Endocrine Abstracts

Agouti-related protein (AgRP) is a 132 amino acid protein, expressed in the brain where it antagonises the anorexic effect of alpha-MSH through melanocortin 3 and 4 receptors. In situ hybridisation and RT-PCR have shown that AgRP mRNA is also expressed in the steroidogenic zone of the adrenal cortex where levels are up-regulated following 24 hrs of fasting. However, its physiological significance in the adrenal gland is still unknown.

To investigate the site of cleavage of AgRP (1-132), the protein was purified from cell lysate and media of AgRP-transfected Y1 adrenal cortical cells. After Sep-pak extraction and repeated HPLC separations, mass spectrometry of the purified protein demonstrated the presence of a 5192.89 Dalton peptide in both cell lysate and cell media, consistent with cleavage of AgRP at a dibasic site producing a C-terminal fragment AgRP (87-132) with high affinity for the melanocortin 4 receptor.

In order to identify the enzyme responsible for this endogenous cleavage, mRNA expression profiles of prohormone convertase family members (PC1/PC3, PC2, PC4, PC5/PC6, furin, PACE4, PC7/PC8) known to cleave their substrates at a dibasic site were studied in brain, adrenal gland and Y1 adrenal cells. Only PC2, PC5, furin, PC4 and PACE4 were found to be expressed in all three tissues consistent with the AgRP mRNA expression pattern. In situ hybridisation demonstrated that of these five enzymes, only furin, PC4 and PACE4 were found to be expressed throughout the entire rat adrenal cortex where AgRP is expressed. Additionally, semi-quantitative PCR showed that PACE4 was the only cortically expressed transcript to be upregulated in rat adrenals 24 hrs after fasting in line with increases in AgRP levels.

These findings lead us to conclude that PACE4 may be the enzyme responsible for the endogenous cleavage of pro-AgRP to AgRP (87-132).