Biochemical, Biophysical, and Pharmacological Characterization of Bacterially Expressed Human Agouti-Related Protein

Abstract

The agouti-related protein gene (Agrp) is a novel gene implicated in the control of feeding behavior. The hypothalamic expression of Agrp is regulated by leptin, and overexpression of Agrp in transgenic animals results in obesity and diabetes. By analogy with the known actions of agouti, these data suggest a role for the Agrp gene product in the regulation of melanocortin receptors expressed in the central nervous system. The availability of recombinant, highly purified protein is required to fully address this potential interaction. A nearly full-length form of AGRP (MKd5-AGRP) was expressed in the cytosolic or soluble fraction of Escherichia coli and appeared as large intermolecular disulfide-bonded aggregates. Following oxidation, refolding, and purification, this protein was soluble, and eluted as a single symmetric peak on RP-HPLC. Circular dichroism studies indicated that the purified protein contains primarily random coil and beta-sheet secondary structure. Sedimentation velocity studies at neutral pH demonstrated that MKd5-AGRP is monomeric at low micromolar concentrations. Mobility shifts observed using SDS-PAGE under reducing and nonreducing conditions for bacterially expressed and mammalian expressed AGRP were identical, an indication of a similar disulfide structure. The purification to homogeneity of a second, truncated form of AGRP (Md65-AGRP) which was expressed in the insoluble or inclusion body fraction is also described. Both forms act as competitive antagonists of alpha-melanocyte stimulating hormone (alpha-MSH) at melanocortin-3 (MC-3) and melanocortin-4 receptors (MC-4). The demonstration that AGRP is an endogenous antagonist with respect to these receptors is a unique mechanism within the central nervous system, and has important implications in the control of feeding.

Functional analysis of the Ala67Thr polymorphism in agouti related protein associated with anorexia nervosa and leanness

“…AgRP 83-132 was a more potent antagonist than the full-length AgRP proteins. This is consistent with the report of Ollmann et al [1] in which the shorter form C of recombinant AgRP was more potent in a melanophore assay than the longer forms A + B, but not with the report of Rosenfeld et al [14] in which recombinant AgRP 86-132 and MKd5-AgRP were equally potent at inhibiting hMC4R in a reporter gene assay in CHO cells. Thus, the effects of AgRP are dependent on the type of assay and cells used.…”

“…AgRP missing the 20 amino acid signal sequence as well as the first five amino acids of the mature protein and including Met-Lys at the N-terminus (MKd5-AgRP [14]) was created from AgRP (1-132) plasmid in the bluescript vector (kindly provided by Dr. G.S. Barsh) with a pair of synthetic oligonucleotides (5 0 -ACTGTAAGCTCATATGA-AAGCCCCCATGGAGGGC-3 0 and 5 0 -CGCTCTAGAAC-TAGTGGATCCTGTTGG-3 0 ) encoding a BamHI and NdeI cleavage site and Met and Lys residues on the N-terminus.…”

“…Both the fulllength protein as well as several fragments containing the cysteine rich C-terminal part of the protein function as competitive antagonists at the melanocortin receptors (MCRs) 3 and 4 [1,[11][12][13][14][15]. More recently, it has been demonstrated that the AgRP 83-132 fragment, and also the smaller AgRP 87-120, functions as an inverse agonist at the hMC3R and hMC4R, suppressing the constitutive activation of adenylate cyclase by these receptors [16][17][18].…”

Agouti-related protein (83-132) is a competitive antagonist at the human melanocortin-4 receptor: no evidence for differential interactions with pro-opiomelanocortin-derived ligands

“…Most studies indicate that AGRP acts as a classic competitive antagonist (i.e. orthosterically) (Rosenfeld et al 1998, Yang et al 1999b) rather than as a non-competitive antagonist (i.e. allosterically) (Ollmann et al 1997).…”

The agouti-related protein and body fatness in humans

“…[1][2][3] or when overexpressed in transgenic mice. 4 AgRP is expressed in the arcuate nucleus of the hypothalamus, the testes, and the adrenal gland, and is upregulated in obese and diabetic mice.…”