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Down-regulation of Melanocortin Receptor Signaling Mediated by the Amino Terminus of Agouti Protein in XenopusMelanophores

1999; Elsevier BV; Volume: 274; Issue: 22 Linguagem: Inglês

10.1074/jbc.274.22.15837

ISSN

1083-351X

Autores

Michael M. Ollmann, Gregory S. Barsh,

Tópico(s)

melanin and skin pigmentation

Resumo

Agouti protein and Agouti-related protein (Agrp) regulate pigmentation and body weight, respectively, by antagonizing melanocortin receptor signaling. A carboxyl-terminal fragment of Agouti protein, Ser73-Cys131, is sufficient for melanocortin receptor antagonism, but Western blot analysis of skin extracts reveals that the electrophoretic mobility of native Agouti protein corresponds to the mature full-length form, His23-Cys131. To investigate the potential role of the amino-terminal residues, we compared the function of full-length and carboxyl-terminal fragments of Agrp and Agouti protein in a sensitive bioassay based on pigment dispersion in Xenopusmelanophores. We find that carboxyl-terminal Agouti protein, and all forms of Agrp tested, act solely by competitive antagonism of melanocortin action. However, full-length Agouti protein acts by an additional mechanism that is time- and temperature-dependent, depresses maximal levels of pigment dispersion, and is therefore likely to be mediated by receptor down-regulation. Apparent down-regulation is not observed for a mixture of amino-terminal and carboxyl-terminal fragments. We propose that the phenotypic effects of Agouti in vivo represent a bipartite mechanism: competitive antagonism of agonist binding by the carboxyl-terminal portion of Agouti protein and down-regulation of melanocortin receptor signaling by an unknown mechanism that requires residues in the amino terminus of the Agouti protein. Agouti protein and Agouti-related protein (Agrp) regulate pigmentation and body weight, respectively, by antagonizing melanocortin receptor signaling. A carboxyl-terminal fragment of Agouti protein, Ser73-Cys131, is sufficient for melanocortin receptor antagonism, but Western blot analysis of skin extracts reveals that the electrophoretic mobility of native Agouti protein corresponds to the mature full-length form, His23-Cys131. To investigate the potential role of the amino-terminal residues, we compared the function of full-length and carboxyl-terminal fragments of Agrp and Agouti protein in a sensitive bioassay based on pigment dispersion in Xenopusmelanophores. We find that carboxyl-terminal Agouti protein, and all forms of Agrp tested, act solely by competitive antagonism of melanocortin action. However, full-length Agouti protein acts by an additional mechanism that is time- and temperature-dependent, depresses maximal levels of pigment dispersion, and is therefore likely to be mediated by receptor down-regulation. Apparent down-regulation is not observed for a mixture of amino-terminal and carboxyl-terminal fragments. We propose that the phenotypic effects of Agouti in vivo represent a bipartite mechanism: competitive antagonism of agonist binding by the carboxyl-terminal portion of Agouti protein and down-regulation of melanocortin receptor signaling by an unknown mechanism that requires residues in the amino terminus of the Agouti protein. Studies of the mouse coat color Agouti gene have led to the identification of a novel pair of secreted signaling molecules which regulate mammalian pigmentation and body weight.Agouti encodes a 131-amino acid secreted protein that is expressed in the skin, where it induces the production of yellow pigment (pheomelanin) in hair follicle melanocytes (Refs. 1Miller M.W. Duhl D.M.J. Vrieling H. Cordes S.P. Ollmann M.M. Winkes B.M. Barsh G.S. Genes Dev. 1993; 7: 454-467Crossref PubMed Scopus (384) Google Scholar and 2Bultman S.J. Michaud E.J. Woychik R.P. Cell. 1992; 71: 1195-1204Abstract Full Text PDF PubMed Scopus (701) Google Scholar, and reviewed in Refs. 3Jackson I.J. Annu. Rev. Genet. 1994; 28: 189-217Crossref PubMed Google Scholar and 4Siracusa L.D. Trends Genet. 1994; 10: 423-428Abstract Full Text PDF PubMed Scopus (109) Google Scholar). Agouti protein is secreted but has a small sphere of action; its localized expression is thought to give rise to characteristic white or yellow markings found in many different mammals such as chinchillas and the Doberman breed of domestic dogs (Ref. 5Millar S.E. Miller M.W. Stevens M.E. Barsh G.S. Development. 1995; 121: 3223-3232PubMed Google Scholar, and reviewed in Ref. 6Searle A.G. Comparative Genetics of Coat Color in Mammals. Academic Press, New York1968Google Scholar). In the dominant mutationsAy and Avy or in transgenic mice, ectopic expression of transcripts encoding Agouti protein results in yellow hair, obesity, hyperinsulinemia, and increased body length (Refs. 7Klebig M.L. Wilkinson J.E. Geisler J.G. Woychik R.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4728-4732Crossref PubMed Scopus (244) Google Scholar, 8Duhl D.M.J. Vrieling H. Miller K.A. Wolff G.L. Barsh G.S. Nat. Genet. 1994; 8: 59-65Crossref PubMed Scopus (381) Google Scholar, 9Michaud E.J. Vanvugt M.J. Bultman S.J. Sweet H.O. Davisson M.T. Woychik R.P. Genes Dev. 1994; 8: 1463-1472Crossref PubMed Scopus (231) Google Scholar, and reviewed in Refs. 4Siracusa L.D. Trends Genet. 1994; 10: 423-428Abstract Full Text PDF PubMed Scopus (109) Google Scholar and 10Perry W.L. Copeland N.G. Jenkins N.A. Bioessays. 1994; 16: 705-707Crossref PubMed Scopus (47) Google Scholar). The nonpigmentary effects of ectopic Agouti expression likely reflect the normal function of Agouti-related protein (Agrp), 1The abbreviations used are: Agrp, Agouti-related protein; Mcr, melanocortin receptor; PAGE, polyacrylamide gel electrophoresis; α-MSH, α-melanocyte stimulating hormone; Mcr, melanocortin receptor; Bicine, N,N-bis(2-hydroxyethyl)glycine; PIPES, 1,4-piperazinediethanesulfonic acid; NDP, nucleoside diphosphate a protein expressed in the hypothalamus and adrenal gland that is similar to Agouti protein in size, sequence, and biochemical activity (11Ollmann M.M. Wilson B.D. Yang Y.K. Kerns J.A. Chen Y. Gantz I. Barsh G.S. Science. 1997; 278: 135-138Crossref PubMed Scopus (1547) Google Scholar, 12Shutter J.R. Graham M. Kinsey A.C. Scully S. Luthy R. Stark K.L. Genes Dev. 1997; 11: 593-602Crossref PubMed Scopus (556) Google Scholar). Ubiquitous expression of Agrp transcripts causes obesity and increased body length but does not alter pigmentation (11Ollmann M.M. Wilson B.D. Yang Y.K. Kerns J.A. Chen Y. Gantz I. Barsh G.S. Science. 1997; 278: 135-138Crossref PubMed Scopus (1547) Google Scholar, 13Graham M. Shutter J.R. Sarmiento U. Sarosi I. Stark K.L. Nat. Genet. 1997; 17: 273-274Crossref PubMed Scopus (323) Google Scholar). Agouti protein and Agrp act by antagonism of melanocortin receptors, a family of G-protein-coupled receptors responsive to endocrine peptides such as α-melanocyte stimulating hormone (α-MSH) and adrenocorticotrophic hormone (ACTH) (reviewed in Refs. 14Cone R.D. Lu D. Koppula S. Vage D.I. Klungland H. Boston B. Chen W. Orth D.N. Pouton C. Kesterson R.A. Recent Prog. Horm. Res. 1996; 51: 287-318PubMed Google Scholar and 15Eberle A.N. The Melanotropins. Chemistry, Physiology and Mechanism of Action. Karger, Basel1988Google Scholar). Genetic and biochemical studies indicate that Agouti protein alters pigmentation by antagonism of the melanocortin 1 receptor (Mc1r) expressed on melanocytes (16Robbins L.S. Nadeau J.H. Johnson K.R. Kelly M.A. Roselli-Rehfuss L. Baack E. Mountjoy K.G. Cone R.D. Cell. 1993; 72: 827-834Abstract Full Text PDF PubMed Scopus (775) Google Scholar, 17Lu D.S. Willard D. Patel I.R. Kadwell S. Overton L. Kost T. Luther M. Chen W.B. Woychik R.P. Wilkison W.O. Cone R.D. Nature. 1994; 371: 799-802Crossref PubMed Scopus (936) Google Scholar), whereas Agrp affects body weight and length by antagonism of the Mc4r and/or Mc3r expressed in the hypothalamus and other regions of the central nervous system (11Ollmann M.M. Wilson B.D. Yang Y.K. Kerns J.A. Chen Y. Gantz I. Barsh G.S. Science. 1997; 278: 135-138Crossref PubMed Scopus (1547) Google Scholar,18Roselli-Rehfuss L. Mountjoy K.G. Robbins L.S. Mortrud M.T. Low M.J. Tatro J.B. Entwistle M.L. Simerly R.B. Cone R.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8856-8860Crossref PubMed Scopus (671) Google Scholar, 19Huszar D. Lynch C.A. Fairchild-Huntress V. Dunmore J.H. Fang Q. Berkemeier L.R. Gu W. Kesterson R.A. Boston B.A. Cone R.D. Smith F.J. Campfield L.A. Burn P. Lee F. Cell. 1997; 88: 131-141Abstract Full Text Full Text PDF PubMed Scopus (2559) Google Scholar, 20Gantz I. Miwa H. Konda Y. Shimoto Y. Tashiro T. Watson S.J. DelValle J. Yamada T. J. Biol. Chem. 1993; 268: 15174-15179Abstract Full Text PDF PubMed Google Scholar, 21Gantz I. Konda Y. Tashiro T. Shimoto Y. Miwa H. Munzert G. Watson S.J. DelValle J. Yamada T. J. Biol. Chem. 1993; 268: 8246-8250Abstract Full Text PDF PubMed Google Scholar). Most evidence suggests Agouti protein and Agrp act as competitive antagonists of melanocortin receptors (17Lu D.S. Willard D. Patel I.R. Kadwell S. Overton L. Kost T. Luther M. Chen W.B. Woychik R.P. Wilkison W.O. Cone R.D. Nature. 1994; 371: 799-802Crossref PubMed Scopus (936) Google Scholar, 22Blanchard S.G. Harris C.O. Ittoop O.R.R. Nichols J.S. Parks D.J. Truesdale A.T. Wilkison W.O. Biochemistry. 1995; 34: 10406-10411Crossref PubMed Scopus (62) Google Scholar, 23Willard D.H. Bodnar W. Harris C. Kiefer L. Nichols J.S. Blanchard S. Hoffman C. Moyer M. Burkhart W. Weiel J. Luther M.A. Wilkison W.O. Rocque W.J. Biochemistry. 1995; 34: 12341-12346Crossref PubMed Scopus (105) Google Scholar), meaning that their effects are due solely to their ability to inhibit binding of melanocortin receptor agonists such as α-MSH. In agreement with this model, Agouti protein and α-MSH inhibit the binding of each other to the Mc1r (17Lu D.S. Willard D. Patel I.R. Kadwell S. Overton L. Kost T. Luther M. Chen W.B. Woychik R.P. Wilkison W.O. Cone R.D. Nature. 1994; 371: 799-802Crossref PubMed Scopus (936) Google Scholar, 24Yang Y.K. Ollmann M.M. Wilson B.D. Dickinson C. Yamada T. Barsh G.S. Gantz I. Mol. Endocrinol. 1997; 11: 274-280Crossref PubMed Scopus (122) Google Scholar, 25Ollmann M.M. Lamoreux M.L. Wilson B.D. Barsh G.S. Genes Dev. 1998; 12: 316-330Crossref PubMed Scopus (190) Google Scholar). Additional findings, however, have suggested that Agouti protein and possibly Agrp inhibit melanocortin receptor signaling by mechanisms besides simple competitive antagonism of α-MSH binding (26Sakai C. Ollmann M. Kobayashi T. Abdel-Malek Z. Muller J. Vieira W.D. Imokawa G. Barsh G.S. Hearing V.J. EMBO J. 1997; 16: 3544-3552Crossref PubMed Scopus (86) Google Scholar, 27Siegrist W. Willard D.H. Wilkison W.O. Eberle A.N. Biochem. Biophys. Res. Commun. 1996; 218: 171-175Crossref PubMed Scopus (39) Google Scholar, 28Siegrist W. Drozdz R. Cotti R. Willard D.H. Wilkison W.O. Eberle A.N. Recept. Signal Transduct. 1997; 17: 75-98Crossref Scopus (79) Google Scholar, 29Jones B.H. Kim J.H. Zemel M.B. Woychik R.P. Michaud E.J. Wilkison W.O. Moustaid N. Am. J. Physiol. 1996; 33: E192-E196Google Scholar, 30Kim J.H. Mynatt R.L. Moore J.W. Woychik R.P. Moustaid N. Zemel M.B. FASEB J. 1996; 10: 1646-1652Crossref PubMed Scopus (65) Google Scholar, 31Mynatt R.L. Miltenberger R.J. Klebig M.L. Zemel M.B. Wilkinson J.E. Wilkinson W.O. Woychik R.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 919-922Crossref PubMed Scopus (62) Google Scholar, 32Zemel M.B. Kim J.H. Woychik R.P. Michaud E.J. Kadwell S.H. Patel I.R. Wilkison W.O. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4733-4737Crossref PubMed Scopus (128) Google Scholar, 33Vage D.I. Lu D.S. Klungland H. Lien S. Adalsteinsson S. Cone R.D. Nat. Genet. 1997; 15: 311-315Crossref PubMed Scopus (183) Google Scholar). Using a sensitive bioassay based on α-MSH-induced pigment dispersion in Xenopus melanophores, we have previously shown that inhibition of melanocortin signaling by Agouti protein is increased significantly by preincubating melanophores in Agouti protein for several hours prior to the addition of α-MSH (25Ollmann M.M. Lamoreux M.L. Wilson B.D. Barsh G.S. Genes Dev. 1998; 12: 316-330Crossref PubMed Scopus (190) Google Scholar). This observation suggested that Agouti protein induces melanocortin receptor down-regulation in addition to its ability to inhibit α-MSH binding. Our previous studies utilized a full-length (His23-Cys131) recombinant form of Agouti protein generated in a baculovirus expression system (11Ollmann M.M. Wilson B.D. Yang Y.K. Kerns J.A. Chen Y. Gantz I. Barsh G.S. Science. 1997; 278: 135-138Crossref PubMed Scopus (1547) Google Scholar). Here we demonstrate that the related cysteine-rich carboxyl-terminal domains of Agouti protein and Agrp are sufficient for competitive antagonism inXenopus melanophores. However, the amino-terminal residues of Agouti protein are required for additional effects likely due to down-regulation of melanocortin receptor signaling. To investigate whether Agouti protein was proteolytically cleaved to smaller formsin vivo, extracts of skin were examined by Western blot analysis using antisera that detected epitopes in both the amino and carboxyl termini. We found little, if any, post-translational proteolysis of full-length (His23-Cys131) Agouti protein. These findings demonstrate that Agouti protein alters melanocortin signaling by two mechanisms mediated by distinct domains within the native protein. Production and characterization of recombinant mouse Agouti protein and Agrp using the baculovirus system has been previously described (11Ollmann M.M. Wilson B.D. Yang Y.K. Kerns J.A. Chen Y. Gantz I. Barsh G.S. Science. 1997; 278: 135-138Crossref PubMed Scopus (1547) Google Scholar, 25Ollmann M.M. Lamoreux M.L. Wilson B.D. Barsh G.S. Genes Dev. 1998; 12: 316-330Crossref PubMed Scopus (190) Google Scholar). In brief, cation followed by anion exchange chromatography resolves mouse Agouti protein to >99% purity as determined by analysis of silver-stained PAGE, mass spectroscopy, and amino-terminal sequencing. Blue-Sepharose (Amersham Pharmacia Biotech) followed by anion exchange chromatography resolves multiple forms of Agrp into two pools. One pool contains form A, a single species processed by signal peptidase cleavage after residue 20, in an approximately equimolar ratio with form B, a mixture of three species processed by cleavage after residues 46, 48, or 50, present in a 15:1:4 ratio, respectively. The second pool contains form C, a mixture of two species processed by cleavage after residues 69 or 71, present in a 1:4 ratio, respectively. Relative purity of Agrp preparations was estimated by scanning densitometry of a 10-μg sample loaded on a 10% Tricine gel stained with ProBlue (Integrated Separation Systems, MA) and was used to calculate 60% purity for forms A+B and 20% purity for form C. Polyclonal antibodies to full-length (His23-Cys131) mouse Agouti were generated in rabbits by standard procedures (37Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley, New York1998Google Scholar); 100–200 μg of protein at approximately 90% purity was used for each of five injections; antisera were recovered at day 70. Following SDS-PAGE carried out under nonreducing conditions and Western blotting, the antiserum detects 1 ng/lane of the immunogen against which it was raised, as well as 1 ng/lane carboxyl-terminal (Ser73-Cys131) Agouti protein. Disulfide bond reduction of the antigen prior to SDS-PAGE slightly reduces sensitivity of the antiserum for detection of the full-length protein ( 99% purity. Active fractions were pooled, dialyzed into 20 mmPIPES, pH 6.8, 50 mm NaCl, flash frozen, and stored at −70 °C. The data shown utilized a preparation of carboxyl-terminal Agouti protein that was made by cleaving a full-length hemagglutinin-tagged Agouti protein (25Ollmann M.M. Lamoreux M.L. Wilson B.D. Barsh G.S. Genes Dev. 1998; 12: 316-330Crossref PubMed Scopus (190) Google Scholar) (the hemagglutinin tag was inserted in the amino terminus of the Agouti protein and is therefore not present in the Ser73-Cys131 fragment). Identical results were observed using a preparation of carboxyl-terminal Agouti protein made from recombinant Agouti protein His23-Cys131. To test the effects of the amino terminus in trans, a mixture of His23-Arg70 or Arg72 and Ser73-Cys131 was made by digesting recombinant mouse Agouti protein (99% purity) with Kex-2 as above. SDS-PAGE, mass spectroscopy, and amino-terminal sequencing revealed the presence of equimolar amounts of His23-Arg70 or Arg72 and Ser73-Cys131, along with a minor amount (<2% of total protein) of uncut full-length Agouti protein. Xenopus melanophores were grown at 27 °C in 50% L-15 medium (Specialty Media, Lavallete, NJ), supplemented with 20% heat-inactivated fetal calf serum, 1 mml-glutamine, penicillin, and streptomycin; the medium had been previously conditioned using Xenopus fibroblasts as described by Potenza and Lerner (48Potenza M.N. Lerner M.R. Pigment Cell. Res. 1992; 5: 372-378Crossref PubMed Scopus (75) Google Scholar). The pigment dispersion assay developed by Potenza and Lerner (48Potenza M.N. Lerner M.R. Pigment Cell. Res. 1992; 5: 372-378Crossref PubMed Scopus (75) Google Scholar) is based on the ability of agents that cause a decrease or increase in intracellular cAMP levels to produce a dose-dependent aggregation or dispersion, respectively, of intracellular pigment granules. Because pigment granules are neither fully aggregated nor dispersed in the absence of any drug, pretreatment of the cells with melatonin to aggregate pigment granules increases the range and sensitivity of the assay for detecting agents such as α-MSH that disperse pigment granules. For a typical assay, cells were plated 24–48 h beforehand in 96-well plates at 25,000 cells/well, washed briefly with 250 μl/well assay buffer (70% L-15 medium; 0.1% bovine serum albumin), and 40 μl/well assay buffer was then added, followed by 40 μl/well assay buffer that contained 2 nm melatonin (Sigma) to provide a final melatonin concentration of 1 nm. After a 45-min incubation to aggregate pigment granules, the optical density of each well was measured at 650 nm (ODinitial) to provide a base-line optical density reading. Test samples (Agouti protein, Agrp, or control buffer) were then added at 40 μl/well, followed by the addition of various concentrations of α-MSH or NDP-MSH at 40 μl/well. All additions were made in assay buffer supplemented with 1 nmmelatonin to maintain a constant concentration of melatonin during the assay. Optical density at 650 nm was then determined at multiple time points from 30 to 420 min (ODfinal). Unless stated otherwise, the entire assay was carried out at 22 °C in triplicate, and assay points represent the mean ± S.E. of the mean. A unitless parameter, degree of pigment dispersion, was calculated as described by Potenza and Lerner (48Potenza M.N. Lerner M.R. Pigment Cell. Res. 1992; 5: 372-378Crossref PubMed Scopus (75) Google Scholar), (ODfinal − ODinitial)/ODfinal, which creates an internal standard for each well (ODinitial) and scales the maximal degree of pigment dispersion to 1. The effects of 1 mmmelatonin occasionally increase during the course of the assay, which gives rise to negative values for the degree of pigment dispersion. Optical density at 650 nm melanophores was measured with aV max kinetic microplate reader (Molecular Devices, Menlo Park, CA) in end point mode, and data were transferred electronically to a Microsoft Excel spreadsheet for analysis. Graphing and curve fitting of dose-response curves were carried out with DeltaGraph (DeltaPoint, Monterey, CA), using a four-parameter logistic equation, y = a + ((b −a)/(1 + (10 c/10 x) d)); where a = minimum, b = maximum,c = half-maximal value, and d = slope. Dose ratios (the amount of α-MSH required for half-maximal response in the presence of Agouti protein/amount of α-MSH required for half-maximal response in the absence of Agouti protein) were determined using the four-parameter logistic equation described above. Within each experiment, individual slopes of the α-MSH dose-response curves were not significantly different and therefore were fixed at the average slope to provide the data for dose-ratio calculations. Schild plots [log(dose ratio − 1)] versuslog([Ser73-Cys131Agouti or Agrp]) were created and analyzed in DeltaGraph (DeltaPoint, Monterey, CA). Linear curve fitting was used to determine the slope and the correlation coefficient. To estimate KB, a second linear curve fitting was performed with the slope fixed at 1. The similar genomic structure of Agouti andAgrp suggests evolution from a common ancestral gene, yet the sequence similarity of the two proteins is confined entirely to the carboxyl-terminal region (Fig. 1). Twenty-one of the final forty-seven residues in Agouti protein and Agrp are identical, of which ten residues are cysteines in a spacing similar to that found in conotoxins and plectoxins from the venoms of cone snails (34Olivera B.M. Rivier J. Scott J.K. Hillyard D.R. Cruz L.J. J. Biol. Chem. 1991; 266: 22067-22070Abstract Full Text PDF PubMed Google Scholar) and spiders (35Quistad G.B. Skinner W.S. J. Biol. Chem. 1994; 269: 11098-11101Abstract Full Text PDF PubMed Google Scholar), respectively. In addition, the amino terminus of the Agouti protein contains several paired basic residues that could serve as potential proteolytic cleavage sites (Fig.1 A). These observations suggested that the Agouti protein might be processed in vivo into an active fragment spanning the cysteine-rich region. In support of this hypothesis, proteolytic fragments containing the carboxyl terminus of either protein retain α-MSH-inhibitory activity (11Ollmann M.M. Wilson B.D. Yang Y.K. Kerns J.A. Chen Y. Gantz I. Barsh G.S. Science. 1997; 278: 135-138Crossref PubMed Scopus (1547) Google Scholar, 23Willard D.H. Bodnar W. Harris C. Kiefer L. Nichols J.S. Blanchard S. Hoffman C. Moyer M. Burkhart W. Weiel J. Luther M.A. Wilkison W.O. Rocque W.J. Biochemistry. 1995; 34: 12341-12346Crossref PubMed Scopus (105) Google Scholar), and a short deletion (Arg64-Lys77) in the amino-terminal half of the mouse Agouti protein does not disrupt activity (36Perry W.L. Nakamura T. Swing D.A. Secrest L. Eagleson B. Hustad C.M. Copeland N.G. Jenkins N.A. Genetics. 1996; 144: 255-264Crossref PubMed Google Scholar). Recombinant mouse Agouti protein produced in insect cells is secreted as a mature form with the signal sequence removed (His23-Cys131) (17Lu D.S. Willard D. Patel I.R. Kadwell S. Overton L. Kost T. Luther M. Chen W.B. Woychik R.P. Wilkison W.O. Cone R.D. Nature. 1994; 371: 799-802Crossref PubMed Scopus (936) Google Scholar, 23Willard D.H. Bodnar W. Harris C. Kiefer L. Nichols J.S. Blanchard S. Hoffman C. Moyer M. Burkhart W. Weiel J. Luther M.A. Wilkison W.O. Rocque W.J. Biochemistry. 1995; 34: 12341-12346Crossref PubMed Scopus (105) Google Scholar, 25Ollmann M.M. Lamoreux M.L. Wilson B.D. Barsh G.S. Genes Dev. 1998; 12: 316-330Crossref PubMed Scopus (190) Google Scholar). However, recombinant proteins secreted by insect cells may undergo altered or incomplete post-translational processing (41Wang W. Yum L. Beinfeld M.C. Peptides (Elmsford). 1997; 18: 1295-1299Crossref PubMed Scopus (12) Google Scholar, 42Misrahi M. Ghinea N. Sar S. Saunier B. Jolivet A. Loosfelt H. Cerutti M. Devauchelle G. Milgrom E. Eur. J. Biochem. 1994; 222: 711-719Crossref PubMed Scopus (109) Google Scholar). To investigate whether the active form of Agouti protein in mouse skin underwent proteolytic cleavage in vivo, we raised polyclonal antibodies against full-length recombinant mouse Agouti protein (His23-Cys131). Western blotting experiments determined that the antiserum detects epitopes in both the amino- and carboxyl-terminal regions of Agouti protein with a sensitivity of 99% purity (determined by amino-terminal sequencing, SDS-PAGE, and mass spectrometry) from both the amino-terminal fragment and a small amount of uncleaved full-length protein. Like full-length Agouti protein, the carboxyl-terminal fragment inhibits α-MSH-induced pigment dispersion in Xenopusmelanophores but has no effect in the absence of α-MSH or other melanocortin peptides (data not shown). By analyzing the effect of the two Agouti peptides on melanocortin receptor signaling over time, however, we discovered a difference in their activities. Addition of α-MSH to melanophores causes a rapid increase in pigment dispersion that reaches equilibrium in 15–30 min. When full-length Agouti protein is added simultaneously or immediately prior to α-MSH, a gradual inhibition of pigment dispersion ensues that increases for several hours (Fig. 3 A). By contrast, under the same conditions, the carboxyl-

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