The orexigenic hormone ghrelin is a 28-amino-acid peptide derived from a 99-amino-acid precursor and acylated at Ser-3, which was initially isolated from rat stomach. In addition to stimulating appetite and growth, it also plays various important roles in energy homeostasis and in the cardiovascular and immune systems. Although analysis of its physiological effects has yielded many significant results, much less information is available on its biosynthesis and the mechanism of its acylation. In this report, we have studied the endoproteolytic processing of this molecule from its precursor (proghrelin) into mature ghrelin in various prohormone convertase null mouse strains generated in our laboratory and have identified the convertase responsible for this event. Using Western blotting, mass spectrometry, and immunocytochemical methods, we have demonstrated that (a) in mouse stomach, prohormone convertase 1/3 (PC1/3) is the endoprotease responsible for the conversion of proghrelin to ghrelin, (b) the acylation of this peptide is processing-independent, and (c) the expression of proghrelin mRNA is increased in the processing-deficient (PC1/3 null) mouse.Ghrelin, a 28-amino-acid peptide, was initially isolated from rat stomach (1) as a ligand of the growth hormone secretagogue receptor (2). Subsequent studies have revealed that it is a potent orexigenic peptide (3). It is also involved in various functions of the heart (4), pancreatic islets, and insulin secretion (5-10) and also in insulin signaling (11), adipose tissue (12), and the immune system (13). The expression of ghrelin is not limited to the stomach. The duodenum also produces large amounts of ghrelin, and it is also expressed at lower levels in other tissues, such as pancreas, brain, liver, and lung, (14).Similar to many other peptide hormones, ghrelin is processed from a 94-amino-acid precursor. Octanoylation at a serine residue (Ser-3) is necessary for its action via the growth hormone secretagogue receptor, although the desoctanoyl peptide may have alternative functions (15). Interestingly, recent research has revealed another bioactive peptide named obestatin within the ghrelin precursor (16). Obestatin is a 23-amino-acid peptide and is a cognate ligand for receptor GPR39, which is a member of a ghrelin receptor subfamily (17, 18). Although derived from the same precursor, in contrast to the effects of ghrelin, obestatin suppresses food intake, inhibits intestinal activity, and decreases weight gain (16). It is well established that many bioactive peptide hormones are generated by limited proteolytic processing by members of a secretory pathway-specialized serine protease family related to yeast Kex2 (the subtilisin-like prohormone convertase family SPCs or PCs), which includes PC1/3, PC2, and PC5/6A (19,20). Much data has accumulated over the past few years on functional aspects of ghrelin. However, biosynthetic studies have been lacking. In this report, we investigate the processing of proghrelin in various prohormone convertase-deficient mouse mode...