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Regulation of melanocortin 1 receptor expression at the mRNA and protein levels by its natural agonist and antagonist¹

Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, 20892 USA

²Correspondence: Laboratory of Cell Biology, Bldg. 37, Room 1B25, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. E-mail: hearingv{at}nih.gov

SPECIFIC AIMS

Pigmentation in mammals is stimulated by -melanocyte-stimulating hormone (MSH), which binds to the melanocortin 1 receptor (Mc1r) and induces activation of melanogenic enzymes through stimulation of adenylate cyclase and protein kinase A. The antagonist agouti signal protein (ASP) interacts with the Mc1r and blocks its stimulation by MSH. We studied the structure and expression of Mc1r transcripts in MSH- or ASP-treated murine melanocytes and to determine the influence of these transcripts on the translation process.

PRINCIPAL FINDINGS

1. Effects of ASP and MSH on Mc1r protein expression
Western blot was used to measure Mc1r protein levels in melan-a black melanocytes treated with ASP or MSH. Immunodetection of Mc1r by PEP19 (a rabbit antibody generated against an Mc1r-specific peptide sequence) revealed a single band at the expected size (35 kDa, Fig. 1 a) for which the intensity varied according to the treatment. It clearly shows that Mc1r expression was enhanced by treatment with MSH and repressed by treatment with ASP. Recessive yellow (e/e) melanocytes, which express a truncated Mc1r in which the epitope recognized by PEP19 is missing, were used as a negative control; as expected, no signal was detected. These results were confirmed by immunofluorescence using a commercially available Mc1r antibody (Fig. 1b ) as melanocytes treated with MSH showed a threefold increase in fluorescence intensity compared with untreated cells. In contrast, treatment with ASP reduced the fluorescence intensity by 20% vs. untreated cells.

View larger version (40K): [in this window] [in a new window]   Figure 1. Mc1r expression after treatment of melanocytes with MSH or ASP. a) Western blot detection of Mc1r protein levels. A unique band of 35 kDa (arrow) whose intensity varies with the treatments, is detected by Western blot using PEP19 antibody and total protein (15 µg/lane) extracted from melanocytes treated for 4 days with buffer (control), MSH, or ASP. No signal was detected in e/e melanocytes in which a truncated Mc1r missing the epitope recognized by PEP19 is produced. GAPDH protein is shown in similar blots as a loading control. b) Immunohistochemical detection of Mc1r protein expression using a commercial antibody to Mc1r. Single labeling indirect immunofluorescence and laser scanning confocal microscopy were used to evaluate the localization and intensity of the staining for Mc1r. Melanocytes show a diffuse reactivity for Mc1r (green) under normal conditions (control, index of fluorescence intensity=916). The intensity of Mc1r reactivity is increased when cells are treated with MSH (IFI=2790), but melanocytes treated with ASP reduce their Mc1r expression levels (IFI=728). Original magnification, x400). c) Northern blot of Mc1r mRNA transcripts reveals one isoform of 1800 nt in untreated melanocytes (filled arrowheads), one of a similar size in ASP-treated melanocytes, and increased levels of two Mc1r transcripts (including a shorter one of 1600 nt, open arrowhead) after 4 days of treatment with MSH. d) Real-time PCR analysis of Mc1r transcripts in melanocytes treated with MSH or ASP compared with untreated cells. Data presented were obtained from 2 independent experiments with 2 points of measurements each and represent means of crossover points ± SD.

2. Influence of ASP and MSH on Mc1r transcript levels
Northern blot was used to detect Mc1r transcripts in MSH-treated, ASP-treated, and untreated control melanocytes (Fig. 1c ); semiquantitative real-time RT-PCR was also used to compare levels of Mc1r mRNA in those cells (Fig. 1d ). A dramatic increase in Mc1r transcripts was observed by both techniques when melanocytes were treated with MSH compared with untreated controls but, surprisingly, no significant change was detected in ASP-treated cells. We quantified the transcripts by real-time RT-PCR, showing that cells treated with MSH have increased levels of Mc1r transcripts of 4.4-fold (±0.6) compared with controls whereas treatment with ASP elicited no significant change (1.3-fold, ±0.2). Northern blot revealed that MSH-treated melanocytes have two distinct mRNA species, one at the size seen in control and ASP-treated melanocytes, as well as a more abundant (by twofold), smaller-sized mRNA seen only in the MSH-treated melanocytes.

3. Characterization of Mc1r transcript structures
RACE-PCR amplifications were performed using two distinct adaptor-ligated cDNA libraries of melanocytes treated with MSH or ASP or untreated as a control. The 3' RACE-PCR products revealed an identical 3' untranslated region of 507 nt. Similar investigations were performed to obtain the 5' untranslated regions (UTR), but different products were detected in the 5' RACE-PCR experiments. The untreated samples revealed a 409 nt 5'UTR whereas only a 361 bp 5'UTR was characterized in ASP-treated cells. In contrast, analysis of fragments amplified for the MSH-treated samples revealed two distinct 5'UTRs of 409 and 207 nt. The 5'UTR detected in the untreated or in the MSH-treated samples and the one found in the ASP-treated cells revealed the presence of 7 AUG codons. However, the MSH-induced T2 transcript had lost its first 202 nt, including two upstream AUGs (uAUG). The difference between the T1 and the T3 ASP-induced transcripts is that T3 is missing its first 48 nt, including a purine tract, but all seven uAUGs are present.

4. Influence of promoter regions of Mc1r on translation efficiency
To measure relative translation efficiency of the transcripts, we cloned the promoters corresponding to the different 5'UTRs of T1, T2, and T3 into the pSEAP2-basic vector (Fig. 2 ), then performed transient transfections into COS7 cells. Expression of the SEAP reporter gene was then monitored by measuring phosphatase activity using luminescence. A negative control was carried out using the pSEAP2-basic vector itself, i.e., the SEAP ORF vector with no promoter sequence (Fig. 2 , #1). A positive control was assessed using the pSEAP2 control vector, which contains the SEAP structural gene under control of the SV40 promoter (#2). The SEAP activity measured in pSEAP2-T1-transfected cells is used as the reference (#3), as T1 is the Mc1r transcript present in untreated melanocytes. Transfection with pSEAP-T3 gave a SEAP decrease of 70% (#4), indicating that the translation efficiency of T3 is weak in ASP-treated melanocytes. We then examined the translation efficiency of T2 (#5) produced in MSH-treated melanocytes and three of its variants (#6–#8), focusing on the role that might be played by upstream uAUGc and uAUGd. We removed uAUGd in T2(2), uAUGc, and uAUGd in T2(3) and introduced an additional uAUG in T2(4). The reporter protein activity measurements revealed a T2-specific increase of 18% corresponding to the influence of this messenger on the translation efficiency of Mc1r under the influence of MSH. The inhibiting effect of uAUGs on the translation process has been confirmed by the 10% and 69% increase induced by the removal of uAUGd and both uAUGc and uAUGd, respectively, compared with T2. This indicates a greater influence of uAUGc than uAUGd on the efficiency of the translation process. The artificial introduction of an additional uAUG in T2(4) induced a dramatic 64% decrease in translation.

View larger version (25K): [in this window] [in a new window]   Figure 2. Relative translation rates of various 5'UTRs of Mc1r. a) Relative translation rates after measurement of luminescence intensities in the culture medium 72 h after transfection with 1: pSEAP2-basic (negative control); 2: pSEAP2-SV40 (positive control); 3: pSEAP2-T1A; 4: pSEAP2-T3A; 5: pSEAP2-T2A; 6: pSEAP2-T2(2); 7: pSEAP2-T2(3); 8: pSEAP2-T2(4). b) Structures of vectors tested for translation efficiency after transfection into COS7 cells. The positions of the primers are reported. Gray squares indicate artificial mutations introduced in the T2A sequence. T2(2) lacks uAUGd, T2(3) lacks uAUGc and uAUGd, and T2(4) bears an additional uAUG.

CONCLUSIONS AND SIGNIFICANCE

In this study, we investigated several levels of regulation that are functional in melanocytes responding to two physiological ligands of the Mc1r (an agonist and an antagonist), as depicted in Fig. 3 . First we showed that the agonist MSH induces an increase of Mc1r expression by melanocytes whereas the antagonist ASP has the opposite effect. A semiquantitative real-time RT-PCR assay demonstrated that Mc1r transcript levels are up-regulated by MSH treatment (fourfold) but were not significantly affected by ASP. Although inducing the switch to pheomelanogenesis, ASP does not turn off the expression of the receptor. In fact, as Mc1r is the only known melanocyte receptor that binds ASP, a minimal Mc1r expression is still required for melanocytes to respond to ASP. Since the eumelanin:pheomelanin switch is reversible, melanocytes need to express a minimum number of melanocortin receptors to allow for MSH binding in order to reinduce eumelanogenesis.

View larger version (23K): [in this window] [in a new window]   Figure 3. Schematic of Mc1r expression and function regulated by MSH or ASP. Under baseline conditions, Mc1r is transcribed as a single mRNA isoform, known as T1; a constitutive number of receptors are expressed on the melanocyte surface (top). Binding of the agonist MSH to the Mc1r (left) induces the synthesis of a second type of transcript (T2), which is twice as abundant as T1. The promoter of T2 is shorter than that of T1 and shows a higher translation efficiency that, when combined with the increase in transcript number, results in a greater number of receptors expressed on the melanocyte surface. The opposite effect is observed when the antagonist ASP binds to the Mc1r (right) and induces the production of a third isoform only (T3), slightly shorter than T1. The translation efficiency (but not quantity) of T3 is severely hampered, which results in a significant decrease in Mc1r synthesis and expression.

Having detected different transcripts by Northern blot, we established their entire structure in ASP- and in MSH-treated melanocytes as well as in untreated melanocytes. Characterization of the 5'UTR revealed different promoters. Untreated melanocytes have only a single Mc1r transcript (T1), which has a 5'UTR of 409 nt identical to the corresponding genomic DNA. The main characteristic of T1 resides in its seven uAUGs (named a through g), which result in six uORFs (as the seventh is terminated after three codons). This is unusual, as <10% of known mammalian genes have uORFs in their 5'UTR. MSH-treated melanocytes revealed the presence not only of T1, but also of T2, a second type of Mc1r transcript that is shorter in that the first 202 nt have been removed, including the first two uAUGs. We determined that in response to MSH, melanocytes transcribe T2 at an 2:1 ratio compared with T1. This suggests that T2 represents a highly suitable answer to the need of increasing gene expression in response to MSH stimulation. In ASP-treated cells, we detected only one unique type of transcript, named T3, which was missing the first 48 bases compared with T1, although it contained all seven uAUGs.

The MSH-induced T2 transcript induces an 18% increase in translation compared with the constitutive T1 transcript, which leads us to conclude that the increase in Mc1r protein elicited by MSH results from the combination of two discrete processes: an increase in mRNA levels and an activation of translation. We showed that uAUGs significantly affect the translation process and suggest that the loss of uAUGa and uAUGb that occurs when the melanocyte switches from T1 to T2 in the presence of MSH might be the reason for the difference observed in translation efficiency between these two transcripts. We showed that ASP does not influence Mc1r expression at the mRNA level; rather, in the presence of ASP, the translation efficiency of the T3 Mc1r transcripts produced in ASP-treated melanocytes is severely repressed, leading to a significant reduction in receptor expression. The difference between T1 and T3 resides in the first 48 nucleotides, which are missing in T3; the 14 nucleotide purine tract located in this region will be the subject of further investigation.

The Mc1r is but one member of the melanocortin receptor family. Four others are known; all are functional in the brain and are regulated by agonists (ACTH) and antagonists (AGRP). It will be interesting to determine whether the expression of these other melanocortin receptors are under controls similar to those shown in this study and whether this might be a regulatory mechanism for genes encoding receptors in general.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0206fje; doi: 10.1096/fj.03-0206fje

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