AGRP Agouti-related protein α-MSH α-melanocyte-stimulating hormone ACTH adrenocorticotropic hormone MC1-R melanocortin receptor 1 ASIP Agouti-signaling protein A y lethal yellow mg mahogany md mahoganoid
The discovery a decade ago of the murine agouti 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, 2) 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) of the melanocortin receptors, a family of G s-coupled receptors, of which five isoforms are presently known (reviewed in Ref. 4). MC1-R is the melanocyte-stimulating hormone receptor expressed in melanocytes and has a physiological role in pigmentation (5). MC4-R is expressed mainly in the brain (6, 7) and has been implicated in the regulation of feeding behavior and metabolism (8). MC3-R is found primarily in the hypothalamic and limbic systems (9); 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, 11). 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, and pseudoagouti 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, 13). 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, 14). 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). This intricate pattern of gene regulation is achieved through the existence of alternatively spliced agouti transcripts 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). 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). In contrast, type II transcripts contain non-coding exons 1A and 1A′ and are under the control of regional (ventral-specific) promoter elements (14). The second class of transcripts is responsible phenotypically for the lighter ventral pigmentation seen in several agouti strains (i.e. white-bellied agouti) (14).
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). Biochemical analysis of the Agouti protein shows that it is highly glycosylated and very stable to thermal denaturation (15). 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).
In vitro studies using recombinant mouse Agouti protein prove that Agouti is a potent melanocortin antagonist (nanomolar range) at MC-R subtypes 1 (K I (app) = 2.6 ± 0.8 n m) and 4 (K I (app) = 54 ± 18 n m), a relatively weak antagonist at MC3-R (K I (app) = 190 ± 74 n m), and a very weak antagonist at MC5-R (K I (app) = 12,000 ± 340 n m) (10, 15, 16). Pharmacological studies of murine Agouti conclude that its mechanism of action is a classical competitive antagonism of melanocortin receptors (10, 15, 16). In addition, a shorter version of Agouti, residues 83–131, is shown to be as potent an antagonist as the full-length protein (15). The Cys-rich C terminus is therefore deemed sufficient for effective antagonism of melanocortin action in vitro as well as in vivo (15, 17). 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).
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 (K I (app) = 0.47 ± 0.06 n m) and MC4-R (K I (app) = 0.14 ± 0.02 n m) and a relatively weak antagonist at MC3 (K I (app) = 6.4 ± 1.1 n m) and MC5-R (K I (app) = 1.16 ± 0.17 n m) (19). Competitive antagonism of ASIP is apparent, however, only toward MC1-R (19). 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). 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). 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). 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). The agouti 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 the A y phenotype in transgenic animals (24).
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) and central administration of MC4-R agonists and antagonists stimulate and inhibit feeding behavior, respectively (25). Several studies, however, have proposed an alternative mechanism for Agouti action that does not involve melanocortin receptor antagonism (reviewed in Ref.26). 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). 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). 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), whereas Agouti is a slow acting, transient activator of the same channels (26). 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). This suggests that modulation of intracellular calcium levels may simply be secondary to Agouti action.
