Common Requirements for Melanocortin-4 Receptor Selectivity of Structurally Unrelated Melanocortin Agonist and Endogenous Antagonist, Agouti Protein*

Julia OosteromDagger , Keith M. GarnerDagger , Wijnand K. den DekkerDagger , Wouter A. J. NijenhuisDagger , Willem Hendrik GispenDagger , J. Peter H. BurbachDagger , Greg S. Barsh§, and Roger A. H. AdanDagger

From the Dagger  Department of Medical Pharmacology, Rudolf Magnus Institute for Neurosciences, University Medical Center Utrecht, P. O. Box 85060, 3508 AB Utrecht, The Netherlands and the § Howard Hughes Medical Institute, Beckman Center B271A, Stanford University School of Medicine, Stanford, California 94305-5323

Received for publication, August 10, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The activity of melanocortin receptors (MCR) is regulated by melanocortin peptide agonists and by the endogenous antagonists, Agouti protein and AgRP (Agouti-related protein). To understand how the selectivity for these structurally unrelated agonists and antagonist is achieved, chimeric and mutants MC3R and MC4R were expressed in cell lines and pharmacologically analyzed. A region containing the third extracellular loop, EC3, of MC4R was essential for selective Agouti protein antagonism. In addition, this part of MC4R, when introduced in MC3R, conferred Agouti protein antagonism. Further mutational analysis of this region of MC4R demonstrated that Tyr268 was required for the selective interaction with Agouti protein, because a profound loss of the ability of Agouti protein to inhibit 125I-labeled [Nle4,D-Phe7]alpha -melanocyte-stimulating hormone (MSH) binding was observed by the single mutation of Tyr268 to Ile. This same residue conferred selectivity for the MC4R selective agonist, [D-Tyr4]MT-II, whereas it inhibited interaction with the MC3R-selective agonist, [Nle4]Lys-gamma 2-MSH. Conversely, mutation of Ile265 in MC3 (the corresponding residue of Tyr268) to Tyr displayed a gain of affinity for [D-Tyr4]MT-II, but not for Agouti protein, and a loss of affinity for [Nle4]Lys-gamma 2-MSH as compared with wild-type MC3R. This single amino acid mutation thus confers the selectivity of MC3R toward a pharmacological profile like that observed for MC4R agonists but not for the antagonist, Agouti protein. Thus, selectivity for structurally unrelated ligands with opposite activities is achieved in a similar manner for MC4R but not for MC3R.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Melanocortin (MC)1 receptors are activated by the POMC (pro-opiomelanocortin)-derived ACTH (adrenocorticotropic hormone) and MSH (melanocyte-stimulating hormone) peptides. MC3R and MC4R are the main MC receptors in the brain, and MC4R is thought to play a prominent role in the regulation of body weight in both rodents and human (1-3). The identification of the endogenous MC receptor antagonists, Agouti protein and Agouti related-protein (AgRP), gave rise to an additional level of regulation of MC receptors, in which ligands with opposite activities control MCR activity (4, 5). It is unknown, however, whether the molecular interaction with the receptor of these oppositely acting ligands is achieved in a similar manner.

Mouse Agouti protein is a 131-amino acid protein normally expressed in hair follicles, which acts on MC1R-expressing melanocytes to regulate pigmentation (6, 7). In mice that ectopically overexpress Agouti protein, chronic MC1R blockade by Agouti protein results in a yellow coat color. In addition, these mice display severe obesity, hyperphagia, and increased plasma levels of the adipose derived satiety factor, leptin (8, 9). In these mice, obesity is thought to be the result of continuous blockade of the hypothalamic MC4R, since recombinant murine Agouti protein has been demonstrated to act as a high affinity antagonist for both mouse MC1R and MC4R in vitro but not for MC3R and MC5R (10-12). This determination is in agreement with the finding that MC4R-/- mice recapitulate the obesity phenotype as observed in yellow obese mice (2).

In wild-type mice, Agouti protein is not expressed in the brain. But its homologue, AgRP, is expressed in the hypothalami of mice, rats, primates, and humans (13-16). Recombinant human AgRP acts as a high affinity antagonist for the MC3R and MC4R, and to a lesser extent for MC5R, but not for MC1R and MC2R (17). Transgenic mice that overexpress AgRP display an obesity phenotype similar to that found in MC4R-/- mice (4). Thus, pharmacological, histochemical, and genetic studies suggest that hypothalamic AgRP is an important endogenous stimulator of feeding and exerts this function by inhibiting MCR signaling.

Even though an important role of MC4R in body weight homeostasis is evident, the role of MC3R in the control of body weight and other processes is ill-defined because of the absence of MC3R selective ligands. Knowledge of the interaction between MCR and its ligands at the level of molecular detail would contribute to the design of selective MC3R and MC4R ligands. This is important not only for the understanding of MCR subtype-specific functions, but in addition, ligands that selectively activate or block MC4R may be therapeutically useful in the treatment of obesity (18) and anorexia (19, 20).

To investigate whether MCR agonists and the structurally unrelated antagonist, Agouti protein, are regulated in a similar manner, the aim of this study was to identify which part of the human MC4R is important for selective Agouti protein interaction and antagonism. To this end, chimeric and mutant MC3R and MC4R were expressed in cell lines and were tested for their interaction with full-length human Agouti protein, [Nle4]Lys-gamma 2-MSH, and [D-Tyr4]MT-II. The results indicate that a single residue in MC4R is required for the selective interaction with Agouti protein. In addition, the results show that selectivity for structurally unrelated agonists and antagonist is achieved in a similar manner.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Peptides-- NDP-alpha -MSH ([Nle4,D-Phe7]-alpha -MSH) and alpha -MSH were purchased from Bachem Feinchemicalien, Bubendorf, Switzerland. [D-Tyr4]MT-II and [Nle4]Lys-gamma 2-MSH were synthesized using solid phase Fmoc chemistry and purified as described previously (21). Full-length human Agouti protein and AgRP were prepared as described previously (17, 22).

Radioiodination of NDP-alpha -MSH-- Iodination was performed exactly as described previously (23). In short, 4 µg of NDP-alpha -MSH was mixed with 1.2 IU bovine lactoperoxidase (Calbiochem) and 1 mCi of Na125I (ICN) in a final volume of 100 µl of 0.05 M phosphate buffer (pH 6.5). Then, 5 µl of 0.003% H2O2 was added every 60 s. After 4 min, 50 µl of 1 mM dithiothreitol was added to stop the reaction. The sample was purified by high pressure liquid chromatography with a µBondapak C18 column, 3.9 × 300 mm (Waters, Div. of Millipore) by elution with a 22-52% acetonitrile gradient in 10 mM ammonium acetate (pH 5.5) in 40 min. The specific activity of 125I-NDP-alpha -MSH was 2.25 × 106 Ci/mol.

Expression of Chimeric and Mutant Receptors-- All chimeric and MCR mutants used in this study were generated by polymerase chain reaction, and the sequences were verified as described previously (23). The chimeric receptors were named after the extracellular receptor domain that was replaced. With "A" being the N-terminal domain (corresponding to residues 1-60 of MC4R), "B" the first extracellular loop (EC1, residues 67-142), "C" the second extracellular loop (EC2, residues 149-195), and "D" the third extracellular loop (EC3, residues 202-332). These receptors include chimera 3AB (MC4R with N-terminal through TM3 of MC3R), 3B (MC4R with IC1 through TM3 of MC3), 3C (MC4R with IC2 through EC2 of MC3), 3D (MC4R with TM5 through C-terminal of MC3R), 4D (the reverse of chimera 3D; MC3R with TM5 through C-terminal of EC3 of MC4R), "Loop" (MC4R with EC3 of MC3R), "1st half" (MC4R with first half of EC3 of MC3R), "2nd half" (MC4R with second half of EC3 of MC3R), and mutant MC4 (Tyr268 right-arrow Ile). In these constructs, EC3 of MC4R runs from residue Phe267 through Ala282, the first half of EC3 runs from Phe267 through Gln273, and the second half of EC3 runs from Val278 through Ala282 (Fig. 1). For the radioligand and adenylate cyclase assays, wild-type rat MC3R (24), human MC4R (25), and chimerae 3AB, 3B, 3C, 3D, and 4D were stably expressed in B16G4F mouse melanoma cells. These cells were grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% fetal calf serum. In addition, human MC4R, loop, 1st half, 2nd half, and MC4 (Tyr268 right-arrow Ile) were expressed in 293 HEK cells and used in the radioligand binding assay. 293 HEK cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum. An MC3R mutant was generated in which Ile265 of MC3R was mutated into Tyr (MC3(Ile265 right-arrow Tyr)). This mutant was made using the QuikChangeTM Site-Directed Mutagenesis Kit (Stratagene). The rat MC3R cloned in pcDNA3.1 was used as template. The sequence of the mutagenic oligonucleotides was 5'-TTCCTCCACCTGGTCCTGTACATCACCTGCCCCACCAACC-3' and its complementary oligonucleotide. For the pharmacological analysis, the rat MC3R (wild type) and MC3(Ile265 right-arrow Tyr) were expressed in BHK (baby hamster kidney) and 293 HEK cells with similar results. These cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.



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Fig. 1.   Model of the human MC4R. The gray circles indicate a stretch of residues with complete homology between MC3R and MC4R that were used as boundaries for construction of the chimeric MC3/MC4 receptors. The larger circles indicates residues belonging to the third extracellular loop (EC3, residues 267-282). The black circles with double-residue symbols (for example, Y/I at position 268) indicate that the first residue (Y) is present in MC4R and the second residue (I) is present in MC3R at the corresponding position. The white circles in EC3 are homologous in MC4R and MC3R. The numbering corresponds to the human MC4R amino acid sequence. The N- and C-terminal residues are not shown.

Adenylate Cyclase Assay-- B16G4F cells, stably expressing human MC4R, rat MC3R, and chimerae 3AB, 3B, 3C, 3D, or 4D were grown in 24-well plates (Corning Costar). Agonist-stimulated adenylate cyclase activity was measured as described previously (26). In short, after prelabeling with 500 µl of [3H]adenine (PerkinElmer Life Sciences) in a concentration of 2 µCi/ml, the cells were incubated for 30 min at 37 °C in Dulbecco's modified Eagle's medium containing 0.1 mM isobutylmethylxanthine, alpha -MSH in a concentration ranging from 0.3 nM to 3 µM, with and without 40 nM Agouti protein. The cells were harvested, and [3H]cAMP formation was calculated as the percentage of [3H]ATP converted into [3H]cAMP. For each curve, 12 duplicate data points were collected. EC50 values were determined by fitting the data to a sigmoidal curve with variable slope using GraphPad Prism 2.01 for Windows 95/NT (GraphPad Software Inc., San Diego, CA). Experiments were performed two times with the same results.

Receptor Binding Assay-- MC receptor expressing B16G4F, 293 HEK cells, or BHK cells were grown in 24-well plates (Corning Costar). The cells were incubated with 100,000 cpm (approx 0.1 nM) of 125I-NDP-alpha -MSH and various concentrations of nonradioactive peptides diluted in binding buffer consisting of Ham's F-10 medium (Life Technologies, Inc.) (pH 7.4), 2.5 mM calcium chloride, 0.25% bovine serum albumin, 10 mM Hepes, and 50 µg/ml aprotinin (Sigma). After a 30-min incubation at room temperature, the cells were washed twice with ice-cold Tris-buffered saline containing 2.5 mM calcium chloride and lysed in 1 M sodium hydroxide. Radioactivity of the lysates was counted in a Packard Cobra gamma -counter. Competition curves were fitted from 12 duplicate data points with GraphPad Prism 2.01 for Windows 95/NT, nonlinear regression, one-site competition. IC50 values were calculated with 99% confidence interval. Experiments were repeated at least two times with the same results.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The affinities (IC50 values, using 125I-NDP-alpha -MSH as radioligand) of full-length human Agouti protein, AgRP, and NDP-alpha -MSH were determined for the wild-type MC4R and MC3R expressed in B16G4F cells. NDP-alpha -MSH and AgRP possessed comparable IC50 values for both MC3R and MC4R (Fig. 2). In contrast, Agouti protein displayed an almost 40-fold lower affinity for MC3R than AgRP and NDP-alpha -MSH, whereas Agouti protein displayed high affinity for MC4R. The IC50 of Agouti protein was 10-fold higher for MC3R than for MC4R.2



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Fig. 2.   Affinity of NDP-alpha -MSH, Agouti protein, and AgRP for the human MC4R and rat MC3R expressed in B16G4F cells. The graphs show competition of 125I-NDP-alpha -MSH by NDP-alpha -MSH (black-square), Agouti protein (open circle ) and AgRP (black-down-triangle ). Data points represent the mean of duplicate measurements ± S.D. Binding curves were fitted with GraphPad Prism, one-site competition. The IC50 values for MC3R and MC4R were, respectively: NDP-alpha -MSH, 2.4 and 11 nM; Agouti protein, 101 and 12 nM; and AgRP, 2.7 and 9.7 nM.

Next, the antagonistic properties of Agouti protein were analyzed. Agouti protein at a concentration of 40 nM was a potent antagonist for MC4R (Fig. 3) and was able to increase the EC50 of alpha -MSH almost 20-fold (Table I). In contrast, the same concentration of Agouti protein was not able to significantly alter the EC50 of alpha -MSH for MC3R.



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Fig. 3.   Agouti protein antagonism for MC4R, MC3R, and chimeric receptors. alpha -MSH-stimulated adenylate cyclase activity in the absence (black-square) and presence (open circle ) of 40 nM Agouti protein at MC4R, MC3R, and chimerae 3AB, 3B, 3C, 3D, and 4D expressed in B16G4F cells. Data points represent the mean of duplicate measurements ± S.D. Curves were fitted with GraphPad Prism, sigmoidal dose-response curve fitting, variable slope.


                              
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Table I
EC50 value (in nM) of alpha -MSH in the absence and presence of 40 nM human Agouti protein for MC3R, MC4R, and chimerae 3AB, 3B, 3C, 3D, and 4D expressed in B16G4F cells
EC50 values were calculated using GraphPad Prism, sigmoidal dose-response curve fitting, variable slope.

In addition, it was investigated as to which region of MC4R is required for the antagonistic properties of Agouti protein. Therefore, the ability of Agouti protein to antagonize alpha -MSH-stimulated adenylate cyclase activity of chimeric MC3/MC4 receptors was analyzed. Agouti protein was able to effectively antagonize alpha -MSH-stimulated adenylate cyclase activity for all chimeric receptors except chimera 3D, which only has the C-terminal portion of MC3R starting at TM5 (Fig. 3 and Table I). Furthermore, the reverse chimera, 4D, which is an MC3R with only the C-terminal portion (starting at TM5) of MC4R, was able to confer Agouti protein antagonism to MC3R. Agouti protein was unable to significantly increase the EC50 of alpha -MSH for chimera 3D as observed for MC3R.

To assess which region of MC4R determined the high affinity of Agouti protein for MC4R, the IC50 values of Agouti protein for chimeric MC3/MC4 receptors was analyzed. Table II summarizes the IC50 values of Agouti protein for all chimeric receptors. Chimerae 3AB, 3B, and 3C all displayed a high affinity for Agouti, which was not significantly different from the IC50 for MC4R. However, chimera 3D (MC4R with the C-terminal portion of MC3R, starting at TM5), displayed a significant reduction in the affinity for Agouti protein. The affinities of Agouti protein for chimera 3D and MC3R were comparable (Fig. 4). Conversely, chimera 4D (the reverse of 3D, MC3R with the C-terminal portion of MC4R) displayed high affinity for Agouti protein, which was not different from wild-type MC4R.


                              
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Table II
Inhibition constants (IC50 in nM) of human Agouti protein for MC3R, MC4R, and chimerae 3AB, 3B, 3C, 3D, and 4D
All receptors were stably expressed in B16G4F cells. IC50 values were determined from competition binding curves using 125I-NDP-alpha -MSH as radioligand, derived from 12 duplicate data points using GraphPad Prism, one site competition.



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Fig. 4.   Affinity of Agouti protein for MC4R, MC3R, and chimerae 3D and 4D. The graph shows competition of 125I-NDP-alpha -MSH by Agouti protein on MC4R (black-down-triangle ), MC3R (black-diamond ), and chimerae 3D () and 4D (down-triangle) expressed in B16G4F cells. Data points represent the mean of duplicate measurements ± S.D. Binding curves were fitted with GraphPad Prism, one-site competition.

Next, the role of EC3 of MC4R in interaction with Agouti protein was further specified. To this end, wild-type MC4R, mutant MC4(Tyr268 right-arrow Ile), and chimeric MC4R with small parts of EC3 of MC3R were expressed in 293 HEK cells. The chimera were: loop (MC4R with EC3 from MC3R), 1st half (MC4R with first half of EC3 from MC3R), and 2nd half (MC4R with second half of EC3 from MC3R). Fig. 5 and Table III show that the affinities of NDP-alpha -MSH for the MC4R, chimeric, and mutant receptors were comparable. However, when EC3 of MC4R was replaced by MC3R sequence (loop), the IC50 for Agouti protein increased more than 4-fold as compared with MC4R. This loss of affinity was also observed when only the first half of EC3 was replaced (1st half). Moreover, when only Tyr268 in the first half of EC3 of MC4R was mutated to Ile, the corresponding residue of MC3R, a more than 10-fold drop in Agouti protein affinity was still observed. In contrast, replacement of the second half of EC3 (2nd half chimera) did not alter the affinity for Agouti protein as compared with wild-type MC4R. Next, MC3(Ile265 right-arrow Tyr), the reverse mutant of MC4 (Tyr268 right-arrow Ile), was analyzed by determining the IC50 values of NDP-alpha -MSH, Agouti protein, [Nle4]Lys-gamma 2-MSH, and [D-Tyr4]MT-II affinity (Fig. 6 and Table IV). The latter two peptides were shown previously to display increased and decreased affinity, respectively, for MC4 (Tyr268 right-arrow Ile) (21). The data show that the IC50 of NDP-alpha -MSH and Agouti protein for MC3R and MC3(Ile265 right-arrow Tyr) did not significantly differ. However, the affinity of [Nle4]Lys-gamma 2-MSH was ~40 times lower for the mutant as compared with MC3R. In contrast, the affinity of [D-Tyr4]MT-II was significantly higher for MC3(Ile265 right-arrow Tyr).



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Fig. 5.   Affinity of NDP-alpha -MSH and Agouti protein for the human MC4R, loop, 1st half, 2nd half, and MC4 (Tyr268 right-arrow Ile) expressed in 293 HEK cells. The graphs show the competition of 125I-NDP-alpha -MSH by NDP-alpha -MSH (black-square) and Agouti protein (open circle ). The data points represent the mean of duplicate measurements ± S.D. Curves were fitted with GraphPad Prism, one-site competition.


                              
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Table III
Inhibition constants (IC50 in nM) of human Agouti protein for the MC4R, chimerae loop, 1st half, 2nd half, and mutant MC4 (Tyr268 right-arrow Ile) expressed in 293 HEK cells
For each ligand, IC50 values were determined from competition binding curves derived from 12 duplicate data points. Curves were fitted with GraphPad Prism, one-site competition using 125I-NDP-alpha -MSH as radioligand (see Fig. 3).



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Fig. 6.   Affinity of NDP-alpha -MSH and Agouti protein for the MC3R and MC3 (Ile265 right-arrow Tyr) expressed in BHK cells. The graphs show the competition of 125I-NDP-alpha -MSH by NDP-alpha -MSH (black-square), Agouti protein (open circle ), [D-Tyr4]MT-II (black-triangle), and [Nle4]Lys-gamma 2-MSH (diamond ). The data points represent the mean of duplicate measurements ± S.D. Curves were fitted with GraphPad Prism, one-site competition.


                              
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Table IV
Inhibition constants (IC50 in nM) of human Agouti protein for the MC3R and MC3(Ile265 right-arrow Tyr) expressed in BHK cells
For each ligand, IC50 values were determined from competition binding curves derived from 12 duplicate data points. Curves were fitted with GraphPad Prism, one-site competition using 125I-NDP-alpha -MSH as radioligand (see Fig. 4).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The melanocortin system is unique in the sense that MCR activity is regulated by two structurally unrelated endogenous peptides with opposing activities: on the one hand, POMC-derived melanocortin agonists, and on the other hand, Agouti protein and AgRP, which act as antagonists for MCR. Currently, selective MCR ligands are necessary to obtain a better understanding of the role of brain MC3R and MC4R in processes like the regulation of body weight. Because Agouti protein is a high affinity antagonist for the MC4R, this study was aimed at characterizing the selective interaction of human Agouti protein with the human MC4R. The pharmacological analysis of chimeric and mutant MC3R and MC4R suggested that a single amino acid residue in the first half of the third extracellular loop of MC4R conferred selectivity for Agouti protein.

The results confirm that human Agouti protein is a high affinity antagonist for MC4R but not for MC3R. In contrast, AgRP does not discriminate between MC3R and MC4R. The selective interaction of Agouti protein with MC4R was studied using chimeric and mutant MC3R and MC4R. Apparently, the lower affinity of Agouti protein for chimera 3D (MC4R with EC3 and surrounding domains of MC3R) and MC3R wild type accounts for the inability of Agouti protein to antagonize alpha -MSH action on these receptors. Chimera 4D was tested for a gain of function for Agouti binding and antagonism to test whether this region (EC3 and surrounding domains of MC4R) alone is sufficient to obtain an MC4R-specific pharmacological profile. Indeed, this EC3-containing region of MC4R fully conferred Agouti protein antagonism to MC3R. Thus, this region changes the MC3R pharmacological profile to that of MC4R.

A more detailed analysis of the role of EC3 in Agouti protein binding showed that the first half of EC3, in particular position Tyr268 of MC4R, was critical for high affinity Agouti protein binding because, when it was replaced by Ile, a loss of affinity for Agouti protein occurred. Previously, it was shown that Tyr268 was also required for the selective interaction of the agonist [D-Tyr4]MT-II with MC4R, whereas it hindered interaction with the MC3R selective agonist [Nle4]Lys-gamma 2-MSH (21). Thus, Tyr268 is required for selective agonist and antagonist binding of the MC4R. This is a striking observation because there exists no obvious amino acid homology between melanocortins and Agouti protein (Fig. 7).



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Fig. 7.   Amino acid sequences of alpha -MSH, [Nle4]Lys-gamma 2-MSH, [D-Tyr4]MT-II, and Agouti-(116-123)*. The amino acid residues present in a loop defined by disulfide-bridged Cys116 and Cys123 were suggested to be involved in the interaction with MCR. Presumably, Phe118 is crucial for selective interaction with MC4R (27).

To test whether Ile265 of MC3R hindered interaction with Agouti protein, Ile265 of MC3R was mutated into Tyr. MC3 (Ile265 right-arrow Tyr) did not show a gain of affinity for Agouti protein, indicating that Tyr268, in the context of the MC3R conformation, does not confer selectivity for antagonists. Surprisingly, MC3 (Ile265 right-arrow Tyr) displayed increased affinity for [D-Tyr4]MT-II and decreased affinity for [Nle4]Lys-gamma 2-MSH. Thus, this residue is able to change the pharmacological profile of MC3R toward that of MC4R for agonists but not for antagonists. These data imply that it may not be feasible to design MC3R-selective antagonists based upon MC3R selective agonists (such as gamma -MSH) and MC4R-selective antagonists. This approach, however, may be applicable for MC4R because the structural requirements for selective agonists and antagonists interaction with MC4R is more alike.

In conclusion, through pharmacological analysis of chimeric and mutant MC3R and MC4R, it was demonstrated that Tyr268 in EC3 of MC4R is critical for selective Agouti protein interaction. This study and previous data show that the mutation of MC4R Tyr268 to Ile decreases affinity for MC4R-selective ligands but increases affinity for the MC3R-selective ligands. Moreover, Tyr268 in MC4R determined both agonist and antagonist selectivity. This is the first report describing details of the molecular recognition of the endogenous MCR antagonist, Agouti protein, by MC4R. This report demonstrates that the selectivity of structurally unrelated peptide ligands with opposite activities (melanocortin peptide agonist versus Agouti protein antagonist) is achieved in a similar manner, strongly suggesting that they use the same binding pocket. An understanding of the molecular basis governing selective ligand interaction with MC receptors contributes to the rational design of new selective ligands.


    FOOTNOTES

* 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: Dept. of Medical Pharmacology, Rudolf Magnus Institute for Neurosciences, University Medical Center Utrecht, P. O. Box 85060, 3508 AB Utrecht, The Netherlands. Tel.: 31(0)30-2538817; Fax: 31(0)30-2539032; E-mail: r.a.h. adan@med.uu.nl.

Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M007261200

2 In this study, the terms "binding" and "affinity" always refer to the ability of a ligand to compete for 125I-NDP-alpha -MSH binding.


    ABBREVIATIONS

The abbreviations used are: MC, melanocortin; MCR, MC receptor; MSH, melanocyte-stimulating hormone; 125I-NDP-alpha -MSH, 125I-labeled [Nle4,D-Phe7]alpha -MSH; AgRP, Agouti-related protein; EC, extracellular loop; BHK cells, baby hamster kidney cells; MT-II, cyclic [Nle4,Asp5,D-Phe7, Lys10]alpha -MSH-(4-10).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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