Analysis of post-translational processing and trafficing of agouti-related protein
LE Pritchard1,2, JWM Creemers3, AC Gyte1,2, N Davies1, P Le Rouzic1, C Lawrence1, S Luckman1, JC Brennand2 & A White1
Agouti-related protein (AGRP) plays a key role in energy homeostasis with the carboxy-terminal domain acting as an endogenous antagonist of the melanocortin-4 receptor (MC4-R). It has been suggested that the amino-terminal domain of AGRP binds to syndecan-3, allowing it to regulate local concentrations of AGRP at the MC4-R. This model assumes that AGRP is secreted as a full-length peptide. However, AGRP contains several consensus prohormone convertase cleavage sites: Lys52-Lys53, Arg79-Glu80-Pro81-Arg82, Arg85-Arg86 and Arg86-Cys87-Val88-Arg89. To investigate whether any of these sites are cleaved, full-length wild type AGRP and several mutated AGRP constructs were transfected into beta TC3 cells (endogenously expressing prohormone convertase 1 (PC1) and 2 (PC2)), alpha TC1 cells (PC2) and AtT20 cells (PC1). These studies demonstrate that AGRP is sorted into secretory granules and post-translational cleavage occurs at Arg79-Glu80-Pro81-Arg82. Cleavage was apparent in all 3 cell lines indicating that both PC1 and PC2 can cleave AGRP. Moreover, RNA interference (RNAi) inhibition of PC1 in AtT20 cells blocks cleavage, demonstrating an important role for PC1 in the AGRP processing pathway. We have shown both PC1 and PC2 are expressed in AGRP hypothalamic neurons by dual in situ hybridisation analysis, suggesting that processing is likely to occur in vivo. Post-translational cleavage may be required to potentiate the effect of AGRP at the MC4R, because AGRP83-132 is at least 6.1-fold more potent an antagonist than full-length AGRP based on Schild analysis. The physiological role, if any, of the amino-terminal domain of AGRP is uncertain as we have found that intracerebroventricular injection of rat AGRP25-51 and AGRP54-82 has no effect on body weight, food intake or core body temperature. The fact that AGRP is cleaved prior to secretion indicates that the role of syndecan-3 in food intake regulation is probably independent of the melanocortin system.