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Expression, Candidate Gene, and Population Studies of the Melanocortin 5 Receptor

2001; Elsevier BV; Volume: 116; Issue: 4 Linguagem: Inglês

10.1046/j.0022-202x.2001.01286.x

1523-1747

Naohito Hatta, Craig Dixon, Amanda Ray, Siôn R. Phillips, W.J. Cunliffe, Mike Dale, Carol Todd, Simon Meggit, Mark A. Birch‐Machin, Jonathan L. Rees,

Regulation of Appetite and Obesity

In mouse the melanocortin 5 receptor is known to regulate sebaceous gland function. To clarify its role in man, we have studied melanocortin 5 receptor expression in skin, and allelic variation at the melanocortin 5 receptor locus in diverse human populations and candidate disease groups. Melanocortin 5 receptor protein and mRNA expression were studied by immunohistochemistry and reverse transcriptase polymerase chain reaction. Melanocortin 5 receptor mRNA was detected in normal skin and cultured keratinocytes but not in cultured fibroblasts or melanocytes. Immunohistochemistry revealed melanocortin 5 receptor immunoreactivity in the epithelium and appendages, including the sebaceous gland, eccrine glands, and apocrine glands, as well as low level expression in the interfollciular epidermis. In order to screen for genetic diversity in the melanocortin 5 receptor that might be useful for allelic association studies we sequenced the entire melanocortin 5 receptor coding region in a range of human populations. One nonsynonymous change (Phe209Leu) and four synonymous changes (Ala81Ala, Asp108Asp, Ser125Ser, and Thr248Thr) were identified. Similar results were found in each of the populations except for the Inuit in which only the Asp108Asp variant was seen. The apparent "global distribution" of melanocortin 5 receptor variants may indicate that they are old in evolutionary terms. Variation of melanocortin 5 receptor was examined in patients with acne (n = 21), hidradenitis supprativa (n = 4), and sebaceous gland lesions comprising sebaceous nevi, adenomas, and hyperplasia (n = 13). No additional mutations were found. In order to determine the functional status of the Phe209Leu change, increase in cAMP in response to stimulation with α-melanocyte-stimulating hormone was measured in HEK-293 cells transfected with either wild-type or the Phe209Leu variant. The variant melanocortin 5 receptor was shown to act in a concentration-dependent manner, which did not differ from that of wild type. We have therefore found no evidence of a causative role for melanocortin 5 receptor in sebaceous gland dysfunction, and in the absence of any association between variation at the locus and disease group, the pathophysiologic role of the melanocortin 5 receptor in man requires further study. In mouse the melanocortin 5 receptor is known to regulate sebaceous gland function. To clarify its role in man, we have studied melanocortin 5 receptor expression in skin, and allelic variation at the melanocortin 5 receptor locus in diverse human populations and candidate disease groups. Melanocortin 5 receptor protein and mRNA expression were studied by immunohistochemistry and reverse transcriptase polymerase chain reaction. Melanocortin 5 receptor mRNA was detected in normal skin and cultured keratinocytes but not in cultured fibroblasts or melanocytes. Immunohistochemistry revealed melanocortin 5 receptor immunoreactivity in the epithelium and appendages, including the sebaceous gland, eccrine glands, and apocrine glands, as well as low level expression in the interfollciular epidermis. In order to screen for genetic diversity in the melanocortin 5 receptor that might be useful for allelic association studies we sequenced the entire melanocortin 5 receptor coding region in a range of human populations. One nonsynonymous change (Phe209Leu) and four synonymous changes (Ala81Ala, Asp108Asp, Ser125Ser, and Thr248Thr) were identified. Similar results were found in each of the populations except for the Inuit in which only the Asp108Asp variant was seen. The apparent "global distribution" of melanocortin 5 receptor variants may indicate that they are old in evolutionary terms. Variation of melanocortin 5 receptor was examined in patients with acne (n = 21), hidradenitis supprativa (n = 4), and sebaceous gland lesions comprising sebaceous nevi, adenomas, and hyperplasia (n = 13). No additional mutations were found. In order to determine the functional status of the Phe209Leu change, increase in cAMP in response to stimulation with α-melanocyte-stimulating hormone was measured in HEK-293 cells transfected with either wild-type or the Phe209Leu variant. The variant melanocortin 5 receptor was shown to act in a concentration-dependent manner, which did not differ from that of wild type. We have therefore found no evidence of a causative role for melanocortin 5 receptor in sebaceous gland dysfunction, and in the absence of any association between variation at the locus and disease group, the pathophysiologic role of the melanocortin 5 receptor in man requires further study. melanocortin receptor melanocyte-stimulating hormone pro-opiomelanocortin The pro-opiomelanocortin (POMC) derived peptides, including adrenocorticotropic hormone (ACTH) and α-melanocyte-stimulating hormone (α-MSH) have been implicated in a range of skin functions, including sebogenesis and pigmentation (Thody and Shuster, 1989Thody A.J. Shuster S. Control and function of sebaceous glands (Review – 315 refs).Physiol Rev. 1989; 69: 383-416Crossref PubMed Scopus (291) Google Scholar;Rees et al., 1999Rees J.L. Birch-Machin M. Flanagan N. Healy E. Phillips S. Todd C. Genetic studies of the human melanocortin 1 receptor (MC1R).Ann NY Acad Sci. 1999; 885: 134-142Crossref PubMed Scopus (34) Google Scholar;Rees, 2000Rees J.L. The melancortin 1 receptor (MC1R); more than just red hair.Pigment Cell Res. 2000; 13: 135-140Crossref PubMed Scopus (150) Google Scholar). Pituitary-derived α-MSH is a major sebotrophic hormone in rodents (Thody and Shuster, 1989Thody A.J. Shuster S. Control and function of sebaceous glands (Review – 315 refs).Physiol Rev. 1989; 69: 383-416Crossref PubMed Scopus (291) Google Scholar). Removal of the source of α-MSH, the pars intermedia in rats, leads to reduced sebum secretion, a change reversed by subsequent administration of α-MSH. α-MSH and testosterone produce a synergistic increase in sebum production with α-MSH specifically involved in the stimulation of wax and sterol ester biosynthesis (Thody and Shuster, 1989Thody A.J. Shuster S. Control and function of sebaceous glands (Review – 315 refs).Physiol Rev. 1989; 69: 383-416Crossref PubMed Scopus (291) Google Scholar). Specific binding of [Nle4D-Phe7]-α-MSH (Tatro and Reichlin, 1987Tatro J.B. Reichlin S. Specific receptors for alpha-melanocyte-stimulating hormone are widely distributed in tissues of rodents.Endocrinology. 1987; 121: 1900-1907Crossref PubMed Scopus (138) Google Scholar) and the trophic action of melanocortins have also been demonstrated in other physiologically cognate execrine glands including the lacrimal, preputial, and prostate glands of rodents (Ebling et al., 1975Ebling F.J. Ebling E. Randall V. Skinner J. The synergistic action of alpha-melanocyte-stimulating hormone and testosterone of the sebaceous, prostate, preputial, Harderian and lachrymal glands, seminal vesicles and brown adipose tissue in the hypophysectomized-castrated rat.J Endocrinol. 1975; 66: 407-412Crossref PubMed Scopus (36) Google Scholar). Until the recent cloning of a family of melanocortin receptors, however, the cellular signaling mechanisms of α-MSH on exocrine function were unknown. Five melanocortin receptor (MC-R) subtypes have been identified: MC1-R (MSH-R) (Chhajlani and Wikberg, 1992Chhajlani V. Wikberg J.E. Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA.FEBS Lett. 1992; 309: 417-420Abstract Full Text PDF PubMed Scopus (563) Google Scholar;Mountjoy et al., 1992Mountjoy K.G. Robbins L.S. Mortrud M.T. Cone R.D. The cloning of a family of genes that encode the melanocortin receptors.Science. 1992; 257: 1248-1251Crossref PubMed Scopus (1417) Google Scholar), MC2-R (ACTH-R) (Mountjoy et al., 1992Mountjoy K.G. Robbins L.S. Mortrud M.T. Cone R.D. The cloning of a family of genes that encode the melanocortin receptors.Science. 1992; 257: 1248-1251Crossref PubMed Scopus (1417) Google Scholar), MC3-R, MC4-R (Gantz et al., 1993Gantz I. Konda Y. Tashiro T. et al.Molecular cloning of a novel melanocortin receptor.J Biol Chem. 1993; 268: 8246-8250Abstract Full Text PDF PubMed Google Scholar), and MC5-R (Gantz et al., 1994Gantz I. Shimoto Y. Konda Y. Miwa H. Dickinson C.J. Yamada T. Molecular cloning, expression, and characterization of a fifth melanocortin receptor.Biochem Biophys Res Commun. 1994; 200: 1214-1220https://doi.org/10.1006/bbrc.1994.1580Crossref PubMed Scopus (279) Google Scholar) (reviewed inCone et al., 1996Cone R.D. Lu D. Koppula S. et al.The melanocortin receptors: agonists, antagonists, and the hormonal control of pigmentation.Rec Prog Horm Res. 1996; 51 (Review): 287-317PubMed Google Scholar). The tissue expression patterns of the various MC-R is, at least in part, in keeping with their known physiologic roles (Cone et al., 1996Cone R.D. Lu D. Koppula S. et al.The melanocortin receptors: agonists, antagonists, and the hormonal control of pigmentation.Rec Prog Horm Res. 1996; 51 (Review): 287-317PubMed Google Scholar). MC1-R is a major pigmentary control point and is expressed on melanocytes and some other cell types (Healy et al., 1999Healy E. Birch-Machin M.A. Rees J.L. Cone R.D. The Human Mc1r. Humana Press, New Jersey1999: 341-360Google Scholar); MC2-R, the physiologic receptor for ACTH, is expressed primarily in the adrenal cortex (Mountjoy et al., 1992Mountjoy K.G. Robbins L.S. Mortrud M.T. Cone R.D. The cloning of a family of genes that encode the melanocortin receptors.Science. 1992; 257: 1248-1251Crossref PubMed Scopus (1417) Google Scholar); whereas MC3-R and MC4-R are expressed mainly in the central nerve system, with MC4-R playing an important role in the regulation of feeding behavior and body weight (Jordan and Jackson, 1998Jordan S.A. Jackson I.J. Melanocortin receptors and antagonists regulate pigmentation and body weight.Bioessays. 1998; 20: 603-606https://doi.org/10.1002/(sici)1521-1878(199808)20:8 3.3.co;2-9Crossref PubMed Scopus (0) Google Scholar;Versteeg et al., 1998Versteeg D.H. Van Bergen P. Adan R.A. De Wildt D.J. Melanocortins and cardiovascular regulation.Eur J Pharmacol. 1998; 360: 1-14https://doi.org/10.1016/s0014-2999(98)00615-3Crossref PubMed Scopus (0) Google Scholar;Yaswen et al., 1999Yaswen L. Diehl N. Brennan M.B. Hochgeschwender U. Obesity in the mouse model of pro-opiomelanocortin deficiency responds to peripheral melanocortin.Nat Med. 1999; 5: 1066-1070https://doi.org/10.1038/12506Crossref PubMed Scopus (791) Google Scholar). The role of MC5-R was until recently unclear. This receptor is expressed at a low level in a range of peripheral tissues (Van der Kraan et al., 1998Van der Kraan M. Adan R.A.H. Entwistle M.L. Gispen W.H. Burbach J.P.H. Tatro J.B. Expression of melanocortin-5 receptor in secretory epithelia supports a functional role in exocrine and endocrine glands.Endocrinology. 1998; 139: 2348-2355Crossref PubMed Scopus (94) Google Scholar) including a variety of exocrine glands. The role of MC5-R in sebaceous gland function in mice was recently shown byChen et al., 1997Chen W.B. Kelly M.A. Opitz-Araya X. Thomas R.E. Low M.J. Cone R.D. Exocrine gland dysfunction in MC5-R-deficient mice: evidence for coordinated regulation of exocrine gland function by melanocortin peptides.Cell. 1997; 91: 789-798Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, who, following targeted disruption of the murine MC5-R gene, noted defective water repulsion and coat drying as a result of a reduction in hair lipid content. Binding of [Nle4D-Phe7]-α-MSH was absent in the sebaceous glands of these MC5-R-/– mice, indicating lack of expression of other α-MSH binding receptor subtypes (MC1-R, MC3-R, and MC4-R) in this tissue. A study of the POMC knockout mouse, the precursor molecule for the putative physiologic ligand for the MC5-R, also showed defects in lipogenesis (Yaswen et al., 1999Yaswen L. Diehl N. Brennan M.B. Hochgeschwender U. Obesity in the mouse model of pro-opiomelanocortin deficiency responds to peripheral melanocortin.Nat Med. 1999; 5: 1066-1070https://doi.org/10.1038/12506Crossref PubMed Scopus (791) Google Scholar). Whereas human mutations in the other melanocortin receptors have been informative in respect of the physiologic function in man, the role of the MC5-R in man is unclear. Mutations in the POMC gene, which might have been informative for the signaling pathway, have been identified in two infants displaying red hair, severe early onset obesity, and adrenal insufficiency (Krude et al., 1998Krude H. Biebermann H. Luck W. Horn R. Brabant G. Grüters A. Severe early-onset obesity, adrenal insufficiency and red hair pigmentation caused by POMC mutations in humans.Nature Genet. 1998; 19: 155-157Crossref PubMed Scopus (1310) Google Scholar). As these individuals are still prepubertal, however, it is difficult to comment on any sebaceous-gland abnormality and no clear MC5-R related phenotype could be observed, e.g., the absence of sebum production, or the complete absence of acne. In order to define the relevance of MC5-R we have studied expression patterns of the MC5-R in human skin, using a combination of immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR), and searched for mutations in a variety of clinical conditions in which we hypothesized that the MC5-R may play a role, including acne and sebaceous hyperplasia and neoplasia. Ten normal human skin specimens (scalp, face, axilla, and leg) were obtained from 10 Caucasian patients undergoing surgery for the excision of skin tumors. Affected skin specimens were also obtained from 10 acne patients with either a comedo or inflamed lesion and from psoriatic plaque. The skin specimens were frozen immediately and used for RNA extraction and immunohistochemistry. Immunocytochemistry was performed on COS-7 cells transfected with the human MC5-R cDNA using a goat polyclonal antibody specific to the carboxyl terminus of MC5-R (C-18, Santa Cruz Biotechnology, Santa Cruz, CA). Cultured cells were grown on glass cover slips, which were then mounted on glass slides. After fixation with cold acetone, and blocking with H2O2 and normal donkey serum, slides were incubated with the primary antibody (1:400) diluted in donkey serum. A peroxidase-conjugated streptavidin-biotin system (Santa Cruz) was used to detect the binding of the primary antibody and the reaction products were visualized using aminoethil-carbasol (Vector Laboratories, Burlingame, CA) as chromogen with hematoxylin counterstaining as required. Normal goat IgG was used as a negative control. Primary cultures of normal human keratinocytes, fibroblasts, and melanocytes were established from the normal human skin samples described above. Fibroblasts and human immortalized keratinocytes (HaCaT) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Paisley, U.K.) supplemented with 10% fetal bovine serum; keratinocytes were cultured in KMCDB 153 medium (Sigma, Dorset, U.K.) and melanocytes in Melanocyte Medium (Sigma). All of the media contained penicillin (50 IU per ml) and streptomycin (50 μg per ml). Cells from the second or third passage were used for experimentation. Total RNA was extracted from frozen skin samples and cultured cells using a silica-gel-based membrane system (RNeasy, Qiagen, West Sussex, U.K.) according to the manufacturer's instructions. As the MC5-R gene contains only one exon with no intervening introns, RNA samples were treated with DNase I for 90 min (Boehringer Mannheim, Mannheim, Germany) to avoid false positives from contaminating DNA. DNase I treatment of 800 ng of genomic DNA was also carried out on each occasion as a control for successful digestion of contaminating DNA. Primers were designed to allow the amplification of a portion of MC5-R RNA (227 bp, base no. 569–795), based on information obtained from the published sequence of the MC5-R gene (Chhajlani et al., 1993Chhajlani V. Muceniece R. Wikberg J.E. Molecular cloning of a novel human melanocortin receptor.Biochem Biophys Res Comm. 1993; 195 ([published erratum appears in Biochem Biophys Res Commun, 218:638, 1996 January 17]): 866-873Crossref PubMed Scopus (310) Google Scholar) (GenBank accession number, z25470). The primer sequences were as follows: forward 5′-AGACATGGGCATTGCTGTGG-3′ and reverse 5′-AGGCGTCTGCTATCACTAGG-3′. Reverse transcription of each of the RNA samples was carried out using oligo-dT primers (Boehringer Mannheim) and Moloney Murine Leukemia Virus reverse transcriptase (Promega, Madison, WI). The quality of the cDNA synthesis was monitored by amplifying the β-actin. Positive (genomic DNA) and negative (DNase I treated genomic DNA and water) controls were included in each set of PCR reactions. Twenty-five microliter PCR reactions contained 1 µl of reverse transcriptase reaction product, 1 × reaction buffer, 0.5 unit of AmpliTaq DNA polymerase (PE Applied Biosystems, Foster City, CA), 0.2 mM dNTPs, 2 mM MgCl2, and 0.2 µM of each primer. The PCR cycle profile was 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min for 34 cycles followed by one cycle of 95°C for 1 min, 60°C for 1 min, and 72°C for 10 min. The PCR products were separated by electrophoresis through 1% agarose gels. The populations studied in this paper are listed in Table I. A description of these samples has been given previously (Harding et al., 2000Harding R.M. Healy E. Ray A.J. et al.Evidence for variable selective pressures at the human pigmentation locus, MC1R.Am J Hum Genet. 2000; 66: 1351-1361Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Genomic DNA was obtained from 21 patients with severe acne and four patients with hidradenitis suppurativa including two individuals with an extensive family history. DNA was also extracted from paraffin-embedded biopsy material taken from patients with sebaceous gland abnormalities (n = 13) comprising sebaceous naevi (n = 6), sebaceous adenomas (n = 4), and sebaceous hyperplasia (n = 3). Paraffin-embedded biopsy material was cut into 5–10 μm slices for microdissection to separate tumor/sebaceous gland and normal tissue. DNA was then extracted from the resulting tissue samples using a Nucleon Hard Tissue Kit (Nucleon Biosciences, Glasgow, U.K.) according to the manufacturer's instructions.Table IFrequencies of MC-5R variant alleles identified in population studyPopulationNo. of subjectsNo. of total alleles% of Consensu allelesNumber of mutated allelesAla81AlaAsp108AspSer125SerPhe209LeuThr248ThrNegro15309011033South Indian18368360066Inuit8166905000Japanese14287523077Polynesian247510011Caucasian44889213155Total10120286111212222 Open table in a new tab The entire MC5-R coding sequence was amplified by PCR using M13 tagged primer (forward, 5′-TGTAAAACGACGGCCAGTGTAGGCTGCTAACCTCTTTGG-3′; reverse, 5′-CAGGAAACAGCTATGACCCAAAGGAGAACAGAGCCACAG-3′). Five hundred nanograms of genomic DNA was amplified using 0.625 µM of each primer, 1 × reaction buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, and 1 unit of Taq DNA polymerase (Bioline, London, U.K.) in a total reaction volume of 50 ml. DNA was amplified using an Applied Biosystems GeneAmp 9700 thermal cycler for two cycles of 1 min at 95°C, 1 min at 59°C, and 1 min 40 s at 72°C followed by 32 cycles of 1 min at 95°C, 1 min at 64°C, and 1 min 40 s at 72°C. Amplification yielded a 1098 bp PCR product including 5′ and 3′ flanking sequences. The DNA extracted from paraffin-embedded tissues was amplified in three sections. For the first portion (start codon to base number 320) the existing forward MC5-R primer and reverse primer 1 (5′-CAGGAAAC AGCTATGACCGCGCACAAAGGCGTCTGCTA-3′) were used, for the second portion (base number 300–630) forward primer 2 (5′-TGTAAA ACGACGGCCAGTCTACTCAACAACAAGCACCTAG-3′) and reverse primer 2 (5′-CAGGAAACAGCTATGACCGCGATCCGCTTGACGTGAGT-3′) were used, and for the third portion (base number 540–978) forward primer 3 (5′-TGTAAAACGACGGCCAGTAGAATCCACCTACGTCATCC-3′) and the existing reverse MC5-R primer were used. All primers were M13 tagged. PCR products were separated by electrophoresis through 1% agarose gels and purified using a Qiagen Gel Extraction Kit (Qiagen). Sequencing was performed using an Applied Biosystems ABI PRISM 373 DNA sequencer. The chimpanzee MC5-R gene was amplified using the same primers and thermal cycling protocol. Both wild-type and a nonsynonymous variant (F209L) of the MC5-R gene and wild-type MC1-R gene were amplified from genomic DNA in 25 ml of reaction mixture [1 µM of each primer, 0.2 mM dNTPs, 1 × Pfu buffer (containing 2 mM MgCl2), 1.25 unit of Pfu DNA polymerase (Stratagene, La Jolla, CA), and 500 ng genomic DNA template]. One of the primers was phosphorylated before the PCR to allow directional cloning. Following amplification, 1 unit of Taq DNA polymerase was added and the mixture was incubated at 72°C for 10 min to add an (A)n overhang to the PCR product. PCR products were separated by electrophoresis through a 1% agarose gel, excised, and then freeze-thawed to obtain a solution containing the desired DNA product. These products were then cloned into the mammalian expression vector pCR 3.1 using a Eukaryotic TA Cloning Kit (Invitrogen, Leek, Netherlands) following the manufacturer's instructions. Selection was with 50 mg per ml of ampicillin (Sigma). MC5-R and MC1-R from the final constructs were re-sequenced following cloning. For receptor expression studies, HEK-293 cells were grown in DMEM with 10% fetal bovine serum, penicillin (50 IU per ml), and streptomycin (50 µg per ml). Constructs containing either the wild-type or the nonsynonymous variant of MC5-R were transfected into 80% confluent HEK-293 cells in serum-free medium (Optimem, Gibco) for 5 h using pfx6 lipid (PerFect transfection reagent, Invitrogen). After transfection, the serum-free medium was replaced by serum-containing medium and the cells were cultured for approximately 48 h. 0.5 mg per ml geneticin (Gibco) was then added to the medium and the cells were cultured for a further 2 wk to select a stable transfected cell line. The same procedure was performed for the MC1-R construct using COS-7 cells. The cAMP assays were performed as described previously (Schioth et al., 1999Schioth H.B. Phillips S. Rudzish R. Birch-Machin M. Wikberg J. Rees J.L. Loss of function mutations of the human melanocortin 1 receptor are common and associated with red hair.Biochem Biophys Res Comms. 1999; 260: 488-491https://doi.org/10.1006/bbrc.1999.0935Crossref PubMed Scopus (204) Google Scholar) with some modifications. Transfected cells were incubated for 30 min with 150 µl serum-free DMEM in 24-well microtiter plates containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX). Appropriate concentrations of α-MSH were added to the cells in 0.05 ml serum-free DMEM containing IBMX and the cells were incubated for a further 25 min. Cell lysates were neutralized with 45 µl of 5 M KOH/1 M Tris. cAMP content was then estimated by adding to each sample 0.14 pmol [3H]-cAMP (approximately 11,000 cpm, specific activity 54 Ci per mmol; Amersham Life Science, Bucks, U.K.) and bovine adrenal binding protein and incubating at 4°C for 120 min. Standards containing nonlabeled cAMP were also assayed concomitantly with the samples. The samples were harvested by filtration on glass fiber filters (Filtermat B, Wallac Oy, Turku, Finland) using a semiautomatic cell harvester. Filters were rinsed with 50 mM Tris/HCl pH 7.4 and then punched out, put into scintillation vials containing scintillation fluid, and counted. The cAMP assays were performed in duplicate wells and each experiment was repeated four times. To confirm the specificity of the C-18 antibody, immunocytochemistry was carried out on COS-7 cells that were transfected with the wild-type MC5-R gene. Untransfected COS-7 cells and COS-7 cells transfected with wild-type MC1-R were also included as negative controls. Cells transfected with MC5-R showed positive staining (Figure 1a) in contrast to the lack of staining observed in both the untransfected cells (Figure 1b) and those transfected with MC1-R (Figure 1c). In normal skin, MC5-R was strongly expressed in arrector pili muscle and the epithelia of apocrine and eccrine glands (Figure 2b, c, d). Epidermal keratinocytes, infiltrating cells of the dermis, and sebaceous glands showed low level expression (Figure 2a). Expression of MC5-R was increased near the surface of the epidermis (Figure 2a). Cultured keratinocytes, melanocytes, and fibroblasts did not demonstrate protein expression of MC5-R (data not shown). Involved psoriatic and acne lesions were also examined and showed no qualitative difference from normal sample MC5-R (data not shown). To confirm MC5-R expression patterns in human skin, RT-PCR was performed using MC5-R specific primers, which amplified a product of 227 bp (base number 569–795). MC5-R RNA was detected in normal human skin (Figure 3, lane 1). When RNA was extracted separately from the upper part of skin (epidermis and upper dermis) or the lower part (lower dermis and subcutaneous tissue) by microdissection techniques, only the lower portion showed MC5-R mRNA expression (Figure 3, lane 3). These results are in keeping with the immunohistochemistry results, showing that there is greater expression in appendages than interfollicular epidermis. RNA extracted from primary cultured normal human keratinocytes, melanocytes, and fibroblasts revealed a low expression of MC5-R mRNA in keratinocytes only (Figure 3, lane 4). The identity of the PCR products was confirmed by direct DNA sequencing and southern blot analysis using an MC5-R sequence-specific probe (data not shown). In studying complex disease states or physiologic variation, such as differences in sebum excretion, multiple alleles for a gene may be present in the human population. Because these states are complex, there may not be a clear one-to-one correlation between the presence of a particular allele and a disease state. In order to gain further insight into structure-function relationships of the MC5-R, we cloned the chimpanzee melanocortin receptor, sequenced MC5-R alleles from a variety of world populations, and carried out functional analysis on one particular nonsynonymous variant detected in human populations. Chimpanzee MC5-R was amplified from genomic DNA using the same primers and PCR profile as for amplification of human MC5-R. The chimpanzee MC5-R sequence differed from the consensus human sequence by two nonsynonymous substitutions (Gly224Ser and Arg272Cys) and six synonymous substitutions (Leu133Leu, Cys173Cys, Pro253Pro, Tyr280Tyr, Ser309Ser, and Ile317Ile) (Figure 4). None of these changes were observed in any of the human sequences, making the consensus sequence also the root sequence. The full sequence for chimpanzee MC5-R has been deposited with the GenBank database (accession number AF208691). In African, Asian, and Caucasian populations one nonsynonymous variant was observed at codon 209 with a phenylalanine (Phe) being replaced by a leucine (Leu). This variant is predicted to lie within the fifth transmembrane (TM) domain of human MC5-R (Chhajlani et al., 1993Chhajlani V. Muceniece R. Wikberg J.E. Molecular cloning of a novel human melanocortin receptor.Biochem Biophys Res Comm. 1993; 195 ([published erratum appears in Biochem Biophys Res Commun, 218:638, 1996 January 17]): 866-873Crossref PubMed Scopus (310) Google Scholar). In addition, four synonymous variants were observed. These were confirmed as Ala81Ala, Asp108Asp, Ser125Ser, and Thr248Thr. These results are summarized in Table I. The Phe209Leu and Thr248Thr variants were not seen independently of each other, suggesting that they may lie on the same chromosome; subsequent cloning of the alleles confirmed this. Similarly, the Ala81Ala variant was always accompanied by the Phe209Leu/Thr248Thr variant. The pattern of MC5-R polymorphism in African, Asian, and Caucasian populations appears to be similar (Figure 5). A relatively low frequency of wild-type MC5-R, however, was observed in the Japanese, Inuit, and Polynesian populations (75%, 69%, and 75%, respectively, vs 92% in Caucasian). These differences may reflect sampling, however, or sample size. In the Inuit population, the Asp108Asp variant was seen in 50% of individuals. The entire coding region of the MC5-R was amplified and sequenced in individuals with severe acne and hidradenitis suppurativa and a range of sebaceous gland abnormalities (Table II). The MC5-R allelic variation seen in these individuals was similar to that in the controlled population, and there was no difference between the germline sequence and the sebaceous gland samples examined.Table IIFrequencies of MC-5R variant alleles in dermatologic disease involving exocrine gland dysfunctionDiagnosisNo. of subjectsNo. of total alleles% of Consensu allelesNumber of mutated allelesAla81AlaAsp108AspSer125SerPhe209LeuThr248ThrSevere grade acne21427722044Hidradenitis suppurativa4810000000Sebaceous neoplasiaaSebaceous naevi (n = 6), adenomas (n = 4), and hyperplasia (n = 3).13268520044a Sebaceous naevi (n = 6), adenomas (n = 4), and hyperplasia (n = 3). Open table in a new tab Because there is not a one-to-one relationship between sequence variation and phenotype for a complex state, the above data do not exclude a functionally significant role for the Phe209Leu variant. HEK-293 cells transfected with both wild-type MC5-R and the Phe209Leu variant responded similarly in a dose-dependent manner in response to our α-MSH with an approximately 6-fold maximum increase in intracellular cAMP (Figure 6). MC5-R is the only MC-R subtype that is widely expressed in peripheral tissue (Gantz et al., 1994Gantz I. Shimoto Y. Konda Y. Miwa H. Dickinson C.J. Yamada T. Molecular cloning, expression, and characterization of a fifth melanocortin receptor.Biochem Biophys Res Commun. 1994; 200: 1214-1220https://doi.org/10.1006/bbrc.1994.1580Crossref PubMed Scopus (279) Google Scholar;Griffon et al., 1994Griffon N. Mignon V. Facchinetti P. Diaz J. Schwartz J.C. Sokoloff P. Molecular cloning and characterization of the rat fifth melanocortin receptor.Biochem Biophys Res Commun. 1994; 200: 1007-1014https://doi.org/10.1006/bbrc.1994.1550Crossref PubMed Scopus (184) Google Scholar;Labbe et al., 1994Labbe O. Desarnaud F. Eggerickx D. Vassart G. Parmentier M. Molecular cloning of a mouse melanocortin 5 receptor gene widely expressed in peripheral tissues.Biochemistry. 1994; 33: 4543-4549Crossref PubMed Scopus (184) Google Scholar). Recently, MC5-R mRNA expression was detected in both the sebaceous glands of mouse skin (Chen et al., 1997Chen W.B. Kelly M.A. Opitz-Araya X. Thomas R.E. Low M.J. Cone R.D. Exocrine gland dysfunction in MC5-R-deficient mice: evidence for coordinated regulation of exocrine gland function by melanocortin peptides.Cell. 1997; 91: 789-798Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar) and the secretory epithelia of exocrine glands in rat skin (Van der Kraan et al., 1998Van der Kraan M. Adan R.A.H. Entwistle M.L. Gispen W.H. Burbach J.P.H. Tatro J.B. Expression of melanocortin-5 receptor in secretory epithelia supports a functional role in exocrine and endocrine glands.Endocrinology. 1998; 139: 2348-2355Crossref PubMed Scopus (94) Google Scholar). The only noticeable phenotype of MC5-R knockout mice was attributed to reduction in coat sebum production. These results suggest a role for the MC5-R in the physiologic control of sebogenesis. The expression of the role of MC5-R in humans, however, had not previously been studied. In this study we show that MC5-R mRNA protein is expressed in human skin. Immunohistochemistry shows strong expression of MC5-R protein in apocrine and eccrine glands and lower level expression in the interfollicular epidermis and sebaceous glands. Microdissection of epidermis suggests that the majority of MC5-R mRNA is expressed in the appendages rather than in the interfollicular epidermis. MC5-R was also expressed at the level of RT-PCR in cultured keratinocytes although immunostaining was not above background, probably due to the low level expression. In contrast to the cultured keratinocytes, immunostaining revealed faint epidermal staining for MC5-R protein in the upper layer. The discrepancy may be explained by the environmental difference between in vivo and in vitro culture (e.g., POMC peptide derived from epidermal keratinocytes). Our attempts to perform in situ hybridization failed, probably due to the relatively low level of MC5-R message. Because the relation between variation at this locus and phenotype may be complex, we cloned the chimpanzee sequence and studied various human populations in order to define allelic variants in man. These studies showed that the chimpanzee differed from the human consensus by two nonsynonymous and six synonymous changes; only one common nonsynonymous change was found in human populations - the Phe209Leu change. Sequencing of a variety of somatic sebaceous gland lesions failed to reveal any specific association with the Phe209Leu change, nor did studies of individuals with acne or hidradenitis suppurativa. Because the power of these studies to exclude a functional role for this change was limited we performed functional studies using transient transfections in HEK cells. The Phe209Leu variant of MC5 did not differ from the results obtained with the wild-type MC5-R. This result is in keeping with mutrogenesis studies of the melanocortin receptors, which have indicated that the main binding regions are likely to include the TM1, TM2, TM3, TM6, and TM7 domain (Schioth et al., 1996Schioth H.B. Muceniece R. Wikberg J.E. Characterisation of the melanocortin 4 receptor by radioligand binding.Pharmacol Toxicol. 1996; 79: 161-165Crossref PubMed Scopus (123) Google Scholar,Schioth et al., 1997aSchioth H.B. Petersson S. Muceniece R. Szardenings M. Wikberg J.E.S. Deletions of the N-terminal regions of the human melanocortin receptors.FEBS Lett. 1997; 410: 223-228https://doi.org/10.1016/s0014-5793(97)00593-0Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,Schioth et al., 1997bSchioth H.B. Muceniece R. Szard"enings M. et al.Characterisation of D117A and H260A mutations in the melanocortin 1 receptor.Mol Cell Endocrinol. 1997; 126: 213-219https://doi.org/10.1016/s0303-7207(96)03993-7Crossref PubMed Google Scholar,Schioth et al., 1998aSchioth H.B. Fredriksson A. Carlsson C. Yook P. Muceniece R. Wikberg J.E.S. Evidence indicating that the extracellular loops of the mouse MC5 receptor do not participate in ligand binding.Mol Cell Endocrinol. 1998; 139: 109-115https://doi.org/10.1016/s0303-7207(98)00067-7Crossref PubMed Google Scholar,Schioth et al., 1998bSchioth H.B. Yook P. Muceniece R. Wikberg J.E.S. Szardenings M. Chimeric melanocortin MC1 and MC3 receptors: identification of domains participating in binding of melanocyte-stimulating hormone peptides.Mol Pharmacol. 1998; 54: 154-161PubMed Google Scholar), and the Phe209Leu variant is located in the TM5 domain. By contrast one of the nonsynonymous human-chimp changes (Arg272Cys) has been reported to affect the structure of MC5-R using an increased receptor affinity for α-MSH (Frandberg et al., 1997Frandberg P.A. Xu X. Chhajlani V. Glutamine235 and arginine272 in human melanocortin 5 receptor determines its low affinity to MSH.Biochem Biophys Res Comms. 1997; 236: 489-492https://doi.org/10.1006/bbrc.1997.6994Crossref PubMed Scopus (16) Google Scholar). This suggests that the MC5-R function may have altered during the human-chimpanzee divergence. This cysteine residue at codon 272 is also conserved in the murine MC5-R, which has a higher affinity for α-MSH than the human MC5-R. Although these studies have failed to define the exact physiologic role for this receptor-signaling pathway in man, the clear role in mouse suggests that further studies of other candidate diseases are required perhaps with larger allelic association studies. This work was principally funded by the British Skin Foundation and the William Leech Trust. We thank Dr. Niamh Leonard (Royal Victoria Infirmary, Newcastle) for access to pathology specimens.