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

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• 作者：Robert D. Rosenfeld ; Lisa Zeni ; Andrew A. Welcher ; Linda O. Narhi ; Clarence Hale ; Julie Marasco ; John Delaney ; Thomas Gleason ; John S. Philo ; Viswanathan Katta ; John Hui ; Jamie Baumgartner ; Melissa Gr
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：November 17, 1998
• 年：1998
• 卷：37
• 期：46
• 页码：16041 - 16052
• 全文大小：279K
• 年卷期：v.37,no.46(November 17, 1998)
• ISSN：1520-4995

文摘

The agouti-related protein gene (Agrp) is a novel gene implicated in the control of feedingbehavior. The hypothalamic expression of Agrp is regulated by leptin, and overexpression of Agrp intransgenic animals results in obesity and diabetes. By analogy with the known actions of agouti, thesedata suggest a role for the Agrp gene product in the regulation of melanocortin receptors expressed in thecentral nervous system. The availability of recombinant, highly purified protein is required to fully addressthis potential interaction. A nearly full-length form of AGRP (MKd5-AGRP) was expressed in the cytosolicor 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 symmetricpeak on RP-HPLC. Circular dichroism studies indicated that the purified protein contains primarily randomcoil and fchars/beta2.gif" BORDER=0 ALIGN="middle">-sheet secondary structure. Sedimentation velocity studies at neutral pH demonstrated thatMKd5-AGRP is monomeric at low micromolar concentrations. Mobility shifts observed using SDS-PAGE under reducing and nonreducing conditions for bacterially expressed and mammalian expressedAGRP were identical, an indication of a similar disulfide structure. The purification to homogeneity ofa second, truncated form of AGRP (Md65-AGRP) which was expressed in the insoluble or inclusionbody fraction is also described. Both forms act as competitive antagonists of fchars/alpha.gif" BORDER=0>-melanocyte stimulatinghormone (fchars/alpha.gif" BORDER=0>-MSH) at melanocortin-3 (MC-3) and melanocortin-4 receptors (MC-4). The demonstrationthat AGRP is an endogenous antagonist with respect to these receptors is a unique mechanism within thecentral nervous system, and has important implications in the control of feeding.