Agouti and Agouti-related Protein: Analogies and Contrasts
2000; Elsevier BV; Volume: 275; Issue: 10 Linguagem: Inglês
10.1074/jbc.275.10.6695
ISSN1083-351X
AutoresDaniela M. Dinulescu, Roger D. Cone,
Tópico(s)Polysaccharides Composition and Applications
ResumoAgouti-related protein α-melanocyte-stimulating hormone adrenocorticotropic hormone melanocortin receptor 1 Agouti-signaling protein lethal yellow mahogany mahoganoid The discovery a decade ago of the murineagouti gene was intended to bring scientists a step closer to understanding the complexities of mammalian pigmentation. The first obesity gene was also uncovered in the process. What followed was an explosion of major discoveries in murine as well as human obesity and diabetes research. Recently, a new gene, the agouti-related protein(AGRP),1 was discovered and found to share a striking similarity in structure and function with agouti, although their patterns of distribution are completely different. Identification of a hypothalamic melanocortin receptor, MC4-R, together with AGRP as central components of feeding behavior and metabolism has helped build a picture, albeit incomplete, of the neuronal pathways involved in energy homeostasis. This review will compare and contrast Agouti and AGRP structure and function and gene regulation and their interaction with melanocortin receptors (MC1-R and MC4-R) and suppressors (mahogany/mahoganoid). agouti and extension were first described several decades ago (1.Silvers W.K. Russell E.S. J. Exp. Zool. 1955; 130: 199-220Crossref Scopus (70) Google Scholar, 2.Silvers W.K. Tne Coat Colors of Mice : a Model for Mammalian Gene Action and Interaction. Springer-Verlag, New York1979: 6-44Google Scholar) as the genetic loci that control the relative amount and distribution of eumelanin (brown/black) and phaeomelanin (red/yellow) pigments in the mammalian coat.extension encodes a member (MC1-R) (3.Robbins 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 (779) Google Scholar) of the melanocortin receptors, a family of Gs-coupled receptors, of which five isoforms are presently known (reviewed in Ref. 4.Cone R.D. The Melanocortin Receptors. Humana Press, Totowa, NJ2000Crossref Google Scholar). MC1-R is the melanocyte-stimulating hormone receptor expressed in melanocytes and has a physiological role in pigmentation (5.Mountjoy K.G. Robbins L.S. Mortrud M.T. Cone R.D. Science. 1992; 257: 1248-1251Crossref PubMed Scopus (1461) Google Scholar). MC4-R is expressed mainly in the brain (6.Gantz 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, 7.Mountjoy K.G. Mortrud M.T. Low M.J. Simerly R.B. Cone R.D. Mol. Endocrinol. 1994; 8: 1298-1308Crossref PubMed Scopus (1043) Google Scholar) and has been implicated in the regulation of feeding behavior and metabolism (8.Huszar 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 (2575) Google Scholar). MC3-R is found primarily in the hypothalamic and limbic systems (9.Gantz 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); however, no definitive function has been assigned to MC3-R as yet. Melanocortins such as α-melanocyte-stimulating hormone (α-MSH) and adrenocorticotropic hormone (ACTH) are the natural ligands for this family of receptors (α-MSH for MC1-, MC3-, MC4-, and MC5-R and ACTH for MC2-R). Melanocortins are cleaved from a larger polypeptidic precursor termed pro-opiomelanocortin that is produced in the pituitary gland, hypothalamus, brainstem, and peripheral sites such as skin. Agouti is a paracrine-signaling factor that is secreted by dermal papillae cells, adjacent to melanocytes, and acts within the hair follicle microenvironment to block melanocortin action at the MC1-R (10.Lu D. Willard D. Patel I.R. Kadwell S. Overton L. Kost T. Luther M. Chen W. Woychik R.P. Wilkison W.O. Cone R.D. Nature. 1994; 371: 799-802Crossref PubMed Scopus (939) Google Scholar, 11.Bultman S.J. Michaud E.J. Woychik R.P. Cell. 1992; 71: 1195-1204Abstract Full Text PDF PubMed Scopus (704) Google Scholar). Binding of α-MSH to the receptor triggers elevation of cAMP levels and activation of tyrosinase, the rate-limiting enzyme of melanogenesis, and results in eumelanin production. In the presence of Agouti the opposite is true; eumelanin synthesis is shut down and the default pathway that has phaeomelanin as the final product is activated. There are now more than 20 dominant, recessive, andpseudoagouti alleles that have been identified in rodent, fox, and cattle, with interesting functional variations from species to species. In rodents agouti is expressed in skin only. In humans, however, agouti has a wider pattern of distribution, being expressed in adipose tissue, testis, ovary, heart, and at lower levels in foreskin, kidney, and liver (12.Kwon H.Y. Bultman S.J. Loffler C. Chen W.J. Furdon P.J. Powell J.G. Usala A.L. Wilkison W. Hansmann I. Woychik R.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9760-9764Crossref PubMed Scopus (149) Google Scholar, 13.Wilson B.D. Ollmann M.M. Kang L. Stoffel M. Bell G.I. Barsh G.S. Hum. Mol. Genet. 1995; 4: 223-230Crossref PubMed Scopus (161) Google Scholar). Agouti does not appear to play any role in human pigmentation, and its exact biological function in humans remains unknown. Most of our knowledge of Agouti structure and function comes from studies performed on rodents. This review will therefore focus on murine Agouti and AGRP. The genomic organization of the murine agouti gene is complex. It consists of three coding exons designated as 2, 3, and 4, as well as four non-coding exons, 1A, A′, B, and C, located upstream (11.Bultman S.J. Michaud E.J. Woychik R.P. Cell. 1992; 71: 1195-1204Abstract Full Text PDF PubMed Scopus (704) Google Scholar, 14.Vrieling H. Duhl D.M.J. Millar S.E. Miller K.A. Barsh G.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5667-5671Crossref PubMed Scopus (160) Google Scholar). agouti expression is regulated in mice in a regional and temporal manner to create a differential distribution of yellow and black pigment in individual hair shafts and throughout the coat (11.Bultman S.J. Michaud E.J. Woychik R.P. Cell. 1992; 71: 1195-1204Abstract Full Text PDF PubMed Scopus (704) Google Scholar). This intricate pattern of gene regulation is achieved through the existence of alternatively spliced agoutitranscripts that differ in their 5′-untranslated exons and are controlled by two different sets of control elements. Type I transcripts contain non-coding exons 1B or 1C and are regulated by temporal (hair cycle-specific) elements (14.Vrieling H. Duhl D.M.J. Millar S.E. Miller K.A. Barsh G.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5667-5671Crossref PubMed Scopus (160) Google Scholar). Gene expression of transcript I is restricted to the midphase of the hair-growing cycle and is associated phenotypically with subapical yellow banded hairs throughout the body (14.Vrieling H. Duhl D.M.J. Millar S.E. Miller K.A. Barsh G.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5667-5671Crossref PubMed Scopus (160) Google Scholar). In contrast, type II transcripts contain non-coding exons 1A and 1A′ and are under the control of regional (ventral-specific) promoter elements (14.Vrieling H. Duhl D.M.J. Millar S.E. Miller K.A. Barsh G.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5667-5671Crossref PubMed Scopus (160) Google Scholar). The second class of transcripts is responsible phenotypically for the lighter ventral pigmentation seen in several agouti strains (i.e. white-bellied agouti) (14.Vrieling H. Duhl D.M.J. Millar S.E. Miller K.A. Barsh G.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5667-5671Crossref PubMed Scopus (160) Google Scholar). Despite its genetic complexity the agouti locus encodes a small protein of 131 amino acids. Agouti displays the structural characteristics of a secreted protein having a hydrophobic signal sequence and lacking any transmembrane domains. The prominent structural features of the mature protein are a highly basic N-terminal region, a Pro-rich central domain, and a C-terminal domain rich in Cys residues (11.Bultman S.J. Michaud E.J. Woychik R.P. Cell. 1992; 71: 1195-1204Abstract Full Text PDF PubMed Scopus (704) Google Scholar). Biochemical analysis of the Agouti protein shows that it is highly glycosylated and very stable to thermal denaturation (15.Willard D.H. Bodnar W. Harris C. Nichols J. Kiefer L.L. Hoffman C. Moyer M. Burkhart W. Weiel J. Wilkison W.O. Rocque W. Biochemistry. 1995; 34: 12341-12346Crossref PubMed Scopus (106) Google Scholar). The spacing pattern of the 10 Cys residues present in the C terminus is reminiscent of cone snail (conotoxins) and spider toxins (plectoxins), suggesting a conserved three-dimensional motif. Based on this structural similarity it has been postulated that all Cys residues of the Agouti protein are engaged in disulfide bonds (15.Willard D.H. Bodnar W. Harris C. Nichols J. Kiefer L.L. Hoffman C. Moyer M. Burkhart W. Weiel J. Wilkison W.O. Rocque W. Biochemistry. 1995; 34: 12341-12346Crossref PubMed Scopus (106) Google Scholar). In vitro studies using recombinant mouse Agouti protein prove that Agouti is a potent melanocortin antagonist (nanomolar range) at MC-R subtypes 1 (KI (app) = 2.6 ± 0.8 nm) and 4 (KI (app) = 54 ± 18 nm), a relatively weak antagonist at MC3-R (KI (app) = 190 ± 74 nm), and a very weak antagonist at MC5-R (KI (app) = 12,000 ± 340 nm) (10.Lu D. Willard D. Patel I.R. Kadwell S. Overton L. Kost T. Luther M. Chen W. Woychik R.P. Wilkison W.O. Cone R.D. Nature. 1994; 371: 799-802Crossref PubMed Scopus (939) Google Scholar, 15.Willard D.H. Bodnar W. Harris C. Nichols J. Kiefer L.L. Hoffman C. Moyer M. Burkhart W. Weiel J. Wilkison W.O. Rocque W. Biochemistry. 1995; 34: 12341-12346Crossref PubMed Scopus (106) Google Scholar, 16.Kiefer L.L. Veal J.M. Mountjoy K.G. Wilkison W.O. Biochemistry. 1998; 37: 991-997Crossref PubMed Scopus (67) Google Scholar). Pharmacological studies of murine Agouti conclude that its mechanism of action is a classical competitive antagonism of melanocortin receptors (10.Lu D. Willard D. Patel I.R. Kadwell S. Overton L. Kost T. Luther M. Chen W. Woychik R.P. Wilkison W.O. Cone R.D. Nature. 1994; 371: 799-802Crossref PubMed Scopus (939) Google Scholar, 15.Willard D.H. Bodnar W. Harris C. Nichols J. Kiefer L.L. Hoffman C. Moyer M. Burkhart W. Weiel J. Wilkison W.O. Rocque W. Biochemistry. 1995; 34: 12341-12346Crossref PubMed Scopus (106) Google Scholar, 16.Kiefer L.L. Veal J.M. Mountjoy K.G. Wilkison W.O. Biochemistry. 1998; 37: 991-997Crossref PubMed Scopus (67) Google Scholar). In addition, a shorter version of Agouti, residues 83–131, is shown to be as potent an antagonist as the full-length protein (15.Willard D.H. Bodnar W. Harris C. Nichols J. Kiefer L.L. Hoffman C. Moyer M. Burkhart W. Weiel J. Wilkison W.O. Rocque W. Biochemistry. 1995; 34: 12341-12346Crossref PubMed Scopus (106) Google Scholar). The Cys-rich C terminus is therefore deemed sufficient for effective antagonism of melanocortin action in vitro as well as in vivo (15.Willard D.H. Bodnar W. Harris C. Nichols J. Kiefer L.L. Hoffman C. Moyer M. Burkhart W. Weiel J. Wilkison W.O. Rocque W. Biochemistry. 1995; 34: 12341-12346Crossref PubMed Scopus (106) Google Scholar, 17.Perry 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). The basic domain, on the other hand, appears to play key roles in Agouti biogenesis (i.e. protein folding, post-translational processing, sorting, and secretion) and/or in facilitating the interaction with the receptor (18.Miltenberger R.J. Mynatt R.L. Bruce B.D. Wilkison W.O. Woychik R.P. Michaud E.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8579-8584Crossref PubMed Scopus (17) Google Scholar). The mechanism of Agouti action shows interesting variations across species. Functional analysis of recombinant Agouti-signaling protein (ASIP), the human homologue of murine Agouti, indicates a similar pharmacological profile; ASIP is a potent antagonist at human MC1 (KI (app) = 0.47 ± 0.06 nm) and MC4-R (KI (app) = 0.14 ± 0.02 nm) and a relatively weak antagonist at MC3 (KI (app) = 6.4 ± 1.1 nm) and MC5-R (KI (app) = 1.16 ± 0.17 nm) (19.Yang 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). Competitive antagonism of ASIP is apparent, however, only toward MC1-R (19.Yang 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). By contrast, genetic analysis of fox agouti and extension variants suggests that Agouti functions in this case as a negative antagonist (inverse agonist) of MC1-R rather than a classical competitive antagonist (20.Vage D.I. Lu D. Klungland H. Lien S. Adalsteinsson S. Cone R.D. Nat. Genet. 1997; 15: 311-315Crossref PubMed Scopus (184) Google Scholar). Unlike the fox, in the mouse extension is epistatic to agouti, which means that constitutively active receptors encoded by dominant extension alleles cannot be blocked by Agouti action. Once the agouti gene was cloned it became possible to address the molecular basis of A y, a dominant allele at the agouti locus, and its pleiotropic effects. In doing so scientists were able to reveal a much more complex picture of Agouti function than previously thought. It soon became clear that Agouti and its homologues are part of a general signaling system that extends far beyond the hair follicle and the melanogenesis process. Lethal yellow (A y) was identified at the turn of the century (21.Danforth C.H. J. Hered. 1927; 18: 153-162Crossref Scopus (19) Google Scholar). Animals heterozygous for the A y allele are not only characterized by a yellow coat color but also by late onset obesity associated with hyperphagia, increased linear growth, and non-insulin-dependent diabetes as well as an increased propensity for developing tumors (reviewed in Ref. 22.Yen T.T. Gill A.M. Frigeri L.G. Barsh G.S. Wolff G.L. FASEB J. 1994; 8: 479-488Crossref PubMed Scopus (278) Google Scholar). Genetic analysis shows that A y is in fact the result of a chromosomal rearrangement in which the promoter and the first non-coding exon of a closely linked gene, Raly, get spliced to exons of the wild-type agouti gene (23.Michaud E.J. Bultman S.J. Klebig M.L. van Vugt M.J. Stubbs L.J. Russell L.B. Woychik R.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2562-2566Crossref PubMed Scopus (167) Google Scholar). Theagouti gene, now under the control of the relaxed Raly promoter and devoid of temporal and regional restrictions, becomes ectopically expressed. Overexpression of Agouti in multiple tissues is therefore the cause of the A y phenotype. This conclusion is further supported by the ability of agouti, under the control of a β-actin promoter, to recapitulate theA y phenotype in transgenic animals (24.Klebig M.L. Wilkinson J.E. Geisler J.G. Woychik R.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4728-4732Crossref PubMed Scopus (245) Google Scholar). Pharmacological characterization of murine Agouti makes it now easy to understand why ectopic Agouti expression per se is responsible for the A y phenotype. Chronic antagonism of the cutaneous MC1-R by Agouti results in yellow fur whereas Agouti competition at the hypothalamic MC4-R results in obesity. This conclusion is supported by most of the experimental data available to date. The most compelling evidence are the recent findings that targeted disruption of MC4-R signaling results in knockout mice (MC4-R KO) with a phenotype similar to the A y syndrome (8.Huszar 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 (2575) Google Scholar) and central administration of MC4-R agonists and antagonists stimulate and inhibit feeding behavior, respectively (25.Fan W. Boston B.A. Kesterson R.A. Hruby V.J. Cone R.D. Nature. 1997; 385: 165-168Crossref PubMed Scopus (1670) Google Scholar). Several studies, however, have proposed an alternative mechanism for Agouti action that does not involve melanocortin receptor antagonism (reviewed in Ref.26.Zemel M.B. Mol. Cell. Biochem. 1998; 188: 129-136Crossref PubMed Scopus (174) Google Scholar). On the basis of structural similarities between Agouti and invertebrate neurotoxins, which are known to block calcium channels, it has been speculated that Agouti might in fact regulate lipogenesis and insulin release via a calcium-mediated mechanism (26.Zemel M.B. Mol. Cell. Biochem. 1998; 188: 129-136Crossref PubMed Scopus (174) Google Scholar). In support of this model, murine and human Agouti proteins were shown to cause dose-dependent increases in calcium influx in both adipocytes and pancreatic β-cells (26.Zemel M.B. Mol. Cell. Biochem. 1998; 188: 129-136Crossref PubMed Scopus (174) Google Scholar). Therefore, it may seem that Agouti, like invertebrate toxins, is able to modulate intracellular calcium levels by directly regulating calcium channels. There are striking differences, however, between their mechanisms of action and potency and kinetics. Conotoxins, for example, are rapid and irreversible calcium channel inactivators (27.Olivera 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), whereas Agouti is a slow acting, transient activator of the same channels (26.Zemel M.B. Mol. Cell. Biochem. 1998; 188: 129-136Crossref PubMed Scopus (174) Google Scholar). Furthermore, the increase in lipogenesis that is seen upon transfection of Agouti protein in adipocytes can be elicited in the absence of Agouti through activation of calcium channels (26.Zemel M.B. Mol. Cell. Biochem. 1998; 188: 129-136Crossref PubMed Scopus (174) Google Scholar). This suggests that modulation of intracellular calcium levels may simply be secondary to Agouti action. According to the "calcium hypothesis" development ofagouti-induced obesity is elicited by a combination of factors that include Agouti expression in adipose tissue and hyperinsulinemia (26.Zemel M.B. Mol. Cell. Biochem. 1998; 188: 129-136Crossref PubMed Scopus (174) Google Scholar). This conclusion is, however, inconsistent with the finding that the MC4-R KO phenotype is more profound than theA y syndrome suggesting that disruption of MC4-R signaling is necessary and sufficient for triggering all aspects ofagouti-induced obesity. Furthermore, a recent study indicates that Agouti binds directly to MC1-R and that a functional MC1-R is required for Agouti signaling (28.Ollmann M.M. Lamoreux M.L. Wilson B.D. Barsh G.S. Genes Dev. 1998; 12: 316-330Crossref PubMed Scopus (190) Google Scholar). In light of these new findings direct regulation of calcium channels by Agouti seems less likely. It is still possible, however, that Agouti binding to MC1-R results in modulation of calcium metabolism via an indirect mechanism. Two lines of evidence suggested the existence in the brain of an "Agouti-like" protein that would block signaling at central melanocortin receptors, MC3-R and MC4-R. First, in vitropharmacology studies found that Agouti was a highly specific MC4-R antagonist even though it was normally expressed only in hair follicles (10.Lu D. Willard D. Patel I.R. Kadwell S. Overton L. Kost T. Luther M. Chen W. Woychik R.P. Wilkison W.O. Cone R.D. Nature. 1994; 371: 799-802Crossref PubMed Scopus (939) Google Scholar, 15.Willard D.H. Bodnar W. Harris C. Nichols J. Kiefer L.L. Hoffman C. Moyer M. Burkhart W. Weiel J. Wilkison W.O. Rocque W. Biochemistry. 1995; 34: 12341-12346Crossref PubMed Scopus (106) Google Scholar, 16.Kiefer L.L. Veal J.M. Mountjoy K.G. Wilkison W.O. Biochemistry. 1998; 37: 991-997Crossref PubMed Scopus (67) Google Scholar). Second, central administration of synthetic MC3-R and MC4-R antagonists uncovered a functional role for melanocortin antagonists in vivo, namely stimulation of feeding behavior (25.Fan W. Boston B.A. Kesterson R.A. Hruby V.J. Cone R.D. Nature. 1997; 385: 165-168Crossref PubMed Scopus (1670) Google Scholar). The agouti-related protein (AGRP) gene, was isolated in 1997 based on its homology to Agouti (29.Shutter J.R. Graham M. Kinsey A.C. Scully S. Luthy R. Stark K. Genes Dev. 1997; 11: 593-602Crossref PubMed Scopus (560) Google Scholar, 30.Ollmann 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 (1558) Google Scholar). Likeagouti, AGRP contains three coding exons; however, depending on the site of expression (central versusperipheral) AGRP may or may not contain an upstream non-coding exon (29.Shutter J.R. Graham M. Kinsey A.C. Scully S. Luthy R. Stark K. Genes Dev. 1997; 11: 593-602Crossref PubMed Scopus (560) Google Scholar). The genomic organization of the coding exons is similar between agouti and AGRP despite differences in intron/exon junctions (29.Shutter J.R. Graham M. Kinsey A.C. Scully S. Luthy R. Stark K. Genes Dev. 1997; 11: 593-602Crossref PubMed Scopus (560) Google Scholar). Both Agouti and AGRP are 131-amino acid proteins with putative signal peptide sequences and Cys-rich C-terminal domains (Fig.1). Unlike Agouti, AGRP lacks the large number of basic residues in the N-terminal region and the Pro-rich central domain (29.Shutter J.R. Graham M. Kinsey A.C. Scully S. Luthy R. Stark K. Genes Dev. 1997; 11: 593-602Crossref PubMed Scopus (560) Google Scholar, 30.Ollmann 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 (1558) Google Scholar). The strongest homology between the two proteins is within the poly-Cys domain of the C terminus (Fig.2). Both Agouti and AGRP contain 10 Cys residues, 9 of which are spatially conserved (29.Shutter J.R. Graham M. Kinsey A.C. Scully S. Luthy R. Stark K. Genes Dev. 1997; 11: 593-602Crossref PubMed Scopus (560) Google Scholar, 30.Ollmann 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 (1558) Google Scholar). Like Agouti, all 10 of the AGRP Cys residues form disulfide bridges (15.Willard D.H. Bodnar W. Harris C. Nichols J. Kiefer L.L. Hoffman C. Moyer M. Burkhart W. Weiel J. Wilkison W.O. Rocque W. Biochemistry. 1995; 34: 12341-12346Crossref PubMed Scopus (106) Google Scholar, 31.Bures E.J. Hui J.O. Young Y. Chow D.T. Katta V. Rohde M.F. Zeni L. Rosenfeld R.D. Stark K.L. Haniu M. Biochemistry. 1998; 37: 12172-12177Crossref PubMed Scopus (72) Google Scholar) that are essential for their structural stability and biological function. Biochemical studies indicate also that AGRP is very stable to thermal denaturation (similar to Agouti) as well as acid degradation (32.Rosenfeld R.D. Zeni L. Welcher A.A. Narhi L.O. Hale C. Marasco J. Delaney J. Gleason T. Philo J.S. Katta V. Hui J. Baumgartner J. Graham M. Stark K.L. Karbon W. Biochemistry. 1998; 37: 16041-16052Crossref PubMed Scopus (50) Google Scholar). In addition, the biophysical characterization of Agouti and AGRP shows similar CD spectra for the two proteins, their secondary structure consisting of mainly random coils and β-sheets (15.Willard D.H. Bodnar W. Harris C. Nichols J. Kiefer L.L. Hoffman C. Moyer M. Burkhart W. Weiel J. Wilkison W.O. Rocque W. Biochemistry. 1995; 34: 12341-12346Crossref PubMed Scopus (106) Google Scholar, 32.Rosenfeld R.D. Zeni L. Welcher A.A. Narhi L.O. Hale C. Marasco J. Delaney J. Gleason T. Philo J.S. Katta V. Hui J. Baumgartner J. Graham M. Stark K.L. Karbon W. Biochemistry. 1998; 37: 16041-16052Crossref PubMed Scopus (50) Google Scholar).Figure 2Disulfide arrangement of murine Agouti, AGRP , and conotoxin GVIA (Conus glorimaris).Cys residues are depicted in red. The Cys-spacing pattern and disulfide connectivity of both Agouti and AGRP strongly resemble conotoxins.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Both Agouti and AGRP are competitive antagonists of α-MSH action at melanocortin receptors (32.Rosenfeld R.D. Zeni L. Welcher A.A. Narhi L.O. Hale C. Marasco J. Delaney J. Gleason T. Philo J.S. Katta V. Hui J. Baumgartner J. Graham M. Stark K.L. Karbon W. Biochemistry. 1998; 37: 16041-16052Crossref PubMed Scopus (50) Google Scholar, 33.Fong T.M. Mao C. MacNeil T. Kalyani R. Smith T. Weinberg D. Tota M.R. Van der Ploeg L.H.T. Biochem. Biophys. Res. Commun. 1997; 237: 629-631Crossref PubMed Scopus (199) Google Scholar, 34.Yang Y.-K. Thompson D.A. Dickinson C.J. Wilken J. Barsh G.S. Kent S.B.H. Gantz I. Mol. Endocrinol. 1999; 13: 148-155Crossref PubMed Scopus (162) Google Scholar). Likewise, the C terminus of AGRP, residues 83–131, retains the biological activity of the full-length protein in vitro (34.Yang Y.-K. Thompson D.A. Dickinson C.J. Wilken J. Barsh G.S. Kent S.B.H. Gantz I. Mol. Endocrinol. 1999; 13: 148-155Crossref PubMed Scopus (162) Google Scholar) as well as in vivo (35.Rossi M. Kim M.S. Morgan D.G.A. Small C.J. Edwards C.M.B. Sunter D. Abusnana S. Goldstone A.P. Russell S.H. Stanley S.A. Smith D.M. Yagaloff K. Ghatei M.A. Bloom S.R. Endocrinology. 1998; 139: 4428-4431Crossref PubMed Google Scholar). In contrast to Agouti, AGRP is equally potent in inhibiting signaling at the central melanocortin receptors, MC3-R and MC4-R (binding affinity of human AGRP close to 1 nm for both receptors), very little inhibition is detected at the MC5-R, and virtually no activity is detected at MC1-R (32.Rosenfeld R.D. Zeni L. Welcher A.A. Narhi L.O. Hale C. Marasco J. Delaney J. Gleason T. Philo J.S. Katta V. Hui J. Baumgartner J. Graham M. Stark K.L. Karbon W. Biochemistry. 1998; 37: 16041-16052Crossref PubMed Scopus (50) Google Scholar, 33.Fong T.M. Mao C. MacNeil T. Kalyani R. Smith T. Weinberg D. Tota M.R. Van der Ploeg L.H.T. Biochem. Biophys. Res. Commun. 1997; 237: 629-631Crossref PubMed Scopus (199) Google Scholar, 34.Yang Y.-K. Thompson D.A. Dickinson C.J. Wilken J. Barsh G.S. Kent S.B.H. Gantz I. Mol. Endocrinol. 1999; 13: 148-155Crossref PubMed Scopus (162) Google Scholar). Therefore, AGRP is as potent an antagonist at MC4-R as Agouti and a much stronger antagonist at MC3-R. The tissue distribution of Agouti and AGRP differs greatly. The expression of agouti is normally confined to hair follicles whereas AGRP is expressed primarily in the hypothalamus, adrenal medulla, and at low levels in testis, lung, and kidney (29.Shutter J.R. Graham M. Kinsey A.C. Scully S. Luthy R. Stark K. Genes Dev. 1997; 11: 593-602Crossref PubMed Scopus (560) Google Scholar,30.Ollmann 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 (1558) Google Scholar). Unlike Agouti, the localization pattern of human and murine AGRP is strikingly alike (29.Shutter J.R. Graham M. Kinsey A.C. Scully S. Luthy R. Stark K. Genes Dev. 1997; 11: 593-602Crossref PubMed Scopus (560) Google Scholar), indicating similar roles for AGRP in both species. Brain expression of AGRP mRNA is confined to neuronal cell bodies localized in the arcuate nucleus of the hypothalamus (36.Broberger C. Johansen J. Johansson C. Schalling M. Hokfelt T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15043-15048Crossref PubMed Scopus (685) Google Scholar). These neurons are shown to project to hypothalamic nuclei that receive dense pro-opiomelanocortin innervation and express the two central melanocortin receptors, MC3-R and MC4-R (36.Broberger C. Johansen J. Johansson C. Schalling M. Hokfelt T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15043-15048Crossref PubMed Scopus (685) Google Scholar). The potency of AGRP action at MC3-R and MC4-R together with their similar distribution pattern suggest that AGRP and not Agouti controls their function in vivo. Homeostatic regulation of the central melanocortin system may well be achieved through changes in antagonist (AGRP) rather than agonist (α-MSH) bioavailability. Thus, AGRP may control the activity of central melanocortin receptors in a manner similar to modulation of MC1-R signaling by Agouti in the skin. The presence of AGRP-immunoreactive fibers in a subset of hypothalamic nuclei (i.e. arcuate, paraventricular, dorsomedial) strongly suggests a key role for AGRP and the melanocortin system in the regulation of energy homeostasis. This conclusion is supported by multiple findings. First, central administration of AGRP is shown to mimic the effect of synthetic MC3-R and MC4-R antagonists and stimulate feeding (35.Rossi M. Kim M.S. Morgan D.G.A. Small C.J. Edwards C.M.B. Sunter D. Abusnana S. Goldstone A.P. Russell S.H. Stanley S.A. Smith D.M. Yagaloff K. Ghatei M.A. Bloom S.R. Endocrinology. 1998; 139: 4428-4431Crossref PubMed Google Scholar). In addition, AGRP is able to specifically block the reduction in food intake elicited by administration of α-MSH (25.Fan W. Boston B.A. Kesterson R.A. Hruby V.J. Cone R.D. Nature. 1997; 385: 165-168Crossref PubMed Scopus (1670) Google Scholar). Second, overexpression of AGRP in transgenic animals results in an obesity phenotype strikingly similar to that of the MC4-R KO orA y mice (30.Ollmann 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 (1558) Google Scholar, 37.Graham M. Shutter J.R. Sarmiento U. Sarosi I. Stark K.L. Nat. Genet. 1997; 17: 273-274Crossref PubMed Scopus (324) Google Scholar). In conclusion, melanocortinergic neurons exert a tonic i
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