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Originally published In Press as doi:10.1074/jbc.M201343200 on March 23, 2002

J. Biol. Chem., Vol. 277, Issue 23, 20328-20335, June 7, 2002
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Molecular Determinants of Human Melanocortin-4 Receptor Responsible for Antagonist SHU9119 Selective Activity*

Yingkui YangDagger , Min ChenDagger , Yumei Lai§, Ira Gantz§, Keith E. GeorgesonDagger , and Carroll M. HarmonDagger

From the Dagger  Department of Surgery, University of Alabama at Birmingham, Alabama 35233 and the § Department of Surgery, University of Michigan, Ann Arbor, Michigan 48109

Received for publication, February 8, 2002, and in revised form, March 13, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The hypothalamic melanocortin-4 receptor (MC4R), a seven transmembrane G-protein-coupled receptor, plays an important role in the regulation of body weight. The synthetic melanocortin analog SHU9119 has been widely used to characterize the physiological role of MC4R in feeding behavior and energy homeostasis. Previous studies indicated that SHU9119 is an agonist at the melanocortin-1 receptor (MC1R) but an antagonist at the MC4R. However, the molecular basis of the interaction between hMC4R and SHU9119 has not been clearly defined. To gain insight into the molecular determinants of hMC4R in the selectivity of SHU9119 chimeras and mutants hMC1R and hMC4R were expressed in cell lines and pharmacologically analyzed. A region of receptor containing the third transmembrane of hMC4R was found to be required for selective SHU9119 antagonism. Further mutagenesis studies of this region of hMC4R demonstrated that the amino acid residue leucine 133 in the third transmembrane was critical for the selective antagonist activity of SHU9119. The single substitution of leucine 133 to methionine did not affect SHU9119 binding to hMC4R. However, this substitution did convert SHU9119 from an antagonist to an agonist. Conversely, exchange of Met128 in hMC1R to Leu, the homologous residue 133 of hMC4R, displayed a reduction in SHU9119 binding affinity and potency. This report provides the details of the molecular recognition of SHU9119 antagonism at hMC4R and shows that amino acid Leu133 of hMC4R plays a key role in melanocortin receptor subtype specificity.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The melanocortin-4 receptor (MC4R)1 is a seven transmembrane G-protein-coupled receptor, principally expressed in the hypothalamic nucleus. This receptor plays an important role in the regulation of body weight in rodents and humans (1-3). When stimulated by its putative physiological agonist, alpha -melanocyte-stimulating hormone (alpha -MSH) inhibits feeding in mice through MC4R. Furthermore, mice with MC4R deletion develop hyperphagia, hyperinsulinism, and obesity (4). Recently, mutations in the MC4R have been reported as the most common causes of monogenic human obesity (5-7). MC4R mutations may play a particularly important role in the early onset of childhood obesity, resulting in more severe obesity-related complications, such as hypertension and diabetes, when compared with late onset obesity found in adults (8, 9). In light of these findings, the molecular basis of the role that MC4R plays in obesity has been the subject of intense investigation.

Many new, potent, and enzyme-resistant analogs of melanocortin peptides have been developed based on the extensive studies of the melanocortin peptide, alpha -MSH (10-12). These include NDP-MSH ([Nle4-D-Phe7]alpha -MSH) and melanotan II (MTII, the lactam Ac-Nle4 cycle[Asp-His6-D-Phe7-Arg8-Trp9-Lys10]amide), which have been identified as potent, non-selective agonists at human melanocortin-1, -3, -4, and -5 receptors. In addition, the analog, SHU9119, a synthetic peptide with a beta -(2-naphthyl)-D-alanine (D-Nal) substituted in position 7 of MTII, has been found to be a potent but non-selective antagonist for the MC3 and MC4 receptors (13, 14). Intracerebroventricular administration of MTII has been found to induce weight loss, whereas SHU9119 has been found to increase animal food intake and body weight (15, 16).

Because of potentially important therapeutic implications in the treatment of obesity and perhaps anorexia syndromes, determination of the molecular basis for ligand-receptor interaction between hMC4R and melanocortin peptides is valuable (17, 18). Structure-function studies of NDP-MSH have demonstrated that D-Phe-Arg-Trp of NDP-MSH is the minimal peptide required for hMC4R binding and activation. Furthermore, the conserved residues in the third transmembrane (TM3) and the sixth transmembrane (TM6) domains of melanocortin receptors have been found to be involved in NDP-MSH and MTII binding (18). Mutation of the conserved MC4R TM3 residues aspartic acids 122 and 126 and TM6 residues phenylalanine 261 and histidine 264 significantly decrease the binding affinity and potency of NDP-MSH and MTII (18). Little information is available, however, regarding the molecular basis of hMC4R interaction with the antagonist SHU9119. Whether SHU9119 utilizes the same hMC4R-binding site as NDP-MSH and MTII is unknown. To gain insight into the molecular determinants of hMC4R responsible for SHU9119 selectivity, and particularly to define hMC4R residues essential for SHU9119 antagonist selectivity, we have taken a broad approach to investigating the role of the transmembrane amino acid residues of MC4R in discriminating between MTII and SHU9119 receptor activation. Chimeras and mutants of MC4R were constructed and studied. We hope this approach will provide valuable insight into the molecular basis of ligand-receptor interaction between hMC4R as well as SHU9119 and provide new information for rational drug design of selective MC4R agonists and antagonists.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Peptides-- NDP-MSH, MTII, and SHU9119 were obtained from Peninsula Laboratories, Inc. (Belmont, CA) (Fig. 1). NDP-MSH is a linear 13 amino acid peptide. MTII and SHU9119 are cyclic peptides.


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  		Fig. 1.   Sequences of melanocortin peptide NDP-MSH, MTII, and SHU9119. NDP-MSH differs in primary sequence from the native hormone, alpha -MSH, by Nle replacing Met at position 4 and inversion of chirality of the alpha  carbon of phenylalanine at position 7 to D-Phe. MTII is a cyclic peptide that shares core sequence His-D-Phe-Arg-Trp with NDP-MSH. SHU9119 is a peptide with a beta -(2-naphthyl)-D-alanine (D-Nal) substituted in position 7 of MTII.

Construction of Melanocortin Receptor Chimeras-- The amino acid sequences of the human MC1R and MC4R were examined by both hydrophobicity plotting and by manually comparing their sequences to the previously published alignment of the seven transmembrane G-protein-coupled receptor alpha -helices (18). The chimeras utilized in these studies are schematically depicted in Fig. 2. They were constructed by the polymerase chain reaction using Pfu polymerase (Stratagene, La Jolla, CA) and primer oligonucleotides consisting of the transmembrane domains of interest. The PCR products were subcloned into the eukaryotic expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA) and then sequenced to confirm that the desired sequences were present and that no sequence errors had been introduced. The sequence of the wild-type hMC4R used in these studies can be found in GenBankTM under accession number L08603. The sequence of the wild-type hMC1R used in these studies can be found in GenBankTM under accession number X65634, with the exception that amino acid residue 163 is Arg and 164 is Gln.


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  		Fig. 2.   Schematic representation of the chimeric human melanocortin receptors utilized in these studies. Panel A schematically depicts the seven transmembrane structures of the wild-type (WT) MC4R (heavy lines) and MC1R (thin lines). Panel B depicts the structure of the chimeric MC4R with the substituted TMs of the MC1R. The amino acid sequences of the third, fourth, fifth, and sixth transmembrane domains of the hMC1R and hMC4R are shown for comparison. The non-conserved amino acids between hMC1R and hMC4R are highlighted in bold font.

Site-directed Mutagenesis-- Mutants were constructed by the polymerase chain reaction using Pfu polymerase (Stratagene, La Jolla, CA) and primer oligonucleotides, which consisted of the coding region of the amino acid residues of interest. The homologous amino acid residues of hMC4R were substituted with the corresponding residues found in hMC1R. The presence of the desired mutations was confirmed by single strand sequencing using Sequenase version 2.0 (Life Science, Cleveland, OH). The mutant receptors were subcloned into the eukaryotic expression vector pCDNA3.1 (Invitrogen).

Cell Culture and Transfection-- The coding regions of the genes for the hMC4R wild-type, chimeras, and mutants were subcloned into the pCDNA3.1. Transfection of cells was accomplished by using LipofectAMINE (Invitrogen), and permanently transfected clonal cell lines were selected by resistance to the neomycin analog G418 (17).

Binding Assays-- After removal of medium, cells were incubated with various non-radioligands in 0.5 ml of minimum Eagle's medium (Invitrogen) containing 0.2% bovine serum albumin and radioligand. Binding experiments were performed using conditions previously described (19). Briefly, 2 × 105 cpm of 125I-NDP-MSH (Amersham Biosciences) was used in combination with non-radiolabeled ligands, NDP-MSH, MTII, or SHU9119. Binding reactions were terminated by removing the medium and washing the cells twice with minimum Eagle's medium containing 0.2% bovine serum albumin. The cells were lysed with 0.2 N NaOH, and the radioactivity in the lysate was quantified in an analytical gamma counter. Non-specific binding was determined by measuring the amount of 125I-label bound in the presence of 10-6 M unlabeled ligand. Specific binding was calculated by subtracting non-specifically bound radioactivity from total bound radioactivity.

cAMP Assay-- cAMP generation was measured using a competitive binding assay kit (TRK 432, Amersham Biosciences). Briefly, HEK cell lines stably expressing the human melanocortin receptors, as previously described, were used in these assays (19). Cell culture medium was removed, and cells were incubated with 0.5 ml of Earle's Balanced Salt solution containing melanocortin agonist NDP-MSH, MTII, or antagonist SHU9119 (10-10-10-6 M), for 30 min at 37 °C in the presence of 10-3 M isobutylmethylxanthine. The reaction was stopped by adding ice-cold 100% ethanol (500 µl/well). The cells in each well were scraped, transferred to a 1.5-ml tube, and centrifuged for 10 min at 1900 × g, and the supernatant was evaporated in a 55 °C water bath with pre-purified nitrogen gas. cAMP content was measured according to the instructions accompanying the assay kit. Each experiment was performed a minimum of three times with duplicate wells. The mean value of the dose-response data was fit to a sigmoid curve with a variable slope factor using non-linear squares regression analysis (GraphPad Prism, GraphPad Software, San Diego, CA). All statistical analyses represent the mean of the data ± S.E.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of the hMC4R/hMC1R Chimera with 125I-NDP-MSH Binding-- To investigate the molecular determinant of hMC4R responsible for SHU9119 binding and specific activity, a domain-swapping strategy was used to localize regions of the receptor responsible for ligand receptor selectivity. Cassette substitutions of the third, fourth, fifth, and sixth transmembrane regions of the hMC4R with homologous regions of the hMC1R were constructed. The first, second, and seventh transmembranes were not chosen for investigation because computer modeling of ligand receptor interaction and our previous data suggested that they were not important in NDP-MSH binding (18, 19). The binding affinities of NDP-MSH, MTII, and SHU9119 at wild-type hMC4 and hMC1 receptors were first examined. As shown in Fig. 3, NDP-MSH, MTII, and SHU9119 possessed high affinity binding at both wild-type hMC4R and hMC1R as indicated by dose-dependent displacement of 125I-NDP-MSH binding (Fig. 3, panels A and C). Examination of cells transfected with chimeric receptors indicate that each chimeric receptor was functionally expressed and that the expression levels showed no significant variation when compared with wild-type receptor expression (Table I). To assess the binding affinity of ligands at these chimeric receptors, displacement experiments of labeled 125I-NDP-MSH with unlabeled NDP-MSH, MTII, and SHU9119 were performed (Fig. 4), and the IC50 values of NDP-MSH, MTII and SHU9119 were analyzed and are summarized in Table I.


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  		Fig. 3.   Binding affinity and potency of NDP-MSH, MTII, and SHU9119 at the wild-type hMC1R and hMC4R. Cells transfected with hMC1R or hMC4R were incubated with 125I-NDP-MSH at 37 °C for 1 h in the presence of the indicated amounts of unlabeled ligands. The total 125I-NDP-MSH binding was then determined on duplicate wells as described under "Experimental Procedures." Data points represent the mean ± S.E. of at least three independent experiments. (n >=  3; see Table I for IC50 and EC50 values). Panels A and C depict the binding affinity of three melanocortin peptides, NDP-MSH, MTII, and SHU9119, as determined by inhibition of 125I-NDP-MSH binding. Panels B and D demonstrate the ability of NDP-MSH, MTII, and SHU9119 to stimulate the production of intracellular cAMP. SHU9119 dose-dependently increases cAMP generation at the hMC1R but has no effect at hMC4R.

                              
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Table I
Effect of NDP-MSH analogs on 125I-NDP-MSH binding on HEK cells transfected with chimeras of the hM C 4R


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  		Fig. 4.   Binding affinity and potency of NDP-MSH, MTII, and SHU9119 at the hMC4R chimera. HEK-293 cells transfected with the hMC4R chimera were incubated with 125I-NDP-MSH at 37 °C for 1 h in the presence of the indicated amounts of unlabeled ligands, and total 125I-NDP-MSH binding was determined on duplicate wells as described under "Experimental Procedures." For the cAMP assay, the cells transfected with the hMC4R chimera were incubated with the indicated amounts of NDP-MSH, MTII, or SHU9119 for 30 min, and total cAMP accumulation was determined on duplicate wells. Data points represent the mean ± S.E. of at least three independent experiments. The chimeric receptor hMC4R/3TMhMC1R did not alter NDP-MSH, MTII, and SHU9119 binding affinity but switched SHU9119 from antagonist to agonist.

Characterization of the hMC4R/hMC1R Chimera with cAMP Assays-- To examine the ability of NDP-MSH, MTII, and SHU9119 to activate hMC4/hMC1 chimeric receptors, cAMP production was determined. For wild-type hMC1R and hMC4R, both NDP-MSH and MTII dose-dependently increased cAMP generation as expected. SHU9119 dose-dependently induced cAMP generation at hMC1R but had no effect on cAMP production at hMC4R (Fig. 3, panels B and D). For the hMC4/hMC1 chimeric receptors, both NDP-MSH and MTII also increased cAMP generation in a dose-dependent manner (Fig. 4). SHU9119 had no effect on cAMP levels in cells expressing the chimeric hMC4R/4TMhMC1R, hMC4R/5TMhMC1R, and hMC4R/6TMhMC1R. However, when TM3 of hMC4R was replaced with the corresponding region of hMC1R, SHU9119 switched from antagonist to agonist, resulting in increased cAMP production (Fig. 4). Their EC50 values are shown in Table II. These results identify the TM3 region of both hMC1R and hMC4R as critical molecular determinants of SHU9119 activity.

                              
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Table II
Effect of NDP-MSH analogs on cAMP formation on HEK cells transfected with chimeras of the hMC4R

Identification of Amino Acid Residues in TM3 of hMC4R Responsible for SHU9119 Selectivity-- Because exchange of TM3 of hMC4R with the corresponding region of hMC1R altered SHU9119 specific activity, it was important to determine which amino acid residues of TM3 of hMC4R were responsible for SHU9119 specific activity. The sequence of amino acids of hMC4R TM3 is depicted in Fig. 5. A sequence alignment of hMC1R and hMC4R revealed that the TM3 domain is highly conserved. However, there are nine amino acid differences (Fig. 2). Based on the sequence alignment, seven amino acids candidates of potential importance for antagonist SHU9119 activity were selected for mutation analysis. Leu141 and Ser142 were excluded from mutation analysis because these amino acids are located in the deep intracellular side. To define which amino acid is involved in D-Nal7 of SHU9119 antagonism, the reciprocal exchange of amino acids of TM3 between hMC4R and hMC1R was used. These seven TM3 hMC4R residues were individually replaced by the homogeneous residues of hMC1R, expressed in HEK cells, and evaluated. As shown in Table III, all of the mutant receptors were functionally expressed, and their protein expression level (Bmax) and binding affinity for NDP-MSH, MTII, and SHU9119 were in the range of the wild-type receptor. NDP-MSH, MTII, and SHU9119 dose-dependently displaced 125I-NDP-MSH binding at those mutants. The serine 127 on hMC4R was switched to a valine from the hMC1R, valine 128 to isoleucine, isoleucine 129 to a tyrosine, alanine 135 to serine, and isoleucine 137 to tyrosine. The final exchange of phenylalanine 139 to serine of the TM3 did not influence receptor binding affinity and agonist potency. By marked contrast, swapping the residue Leu133 to methionine led to complete conversion of SHU9119 activity from antagonist to agonist. This effect occurred despite the observation that the binding affinities of NDP-MSH, MTII, and SHU9119 were not altered (Fig. 6 and Table IV).


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  		Fig. 5.   Schematic diagram of hMC4R receptor mutation. Two-dimensional representation of the seven transmembrane structure of the hMC4R. Gray or black highlighting denotes the receptor transmembrane residues mutated in these experiments.

                              
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Table III
Effect of NDP-MSH analogs on 125I-NDP-MSH binding on HEK cells transfected with mutations of the hMC4R


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  		Fig. 6.   Binding affinity and potency of NDP-MSH, MTII, and SHU9119 at the mutant hMC4R L133M. HEK-293 cells transfected with mutant L133M were incubated with 125I-NDP-MSH at 37 °C for 1 h in the presence of the indicated amounts of unlabeled ligands, and total 125I-NDP-MSH binding was determined on duplicate wells. For cAMP assay, the cells were incubated with the indicated amounts of NDP-MSH, MTII, or SHU9119 for 30 min, and total cAMP accumulation was determined on duplicated wells. Data points represent the mean ± S.E. of at least three independent experiments. Panel A shows the binding affinity of different ligands that displace 125I-NDP-MSH. Panel B shows that the above three ligands stimulate cAMP production. HMC4R mutant L133M did not change ligand binding affinity but switched SHU9119 from antagonist to agonist.

                              
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Table IV
Effect of NDP-MSH analog on cAMP formation on HEK cells transfected with mutations of the hMC4R

Effect of Amino Acid Substitution of hMC1R on Ligand SHU9119 Binding and Activation-- Because replacement of Leu133 of the hMC4R with the homologous residue methionine of the hMC1R switched SHU9119 from antagonist to agonist, we hypothesized that the residues of other melanocortin receptor subtypes corresponding to 133 of hMC4R might also be important for selecting agonist or antagonist. To test this hypothesis we made cassette substitutions of the hMC1R residue Met128 with the homologous residue of the hMC4R Leu133. We expected that if the Met128 residue was important for ligand selectivity, the MC1R mutant (M128L), unlike wild-type MC1R, would be able to convert agonist SHU9119 into antagonist or at least reduce MTII and SHU9119 potency. Consistent with our assumption, our results demonstrated that the hMC1R mutant with substitution of Leu of hMC4R dramatically reduced MTII and SHU9119 binding affinity and potency comparable with that of the wild-type hMC1R, though there was no effect on NDP-MSH binding affinity and potency (Fig. 7). However, the substitution of hMC1R did not switch SHU9119 from agonist to antagonist.


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  		Fig. 7.   Binding affinity and potency of NDP-MSH, MTII, and SHU9119 at the mutant hMC1R M128L. HEK-293 cells transfected with mutant M128L were incubated with 125I-NDP-MSH at 37 °C for 1 h in the presence of the indicated amounts of unlabeled ligands. Total 125I-NDP-MSH binding was determined on duplicated wells. For cAMP assay, the cells were incubated with the indicated amounts of NDP-MSH, MTII, or SHU9119 for 30 min, and total cAMP accumulation was determined. Data points represent the mean ± S.E. of at least three independent experiments. Panel A shows the binding affinity of different ligands to displace 125I-NDP-MSH binding. Panel B shows the effect of the above three ligands on cAMP generation. hMC1R mutant M128L dramatically reduced MTII and SHU9119 binding affinity and potency but did not alter NDP-MSH binding affinity and potency.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hypothalamic MC4R plays an important role in the regulation of animal food intake. The melanocortin receptor agonist, MTII, and antagonist SHU9119 have been widely used in the study of MC receptor functions (20-22). The identification of the essential amino acid residues of human MC4R responsible for SHU9119 antagonism should be important for understanding the signaling events that regulate the melanocortin system under physiologic circumstances. In addition, a molecular understanding of SHU9119 activity at hMC4R may have important implications in the design of drugs that may regulate feeding behavior in conditions such as obesity or anorexia.

The natural melanocortins (alpha -MSH, beta -MSH, gamma -MSH, and adrenocorticotropic hormone (ACTH)) and synthetic melanocortins (NDP-MSH, MTII, and SHU9119) have distinct affinity profiles for each of the five MC receptor subtypes. The natural melanocortins are all agonists for hMCRs with the exception of MC2R, for which only ACTH is a full agonist (23). However, synthetic melanocortins have different pharmacological profiles for the five MC receptor subtypes. For example, NDP-MSH and MTII are agonists for all human MC receptor subtypes except hMC2R (24). The varying pharmacological profiles of the five MC receptor subtypes suggest that they have different ligand-binding sites and/or utilize different residues for binding and functional selectivity. The identification of the role that the amino acid residue Phe7 plays in MSH binding and activity led to the discovery of SHU9119, in which a bulky hydrophobic amino acid in position 7 of MTII converts the peptide from an agonist to an antagonist at the MC3R and MC4R (13). However, the molecular basis responsible for this antagonism is unclear. In the present study, we use a domain-swapping strategy to localize regions of the hMC4R responsible for SHU9119 antagonism. hMC4R and hMC1R were chosen to construct chimeras because the hMC4R and hMC1R belong to the same receptor family, yet SHU9119 is an agonist at MC1R and an antagonist at MC4R (25, 26). Our chimeric receptor studies demonstrated that the TM3 of the hMC4R is responsible for SHU9119 selectivity because the substitution of the TM3 of the hMC4R with the corresponding region of hMC1R (hMC4R/3TMhMC1R) switched SHU9119 from antagonist to agonist but did not alter NDP-MSH, MTII, and SHU9119 binding affinity. Therefore, TM3 of hMC4R plays a critical role in SHU9119 antagonism without affecting SHU9119 binding. This suggests that ligand binding and receptor activation by SHU9119 require different amino acids. To determine which residues of the MC4R TM3 are responsible for SHU9119 specific activity, mutagenesis studies were performed. Sequence analysis studies of hMC4R and hMC1R have indicated that both receptors have conserved residues that are known to be involved in receptor binding and activation of NDP-MSH. Our binding studies reported here show no significant differences in the agonist NDP-MSH binding affinities at all chimeric receptors we studied. This has led us to propose that melanocortin ligand binding affinity is directed by ligand interaction with conserved receptor residues. However, functional selectivity might be directed by ligand interactions with non-conserved receptor residues located at equivalent positions in different receptor subtypes. Moreover, residues located at different positions in different receptors may also contribute to ligand functional selectivity. Because of the findings that the substitution of the TM3 did not alter ligand binding affinity we theorized that SHU9119 might utilize receptor-conserved residues to occupy the receptor-binding sites but that D-Nal, in position 7, could not interact with certain non-conserved residue to activate the receptor. Analysis of the hMC4R sequence indicates that there are nine non-conserved amino acids in TM3 that are different from hMC1R. Amino acid residues Ser127, Val128, Ile129, Leu133, Ala135, Ile137, and Ser139 are potential candidates for critical activation molecules. Leu141 and Ser142 are located on the deep intracellular side of the membrane and are not believed to be involved in ligand binding and receptor activation. To investigate which amino acids might be important for SHU9119 specific activity we individually substituted these seven candidate amino acids of hMC4R with the corresponding amino acid of the hMC1R TM3 region. Our results show that substitution of S127V, V128I, I129T, A135S, I137T, and S139F does not affect NDP-MSH, MTII, and SHU9119 binding affinity. In addition, SHU9119 remains a MC4R antagonist, whereas MTII and NDP-MSH remain agonists. However, when Leu133 is replaced with Met133 SHU9119 switches from antagonist SHU9119 to an agonist. This finding suggests that leucine in position 133 of hMC4R is critical for SHU9119 antagonism because the mutation of hMC4R gains ability for SHU9119 to stimulate cAMP generation. Our results suggest that SHU9119 utilizes conserved receptor residues for ligand binding but non-conserved residues for its specific activity. It has been reported that D-Phe of NDP-MSH is important for hMC4R binding and activation with its activation being dramatically reduced if D-Phe is replaced with D-Ala (18). Combined with our current findings, these data suggest that both the stereochemistry and the bulky naphthyl ring in position 7 of MTII are important for positioning of ligand residues with the responding hMC4R residues but that receptor subtypes, not ligands, determine functional selectivity.

To determine whether residue TM3 leucine 133 of hMC4R is specific for SHU9119 activity or whether it is a critical residue common for other melanocortin receptors, we mutated the homologous residue in TM3 of hMC1R, methionine 128 to leucine 128. Our results indicate that the mutation of M128L does not affect NDP-MSH binding affinity but does dramatically decrease MTII and SHU9119 binding affinity. This finding strongly suggested that NDP-MSH had different binding sites from MTII and SHU9119 at hMC1R and that Met128 is crucial for MTII and SHU9119 binding but is not important for NDP-MSH binding. One possible explanation for this finding might be that NDP-MSH is a linear peptide and can compensate the binding site, whereas MTII and SHU9119 are cyclic peptides. Cyclic peptide structures are more restricted than that of NDP-MSH. Therefore the fact that substitution of Met128 in hMC1R, the amino acid corresponding to Leu133 of hMC4R, did not switch SHU9119 from agonist to antagonist indicates that residue 133 in hMC4R is unique for SHU9119 selectivity. It is not the common nature for all melanocortin receptor subtypes. Our results indicated that more than one amino acid residue is required for SHU9119 activation at the hMC1R. Residue Met128 at the hMC1R is involved in both MTII and SHU9119 binding, but the Leu133 residue at the hMC4R is the only specific residue that differentiates D-Phe and D-Nal activity.

It has been reported that two phenylalanines in positions 254 and 259 in TM6 of mouse MC4R are involved in SHU9119 antagonism (27). However, our data demonstrate that replacement of TM6 of human MC4R with TM6 of human MC1R has no effect on SHU9119 activity. Furthermore, sequence analysis of both hMC1R and hMC4R TM6 indicates that both receptors have two phenylalanine residues at the same position. If phenylalanine in TM6 of hMC4R was crucial for SHU9119 specific activity it would be unlikely that SHU9119 would have a different action at hMC1R compared with hMC4R. One explanation for this discrepancy might be that human MC4R has different binding sites for SHU9119 compared with that of mouse MC4R.

In conclusion, these results provide insight into the molecular features of hMC4R responsible for SHU9119 antagonist specific activity. This study shows that the mutation of hMC4R Leu133 to methionine switches SHU9119 from antagonist to agonist while not changing NDP-MSH, MTII, or SHU9119 binding affinity, and Met128 in hMC1R is important for MTII and SHU9119 binding and activation. This report also suggests that melanocortin peptides utilize conserved residues for receptor binding while utilizing non-conserved residues for receptor functional selectivity.

    FOOTNOTES

* This work was supported by the Research Institute of Alabama Children's Hospital.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Div. of Pediatric Surgery, University of Alabama at Birmingham, 300 ACC, 1600 7th Avenue South, Birmingham, AL 35233. Tel.: 205-939-9688; Fax: 205-975-4972; E-mail: mac.harmon@ccc.uab.edu.

Published, JBC Papers in Press, March 23, 2002, DOI 10.1074/jbc.M201343200

    ABBREVIATIONS

The abbreviations used are: MC4R, melanocortin-4 receptor; MC1R, melanocortin-1 receptor; h, human; TM, transmembrane; MSH, melanocyte-stimulating hormone; NDP-MSH, alpha -[Nle4-D-Phe7]MSH; MTII, melanotan II; MC, melanocortin; D-Nal, beta -(2-naphthyl)-D-alanine; Nle, norleucine; HEK, human embryonic kidney..

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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