Agouti-Related Protein Is Posttranslationally Cleaved by Proprotein Convertase 1 to Generate Agouti-Related Protein (AGRP)83–132: Interaction between AGRP83–132 and Melanocortin Receptors Cannot Be Influenced by Syndecan-3

Abstract

Agouti-related protein (AGRP) plays a key role in energy homeostasis. The carboxyl-terminal domain of AGRP acts 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, thereby modulating the effects of carboxyl-terminal AGRP at the MC4-R. This model assumes that AGRP is secreted as a full-length peptide. In this study we found that AGRP is processed intracellularly after Arg(79)-Glu(80)-Pro(81)-Arg(82). The processing site suggests cleavage by proprotein convertases (PCs). RNA interference and overexpression experiments showed that PC1/3 is primarily responsible for cleavage in vitro, although both PC2 and PC5/6A can also process AGRP. Dual in situ hybridization demonstrated that PC1/3 is expressed in AGRP neurons in the rat hypothalamus. Moreover, hypothalamic extracts from PC1-null mice contained 3.3-fold more unprocessed full-length AGRP, compared with wild-type mice, based on combined HPLC and RIA analysis, demonstrating that PC1/3 plays a role in AGRP cleavage in vivo. We also found that AGRP(83-132) is more potent an antagonist than full-length AGRP, based on cAMP reporter assays, suggesting that posttranslational cleavage is required to potentiate the effect of AGRP at the MC4-R. Because AGRP is cleaved into distinct amino-terminal and carboxyl-terminal peptides, we tested whether amino-terminal peptides modulate food intake. However, intracerebroventricular injection of rat AGRP(25-47) and AGRP(50-80) had no effect on body weight, food intake, or core body temperature. Because AGRP is cleaved before secretion, syndecan-3 must influence food intake independently of the MC4-R.

Role of furin in granular acidification in the endocrine pancreas: Identification of the V-ATPase subunit Ac45 as a candidate substrate

“…Short hairpin RNA (ShRNA) constructs for furin and Ac45 were designed and tested by cotransfection with the corresponding cDNA. Efficient silencing of exogenous furin in ␤TC3 cells has been described previously (32) and silencing of Ac45 is shown in Fig. 5A.…”

“…Regulated secretion experiments were performed essentially as described (8), except that cells were incubated overnight in nonsupplemented serum-free DMEM. Sample preparation and separation by SDS/PAGE have been described previously (32). For Western blotting (49), anti-FLAG antibody M2 (Sigma) was used.…”

“…5A. Regulated secretion was analyzed by using recombinant FLAGtagged Agouti-related protein (AGRPf) because it is a soluble protein, efficiently sorted to the regulated secretory pathway (32). Endogenous expression of furin and Ac45 in ␤TC3 cells was confirmed by reverse transcription PCR (data not shown).…”

New ligands for melanocortin receptors

“…However, the N-terminal sequences of Aisp and Agrp have undergone considerable sequence divergence since duplication. Indeed, a series of studies from our group, 73,76,77 and from White and colleagues, 78,79 reveal that the N-terminal domains of Asip and Agrp have evolved to serve very different functions. The N-terminal domain of Asip contains a glycosylation site and a high density of positively charged residues referred to as the 'basic region.'…”

“…First, the affinity of full-length Agrp for Mc4r is approximately 10-fold less than that of a C-terminal Agrp fragment; 73,78 second, full-length Agrp is post-translationally cleaved by a proprotein convertase such that the C-terminal domain is the active form in vivo. 78 By contrast, the active form of Asip in tissues is the full-length protein. 82 A summary of the similarities and differences in mechanisms of Asip and Agrp action are summarized in Figure 2a.…”