No abstract

Several putative peptide-processing endoproteases have been identified by homology to the yeast Kex2 endoprotease, including furin, PC2, and PC1. However, the question is still open as to which might be involved in peptide posttranslational processing. To enable detailed comparisons of physiological changes in peptide processing with biochemical and molecular biological studies, we cloned rat pituitary cDNAs for PC1 and PC2. The amino acid sequence homologies among rat, human, and mouse PC1, PC2, and furin are consistent with each being a highly conserved but distinct member of a larger family of mammalian subtilisin-like proteases. PC1 and PC2 mRNAs show a restricted distribution among rat tissues and cultured cell lines, consistent with a role in tissue-specific peptide processing; the occurrence of furin mRNA among these tissues and cell lines is much more widespread, being high in many nonneuroendocrine tissues. In the neurointermediate pituitary, PC1 and PC2 mRNAs are strikingly regulated in response to dopaminergic agents, in parallel with mRNAs for POMC, peptidylglycine alpha-amidating monooxygenase, and carboxypeptidase-H. In AtT-20 cells, PC1 mRNA is coregulated with POMC and peptidylglycine alpha-amidating monooxygenase mRNAs in response to CRH and glucocorticoids. When the endogenous PC1 mRNA level in AtT-20 cells is significantly and specifically decreased by stable expression of antisense RNA to PC1, biosynthetic labeling of newly synthesized POMC-derived peptides shows a substantial blockade of normal POMC processing. These data are consistent with a role for PC1 protein in endoproteolysis, either as a processing endoprotease or as the activator of the actual processing endoprotease(s).

The peptide hormone ghrelin is the endogenous ligand for the type 1a growth hormone secretagogue receptor (GHS-R1a) and the only currently known circulating appetite stimulant. GHS-R1a antagonism has therefore been proposed as a potential approach for obesity treatment. More recently, ghrelin has been recognized to also play a role in controlling glucose-induced insulin secretion, which suggests another possible benefit for a GHS-R1a antagonist, namely, the role as an insulin secretagogue with potential value for diabetes treatment. In our laboratories, piperidine-substituted quinazolinone derivatives were identified as a new class of small-molecule GHS-R1a antagonists. Starting from an agonist with poor oral bioavailability, optimization led to potent, selective, and orally bioavailable antagonists. In vivo efficacy evaluation of selected compounds revealed suppression of food intake and body weight reduction as well as glucose-lowering effects mediated by glucose-dependent insulin secretion.

Neuropeptide Y (NPY) plays important roles in the central control of appetite and energy balance, but the receptor subtype responsible for this function has not been cloned. Here we report the cloning by expression of a novel NPY receptor subtype from a rat hypothalamus cDNA library. The novel receptor, referred to as the NPY Y5 receptor, has a transcript of approximately 2.6 kilobases with an open reading frame of 1335 base pairs that encodes a 445-amino acid protein. The amino acid sequence deduced from the rat Y5 cDNA clone shows only 30 -33% identity to other NPY receptors, including Y1, Y2, and Y4/PP1. Using the rat Y5 receptor cDNA probe, the human homologue was obtained by low stringency hybridization. The human Y5 amino acid sequence has 88% identity to the rat Y5 receptor. Importantly, pharmacological analysis shows that the rat and human Y5 receptors have high affinity for the peptides that elicit feeding (e.g. NPY, PYY, (2-36)NPY, and (LP)NPY) and low affinity for nonstimulating peptides (e.g. (13-36)NPY and rat PP), suggesting that it is the NPY feeding receptor subtype.Neuropeptide Y (NPY), 1 peptide YY (PYY), and pancreatic polypeptide (PP) are all members of a common family of hormonally active peptides. NPY is predominantly expressed in the central nervous system and is one of the most abundant neuropeptides in the brain (1). NPY has a broad range of functions, including the regulation of vascular tone, anxiogenesis, and food intake (2). These diverse physiological actions are mediated through distinct NPY receptor subtypes (3-10), three of which, Y1, Y2, and Y4/PP1, have been cloned. Another NPY receptor subtype, Y3, which is characterized by its lower affinity for PYY as compared with NPY has been reported (2). However, the feeding response elicited by NPY and its peptide analogues cannot be fully accounted for by the pharmacological profile of these receptors. Thus, although both (LP)NPY and (2-36)NPY are potent stimulators of feeding (11-14), (LP)NPY has higher affinity for the Y1 than the Y2 receptor, whereas (2-36)NPY exhibits higher affinity for the Y2 than the Y1 subtype, suggesting that neither of these receptor subtypes is exclusively involved in the feeding response (15)(16)(17)(18)(19). Likewise, Y3 receptor pharmacology indicates that it has low affinity for PYY, a potent stimulator of feeding in vivo (2). Rat PP and C-terminal fragments of NPY, on the other hand, fail to elicit feeding when injected intracerebroventricularly into mice and rats (11)(12)(13)20). Because the Y2 receptor has high affinity for such C-terminal peptide fragments (2) and PP binds strongly to the Y4/PP1 receptor (5-7), it is unlikely that these subtypes are mediators of feeding behavior. In this report, we describe the molecular identification of a novel NPY receptor subtype, referred to as the Y5 receptor. The pharmacological profile of this receptor suggests that it is the NPY feeding receptor. EXPERIMENTAL PROCEDURESIsolation of Rat Hypothalamus mRNA and Construction of cDNA Library-Total RNA was obtained ...