations of DTT (dithiothreitol) resulted in a decrease in the binding of [125I]-ACTH (adrenocorticotropic hormone, L-isomer) in an uniphasic manner and a decrease in [125I]-NDP-MSH ([Nle(4),D-Phe(7)]-alpha-melanocyte stimulating hormone; D-isomer) binding in a biphasic manner. Pretreatment of hMC1R with 10 mM DTT resulted in a 36-fold loss of affinity for alpha-MSH (L-isomer) without affecting the affinity of NDP-MSH (D-isomer). To characterize the role of individual cysteine residues, we employed site-directed mutagenesis to substitute cysteine by glycine at all fourteen positions in hMC1R and analysed wild-type and mutant receptors for ligand binding and cAMP signalling. Single point mutation of four cysteine residues in extracellular loops to glycine (C35G, C267G, C273G, and C275G) resulted in a complete loss of binding for [125I]-NDP-MSH. Moreover, mutants with normal ligand binding, at positions C191G (transmembrane segment 5), C215G (third intracellular loop), and C315G (C-terminal loop) failed to generate cAMP signal in response to both agonists alpha-MSH and NDP-MSH. Mutant at position C78G (with wild-type binding to alpha-MSH as well as NDP-MSH) generated a cAMP signal in response to alpha-MSH (identical to wild-type hMC1R) but interestingly could not be stimulated by NDP-MSH. Moreover, this single amino acid substitution converted NDP-MSH from being an agonist to antagonist at the C78G mutant receptor. These findings demonstrate that (i) alpha-MSH and ACTH (L-isomers) are different from D-isomer NDP-MSH in their sensitivity to DTT for receptor binding, (ii) cysteine residues in N-terminus and extracellular loop three make disulfide bridges and are needed for structural integrity of hMC1R, (iii) cysteine residues in transmembrane segments and intracellular loops are required for receptor-G-protein coupling, (iv) C78 in transmembrane segment two is required for generating a functional response by D-isomer agonist (NDP-MSH) but not by L-isomer agonist (alpha-MSH), and (v) wild-type receptor agonist NDP-MSH is an antagonist at the mutant C78G receptor.
Asunto(s)
Cisteína/genética , Receptores de Corticotropina/metabolismo , alfa-MSH/análogos & derivados , Hormona Adrenocorticotrópica/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Unión Competitiva/efectos de los fármacos , Células COS , AMP Cíclico/metabolismo , ADN Recombinante/genética , Ditiotreitol/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Radioisótopos de Yodo , Proteínas de la Membrana/agonistas , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Ensayo de Unión Radioligante , Receptores de Corticotropina/química , Receptores de Corticotropina/genética , Receptores de Melanocortina , Relación Estructura-Actividad , Transfección , alfa-MSH/metabolismo , alfa-MSH/farmacologíaRESUMEN
Neuropeptide Y (NPY), a 36-amino-acid peptide widely expressed in the brain is involved in many physiological responses, including hypothalamic control of food intake and cardiovascular homeostasis. NPY mediates its effects through binding to the Y1, Y2 and Y5 G-protein-coupled receptors. Little is known of the role of the Y2 receptor in mediating the different NPY effects. We inactivated the Y2 receptor subtype in mice and found that these mice developed increased body weight, food intake and fat deposition. The null mutant mice showed an attenuated response to leptin administration but a normal response to NPY-induced food intake and intact regulation of re-feeding and body weight after starvation. An absence of the Y2 receptor subtype also affected the basal control of heart rate, but did not influence blood pressure. These findings indicate an inhibitory role for the Y2 receptor subtype in the central regulation of body weight and control of food intake.
Asunto(s)
Peso Corporal/fisiología , Conducta Alimentaria/fisiología , Neuropéptido Y/farmacología , Proteínas/farmacología , Receptores de Neuropéptido Y/metabolismo , Tejido Adiposo/metabolismo , Animales , Presión Sanguínea , Femenino , Frecuencia Cardíaca , Leptina , Ratones , Ratones Mutantes , Unión Proteica , Receptores de Leptina , Receptores de Neuropéptido Y/genéticaRESUMEN
We have cloned the guinea pig neuropeptide Y (NPY) Y1 receptor and found it to be 92-93% identical to other cloned mammalian Y1 receptors. Porcine NPY and peptide YY (PYY) displayed affinities of 43 pM and 48 pM, respectively. NPY2-36 and NPY3-36 had 6- and 46-fold lower affinity, respectively, than intact NPY. Functional coupling was measured by using a microphysiometer. Human NPY and PYY were equipotent in causing extracellular acidification with EC50 values of 0.59 nM and 0.69 nM, respectively, whereas NPY2-36 and NPY3-36 were about 15-fold and 500-fold less potent, respectively, than NPY. The present study shows that the cloned guinea pig Y1 receptor is very similar to its orthologues in other mammals, both with respect to sequence and pharmacology. Thus, results from previous studies on guinea pig NPY receptors might imply the existence of an additional Y1-like receptor sensitive to B1BP3226.
Asunto(s)
Receptores de Neuropéptido Y/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Células CHO , Cricetinae , AMP Cíclico/biosíntesis , ADN , Cobayas , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Filogenia , Receptores de Neuropéptido Y/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de AminoácidoRESUMEN
The hypothesis that macrophages contain an autocrine circuit based on melanocortin [ACTH and alpha-melanocyte-stimulating hormone (alpha-MSH)] peptides has major implications for neuroimmunomodulation research and inflammation therapy. To test this hypothesis, cells of the THP-1 human monocyte/macrophage line were stimulated with lipopolysaccharide (LPS) in the presence and absence of alpha-MSH. The inflammatory cytokine tumor necrosis factor (TNF)-alpha was inhibited in relation to alpha-MSH concentration. Similar inhibitory effects on TNF-alpha were observed with ACTH peptides that contain the alpha-MSH amino acid sequence and act on melanocortin receptors. Nuclease protection assays indicated that expression of the human melanocortin-1 receptor subtype (hMC-1R) occurs in THP-1 cells; Southern blots of RT-PCR product revealed that additional subtypes, hMC-3R and hMC-5R, also occur. Incubation of resting macrophages with antibody to hMC-1R increased TNF-alpha concentration; the antibody also markedly reduced the inhibitory influence of alpha-MSH on TNF-alpha in macrophages treated with LPS. These results in cells known to produce alpha-MSH at rest and to increase secretion of the peptide when challenged are consistent with an endogenous regulatory circuit based on melanocortin peptides and their receptors. Targeting of this neuroimmunomodulatory circuit in inflammatory diseases in which myelomonocytic cells are prominent should be beneficial.
Asunto(s)
Macrófagos/metabolismo , Monocitos/metabolismo , Receptores de la Hormona Hipofisaria/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , alfa-MSH/metabolismo , Hormona Adrenocorticotrópica/genética , Hormona Adrenocorticotrópica/inmunología , Anticuerpos , Comunicación Autocrina/fisiología , Southern Blotting , Línea Celular , Expresión Génica/inmunología , Humanos , Lipopolisacáridos/farmacología , Macrófagos/química , Macrófagos/inmunología , Monocitos/química , Monocitos/inmunología , Neuroinmunomodulación/efectos de los fármacos , Neuroinmunomodulación/inmunología , Receptores de la Hormona Hipofisaria/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , alfa-MSH/genéticaRESUMEN
To delineate domains essential for G-protein coupling in melanocortin 1 receptor (MC1R), we mutated polar and basic residues to alanine at eleven positions in the putative third intracellular loop and determined consequent changes in the ligand binding and generation of second messenger cAMP. Results demonstrate that ligand binding affinity was not affected by any of the mutations. However, every mutant displayed reduced functional response as compared to the wild type receptor. Replacement of residues (K226, R227, Q228, R229, H232, Q233 and K238) present in second half of third intracellular loop resulted in an almost complete loss of functional response. The results have demonstrated that the amino acid residues present in C-terminal portion of third intracellular loop of MC1R are involved in coupling to G-protein and that a region of four amino acids, K226-R227-Q228-R229 is essential for coupling of MC1R to G-protein.
Asunto(s)
Aminoácidos/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores de Corticotropina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Aminoácidos/genética , Animales , Células COS , Línea Celular , AMP Cíclico/metabolismo , Humanos , Concentración 50 Inhibidora , Ligandos , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Receptores de Corticotropina/química , Receptores de Melanocortina , Transducción de Señal/efectos de los fármacos , Transfección , alfa-MSH/análogos & derivados , alfa-MSH/metabolismo , alfa-MSH/farmacologíaRESUMEN
Corticotropin is produced by keratinocytes and may have an immunoregulatory role in oral mucosa and skin. We have investigated its effects on a human oral keratinocyte cell line and shown that corticotropin, acting via its specific receptor, stimulates a dose-dependent increase in DNA synthesis and induces cell proliferation. When cells were incubated in the presence of increasing concentrations of corticotropin, there were significant increases in intracellular cAMP levels. Corticotropin-stimulated mitogenesis and cell proliferation were attenuated by the adenylyl cyclase inhibitor SQ22,536, but were unaffected by inhibitors of protein kinase C or tyrosine kinase. These data identify corticotropin as a mitogenic regulatory peptide of keratinocytes acting via cAMP.
Asunto(s)
Hormona Adrenocorticotrópica/farmacología , Queratinocitos/citología , Mucosa Bucal/efectos de los fármacos , Receptores de Corticotropina/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Inhibidores de Adenilato Ciclasa , Bucladesina/farmacología , División Celular , Línea Celular , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Queratinocitos/efectos de los fármacos , Mucosa Bucal/citología , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Transducción de SeñalRESUMEN
alpha-Melanocyte stimulating hormone (alpha-MSH) regulates skin and hair pigmentation by modulating the activity of MSH receptor (MC1R). We have identified Arg151Cys variant of human MC1R in genomic DNA isolated from a person with red hair and light skin of type I. The Arg151Cys variant of MC1R binds to radio-labelled analogue of alpha-MSH with identical affinity as wild type MC1R but can not be stimulated to produce cyclic AMP (cAMP). The mutation Arg151Cys renders human MC1R completely nonfunctional, which explains the red hair, light skin and poor tanning ability (skin type I). This is the first report ever describing a nonfunctional MC1R isolated from a human subject.
Asunto(s)
Pigmentación/fisiología , Mutación Puntual/genética , Receptores de la Hormona Hipofisaria/genética , Animales , Unión Competitiva , Células COS , Clonación Molecular , AMP Cíclico/metabolismo , Análisis Mutacional de ADN , Femenino , Humanos , Radioisótopos de Yodo/metabolismo , Receptores de la Hormona Hipofisaria/fisiología , Análisis de Secuencia de ADN , Transfección/genética , alfa-MSH/análogos & derivados , alfa-MSH/metabolismoRESUMEN
Human melanoma-specific HLA-A2 restricted CTLs have recently been shown to recognize antigens expressed by melanoma lines and normal melanocytes, including Melan-A/Mart-1, gp100, gp75, and tyrosinase. Herein, we define HLA-A2-restricted CTL epitopes from a recently cloned melanocortin 1 receptor (MC1R), which belongs to a new subfamily of the G-protein-coupled receptors expressed on melanomas and melanocytes. Thirty-one MC1R-derived peptides were selected on the basis of HLA-A2-specific motifs and tested for their HLA-A2 binding capacity. Of a group of 12 high or intermediate HLA-A2 binding peptides, three nonamers, MC1R244 (TILLGIFFL), MC1R283 (FLALIICNA), and MC1R291 (AIIDPLIYA), were found to induce peptide-specific CTLs from peripheral blood mononuclear cells of healthy HLA-A2+ donors after repeated in vitro stimulation with peptide-pulsed antigen-presenting cells. The CTLs raised against these three HLA-A2+-restricted peptides could recognize naturally processed peptides from HLA-A2+ melanomas and from Cos7 cells cotransfected with MC1R and HLA-A2. CTLs induced by the MC1R291 peptide (but not induced or induced only to a very low extent by the other two MCR1 peptide epitopes) showed cross-reactions with two other members of the melanocortin receptor family, which are more broadly expressed on other tissues. Taken together, our findings have implications in relation both to autoimmunity and immunotherapy of malignant melanomas.
Asunto(s)
Antígenos de Neoplasias/inmunología , Antígeno HLA-A2/inmunología , Melanoma/inmunología , Fragmentos de Péptidos/farmacología , Receptores de Corticotropina/química , Linfocitos T Citotóxicos/efectos de los fármacos , Animales , Presentación de Antígeno , Antígenos de Neoplasias/química , Autoinmunidad , Células COS , Humanos , Inmunoterapia , Melanoma/patología , Fragmentos de Péptidos/síntesis química , Receptores de Melanocortina , Células Tumorales CultivadasRESUMEN
The human melanocortin 5 receptor (hMC5R) in the melanocortin receptor family has been identified as the receptor with low affinity towards alpha-MSH. Here we show that the glutamine at position 235 and arginine at the position 272 in the hMC5R are contributing to the low affinity of this receptor. Glutamine235 and arginine272 in hMC5R were mutated to lysine (Q235K) and cysteine (R272C), respectively, residues which are conserved at these positions in other melanocortin receptor subtypes. Upon these mutations affinity of alpha-MSH for hMC5R was increased 10-fold for Q235K and 690-fold for R272C mutants, respectively. The results explain the unusually low affinity of the hMC5R to the melanocortic ligands and suggest the importance of these conserved residues in maintaining the high affinity form of melanocortin receptors.
Asunto(s)
Receptores de Corticotropina/química , alfa-MSH/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Humanos , Ligandos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Receptores de Corticotropina/metabolismo , Receptores de Melanocortina , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
We describe the immunohistochemical detection of the melanocortin 1 receptor (MC1R) protein in human gonadal tissues using a specific monoclonal antibody. The MC1R was found to be present in Leydig's cells in testis, in lutein cells in the corpus luteum and in the nucleus of the trophoblastic cells of the placenta. Though it has been speculated earlier that MC1R is present in gonadal tissues, this is the first report demonstrating the presence of MC1R protein in these cells.
Asunto(s)
Gónadas/química , Placenta/química , Receptores de Corticotropina/análisis , Anticuerpos Monoclonales , Núcleo Celular/química , Femenino , Gónadas/citología , Humanos , Inmunohistoquímica , Células Intersticiales del Testículo/química , Células Lúteas/química , Masculino , Placenta/citología , Receptores de Corticotropina/inmunología , Receptores de Melanocortina , Trofoblastos/químicaAsunto(s)
Mutación Puntual , Receptores de la Hormona Hipofisaria/genética , Animales , Células COS , Color del Cabello/genética , Humanos , Hormonas Estimuladoras de los Melanocitos/metabolismo , Metionina , Receptores de la Hormona Hipofisaria/metabolismo , Proteínas Recombinantes/metabolismo , Piel/metabolismo , ValinaRESUMEN
Distribution of cDNA for five individual melanocortin receptor subtypes in 20 different human tissues was determined by PCR using subtype specific primers. PCR products were first visualised by agarose gel electrophoresis and ethidium bromide staining, and specific products were identified for melanocortin 1 receptor (MC1R) in pituitary and testis, for MC2R in adrenal gland, for MC3R in heart, for MC5R in adrenal gland, fat cells, kidney, leukocytes, lung, lymph node, mammary gland, ovary, pituitary, testis and uterus. The MC4R cDNA could not be detected by ethidium bromide staining. More tissues were revealed as positive when the DNA from PCR were hybridised with subtype specific radioactive probes. All the subtypes except MC4R could be detected in testis. MC4R could only be detected in pituitary. This is the first report describing the comprehensive distribution analysis of melanocortin receptor subtype cDNAs in human tissues, and provides a link between individual receptor subtype and diverse biological activities of melanocortic peptides in the respective target tissues.
Asunto(s)
Receptores de Corticotropina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Sondas de ADN , ADN Complementario/análisis , Electroforesis en Gel de Agar , Humanos , Datos de Secuencia Molecular , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Receptores de Corticotropina/clasificación , Receptores de Corticotropina/genética , Receptores de MelanocortinaRESUMEN
To investigate whether residues in the extracellular domains of melanocortin 1 receptor (MC1R) are required for ligand binding, a number of mutants were constructed where charged residues were converted to alanine. The residues targeted for mutagenesis were Ser6, Glu102, Arg109, Asp184, Glu269, and Thr272. The mutant receptor DNAs were transiently expressed in COS-1 cells and their ability to bind [N1e4,D-Phe7]-alpha-MSH (NDP-MSH) was evaluated. Substitution of Asp184 by alanine completely abolished the binding of radiolabelled NDP-MSH as well as ACTH, even though the mutated receptor could be detected on cell surface using anti MC1R specific polyclonal antiserum. Mutations of Ser6, Glu269 and Thr272 resulted in a considerable loss of affinity for radiolabelled NDP-MSH as well as the ability of alpha-MSH to displace the bound radiolabelled NDP-MSH. The results demonstrate that the extracellular loops of human MC1R contain important ligand binding epitopes.
Asunto(s)
Estructura Secundaria de Proteína , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , alfa-MSH/análogos & derivados , Marcadores de Afinidad , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Membrana Celular , Chlorocebus aethiops , Clonación Molecular , Cartilla de ADN , Humanos , Cinética , Ligandos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Ensayo de Unión Radioligante , Receptores de Melatonina , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfección , alfa-MSH/metabolismoRESUMEN
This study was conducted to determine the binding properties of recently discovered, putative alpha-MSH antagonist 153N-6 peptide at melanocortin receptor subtypes. The results indicated that 153N-6 peptide can competitively inhibit [125I]NDP-MSH binding from all the receptor subtypes investigated. The relative potency order of 153N-6 for inhibiting [125I]NDP-MSH binding was MC1R (Ki 955 +/- 35.7 nM) = MC4R (Ki 1151 +/- 106 nM) > MC3R (Ki 3229 +/- 637 nM) > MC5R (Ki 6286 +/- 462 nM), which is different than the potency order of either NDP-MSH or alpha-MSH. Substitution of aspartic acid117 and histidine260 by alanine in melanocortin 1 receptor resulted in a 4.75-fold decrease (Ki 4541 +/- 644 nM) and an 11-fold increase (Ki 84.29 +/- 4.53 nM), respectively, in the relative potency of 153N-6 for competitively inhibiting [125I]NDP-MSH binding.
Asunto(s)
Oligopéptidos/metabolismo , Receptores de Corticotropina/metabolismo , alfa-MSH/antagonistas & inhibidores , Marcadores de Afinidad/metabolismo , Animales , Línea Celular , Cricetinae , Cricetulus , Ensayo de Unión Radioligante , Receptores de Melanocortina , alfa-MSH/análogos & derivados , alfa-MSH/metabolismoRESUMEN
The mouse adrenocortical cell line Y1, that expresses ACTH receptors (MC2R), was used to probe the binding of ACTH and MSH peptides by using radio-labelled ACTH (1-39). The Y1 cells were found to bind [125I]-labelled ACTH (1-39) with high affinity (Kd approximately 130 pM). However, none of the melanocortin peptides NDP-MSH, alpha-MSH, beta-MSH or gamma 1-MSH could compete with the binding of the labelled ACTH(1-39). When other MC receptor subtype DNAs (MC1, MC3 and MC4) were transfected into the Y1 cells, characteristic binding of the [125I]NDP-MSH appeared for each of the receptor subtype, but no specific binding was present in non-transfected cells. This is the first report clearly demonstrating that the ACTH receptor binds only ACTH, but not other melanocortin peptides.
Asunto(s)
Receptores de Corticotropina/metabolismo , Corteza Suprarrenal/metabolismo , Hormona Adrenocorticotrópica/metabolismo , Animales , Línea Celular , Humanos , Radioisótopos de Yodo , Hormonas Estimuladoras de los Melanocitos/metabolismo , Ratones , Péptidos/metabolismo , Ensayo de Unión Radioligante , Receptores de MelanocortinaRESUMEN
The expression of growth hormone receptor (GHR) mRNA in an ovine choroid plexus cell line (SCP) was studied. RNA isolated from SCP cells was subjected to reverse transcription followed by PCR using a set of primers, designed on the basis of the ovine liver GHR sequence. A specific product with expected size of 1204 bp was obtained and the nucleotide sequence was found to be identical to that of ovine liver GHR. When the PCR product was used as a probe for Northern blot analysis, a transcript of 4.4 kb was detected in mRNA isolated from the SCP cells. This is the first report demonstrating the presence of mRNA for GHR in the choroid plexus.
Asunto(s)
Plexo Coroideo/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Somatotropina/genética , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , Expresión Génica , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/aislamiento & purificación , OvinosRESUMEN
The localization of the melanocortin 1 (MC1) receptor in brain was investigated by in situ hybridization and immunohistochemistry. In rat periaqueductal gray matter (PAG), a small number of cells which displayed a punctate distribution showed a specific hybridization signal for MC1 receptor mRNA, whereas other regions studied were negative. In human PAG, the immunoreactivity for MC1 receptor was detected in scattered cells. These results indicate that a restricted distribution of MC1 receptor is present in the central nervous system.