Agouti-related protein functions as an inverse agonist at a constitutively active brain melanocortin-4 receptor

doi:10.1016/S0167-0115(01)00234-8

Regulatory Peptides

Volume 99, Issue 1, 5 May 2001, Pages 1-7

https://doi.org/10.1016/S0167-0115(01)00234-8 Get rights and content

Abstract

Agouti-related protein (AGRP) is one of two naturally occurring antagonists of G-Protein coupled receptors (GPCRs) identified to date, and has been physiologically implicated in regulating food intake, body weight, and energy homeostasis. AGRP has been identified in vitro, as competitively antagonizing the brain melanocortin-4 (MC4R) and melanocortin-3 (MC3R) receptors, and when over expressed in transgenic mice, results in an obese phenotype. Emerging data propose that AGRP has additional targets in the hypothalamus and/or physiologically functions via a mechanism in addition to competitive antagonism of α-MSH at the brain melanocortin receptors. We report data herein supporting an alternative mechanism for AGRP involvement in feeding behavior. A constitutively active MC4R has been generated which possess EC₅₀ values for melanocortin agonists (α-MSH, NDP-MSH, and MTII) and a pA₂ value for the synthetic peptide antagonist SHU9119 identical to the wildtype receptor, but increases basal activity to 50% maximal response. AGRP possesses inverse agonist activity at this constitutively active MC4R. These data support the hypothesis for an additional physiological mechanism for AGRP action in feeding behavior and energy homeostasis.

Introduction

The melanocortin system is a unique G-protein coupled receptor (GPCR) pathway that not only includes endogenous agonists and receptors, but also the only two identified naturally occurring antagonists of GPCRs (agouti/ASP and AGRP) [1], [2], and the single transmembrane spanning mahogany protein, which acts upstream of the melanocortin receptors with the ASP, and possibly AGRP [3], [4] (Fig. 1). The melanocortin peptides (α-, β-, γ-melanocyte stimulating hormones and ACTH) are the endogenous agonist ligands for these melanocortin receptors and are derived by post-translational processing of the proopiomelanocortin (POMC) gene transcript. The melanocortin receptor family consists of five isoforms (MC1R–MC5R) identified to date [5], [6], [7], [8], [9], [10], [11] and stimulates the cAMP second messenger signal transduction pathway.

The melanocortin pathway includes five genetic factors that have been linked to energy homeostasis and obesity in mice and humans. The mouse agouti protein (ASP) [12], [13] was first characterized as an antagonist of the skin MC1R and brain MC4R [1] which led to the agouti obesity hypothesis in which the obesity of the agouti (Ay^a) mouse was attributed to chronic antagonism of the MC4R by the agouti protein [1], [14], [15]. The AGRP protein was demonstrated pharmacologically to competitively antagonize the MC3R and MC4R brain melanocortin receptors [16], and when ectopically expressed, resulted in an obese phenotype [2], [17]. The brain melanocortin receptor (MC3R and MC4R) knock out animals have been identified as physiologically participating in the regulation of energy homeostasis [15], [18]. Furthermore, a genetic modification of the gene transcripts for POMC (from which the agonist ligands are derived) [19] and MC4R [20], [21], [22], [23] in obese humans has been identified.

AGRP brain mRNA expression has been identified primarily in the arcuate nucleus of the hypothalamus of the rat and primate [24], [25] with neuronal projections to the paraventricular hypothalamic nucleus (PVN) [25], [26]. Both the arcuate nucleus and the PVN brain nuclei are well recognized for their participation in energy homeostasis. MC4R protein expression has been identified in the PVN [27] as well as MC3R mRNA expression [7]. AGRP is co-localized in neuropeptide Y (NPY) containing neurons [28], [29], and when administered centrally to NPY−/− knockout mice, results in increased food intake [30]. Unexpectedly, the NPY knock out mouse possesses a normal weight phenotype [31] that has subsequently been attributed to a compensatory mechanism involving AGRP [30].

Central administration of AGRP in the MC4R knock out animal resulted in increased food intake [30], which may be attributed to AGRPs antagonism of the MC3R, or via an unidentified mechanism or target [30]. Twenty-four-hour pretreatment of centrally administered AGRP(83–132) followed by administration of the melanocortin agonist MTII [32], resulted in no statistical difference between the animals that had received saline instead of MTII [33]. These data led the authors of those studies to postulate that AGRP may possess an additional physiological mechanism in addition to competitive antagonism.

This study was undertaken to characterize AGRP at a constitutively active MC4R and determine if AGRP possesses inverse agonist activity. Additionally, this study provides experimental evidence that AGRP participates in energy homeostasis via a mechanism in addition to competitive antagonism.

Section snippets

Receptor mutagenesis

Mouse MC4R cDNA (1.6-kb fragment) was subcloned into pBluescript (Stratagene) and was used for mutagenesis. Mutants were prepared by the polymerase chain reaction (PCR) using pfu polymerase (Stratagene) and a complementary set of primers containing the nucleotide mutation(s) resulting in the desired amino acid residue change. After completion of the PCR reaction (95° 30 s, 12 cycles of 95° 30 s, 55° 1 min, 68° 9 min) the product was purified (Qiaquick PCR reaction, Qiagen) and eluted in water.

Results

Table 1 summarizes the primary sequences of the agonists and antagonists used in this study. Table 2 summarizes the pharmacology of these melanocortin ligands at the wildtype and constitutively active mMC4Rs. The agonists and synthetic peptide antagonist SHU9119 had nearly identical stimulatory or inhibitory activities at both the wildtype and constitutively active mMC4Rs (Fig. 2), within experimental error. The synthetic melanocortin antagonist, SHU9119 [37] possess slight partial agonist

Discussion

A constitutively active mouse MC4R has been generated and characterized in vitro to possess an increased basal activity (approximately 50% the maximal response) in the absence of agonist ligand. The melanocortin ligands α-MSH, NDP-MSH, MTII (agonists) and SHU9119 (antagonist) possess pharmacological properties similar to those reported at the wildtype receptor (Table 2) [38]. AGRP(83–132) however, possesses inverse agonist activity at this constitutively active receptor (Fig. 3). These data

Acknowledgements

This work has been funded by NIH Grant RO1-DK57080-01 (C.H.L.), the Howard Hughes Medical Institute Research Resources Program, University of Florida and Carrie Haskell-Luevano is a recipient of a Burroughs Wellcome fund Career Award in the Biomedical Sciences.

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Rapid communicationAgouti-related protein functions as an inverse agonist at a constitutively active brain melanocortin-4 receptor

Abstract

Introduction

Section snippets

Receptor mutagenesis

Results

Discussion

Acknowledgements

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Cell

Cell

Neuron

Brain Res.

Anal. Biochem.

Peptides

J. Biol. Chem.

Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor

Nature

Antagonism of central melanocortin receptors in vitro and in vivo by agouti-related protein

Science

The mouse mahogany locus encodes a transmembrane form of human attractin

Nature

The mahogany protein is a receptor involved in suppression of obesity

Nature

The cloning of a family of genes that encode the melanocortin receptors

Science

Identification of a receptor for g melanotropin and other proopiomelanocortin peptides in the hypothalamus and limbic system

Proc. Natl. Acad. Sci. U. S. A.

Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain

Mol. Endocrinol.

Cloning of the mouse agouti gene predicts a secreted protein ubiquitously expressed in mice carrying the lethal yellow mutation

Genes Dev.

Role of melanocortinergic neurons in feeding and the agouti obesity syndrome

Nature

Characterization of agouti-related protein binding to melanocortin receptors

Mol. Endocrinol.

Overexpression of AGRT leads to obesity in transgenic mice

Nat. Genet.

Rapid communication
Agouti-related protein functions as an inverse agonist at a constitutively active brain melanocortin-4 receptor