BIOCHEMICAL, BIOPHYSICAL, AND PHARMACOLOGICAL CHARACTERIZATION OF BACTERIALLY EXPRESSED HUMAN AGOUTI-RELATED PROTEIN

The agouti-related protein gene (Agrp) is a novel gene implicated in t he 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 re gulation of melanocortin receptors expressed in the central nervous sy stem. The availability of recombinant, highly purified protein is requ ired to fully address this potential interaction. A nearly full-length form of AGRP (MKd5-AGRP) was expressed in the cytosolic or soluble fr action of Escherichia coli and appeared as large intermolecular disulf ide-bonded aggregates. Following oxidation, refolding, and purificatio n, this protein was soluble, and eluted as a single symmetric peak on RP-HPLC. Circular dichroism studies indicated that the purified protei n contains primarily random coil and beta-sheet secondary structure. S edimentation velocity studies at neutral pH demonstrated that MKd5-AGR P is monomeric at low micromolar concentrations. Mobility shifts obser ved using SDS-PAGE under reducing and nonreducing conditions for bacte rially expressed and mammalian expressed AGRP were identical, an indic ation of a similar disulfide structure. The purification to homogeneit y of a second, truncated form of AGRP (Md65-AGRP) which was expressed in the insoluble or inclusion body fraction is also described. Both fo rms act as competitive antagonists of alpha-melanocyte stimulating hor mone (a-MSH) at melanocortin-3 (MC-3) and melanocortin-4, receptors (M C-4). The demonstration that AGRP is an endogenous antagonist with res pect to these receptors is a unique mechanism within the central nervo us system, and has important implications in the control of feeding.