We thank Dr. Geoffrey Murdoch (OHSU) for supplying total RNA from human brain, and Drs. Ri chard Simerly (Oregon Regional Primate Research Center) and Marty Mortrud (OHSU) for val uabl e assistance with i n situ histochemical analysis.

Five novel peptides were identified in the brains of mice lacking active carboxypeptidase E, a neuropeptide-processing enzyme. These peptides are produced from a single precursor, termed proSAAS, which is present in human, mouse, and rat. ProSAAS mRNA is expressed primarily in brain and other neuroendocrine tissues (pituitary, adrenal, pancreas); within brain, the mRNA is broadly distributed among neurons. When expressed in AtT-20 cells, proSAAS is secreted via the regulated pathway and is also processed at paired-basic cleavage sites into smaller peptides. Overexpression of proSAAS in the AtT-20 cells substantially reduces the rate of processing of the endogenous prohormone proopiomelanocortin. Purified proSAAS inhibits prohormone convertase 1 activity with an IC 50 of 590 nM but does not inhibit prohormone convertase 2. Taken together, proSAAS may represent an endogenous inhibitor of prohormone convertase 1.

ProSAAS is a neuroendocrine peptide precursor that potently inhibits prohormone convertase 1 in vitro. To explore the function of proSAAS and its derived peptides, transgenic mice were created which express proSAAS using the beta-actin promoter. The body weight of transgenic mice was normal until approximately 10-12 weeks, and then increased 30-50% over wild-type littermates. Adult transgenic mice had a fat mass approximately twice that of wild-type mice, and fasting blood glucose levels were slightly elevated. In the pituitary, the levels of several fully processed peptides in transgenic mice were not reduced compared with wild-type mice, indicating that the proSAAS transgene did not affect prohormone convertase 1 activity in this tissue.Because the inhibitory potency of proSAAS-derived peptides towards prohormone convertase 1 is much greater in the absence of carboxypeptidase E activity, the pro-SAAS transgene was also expressed in carboxypeptidase E-deficient Cpe fat/fat mice. Although the transgenic mice were born in the expected frequency, 21 of 22 proSAAS transgenic Cpe fat/fat mice died between 11 and 26 weeks of age, presumably due to greatly elevated blood glucose. The levels of several pituitary peptides were significantly reduced in the proSAAS transgenic Cpe fat/fat mice relative to non-transgenic Cpe fat/fat mice, suggesting that the transgene inhibited prohormone convertase 1 in these mice. Taken together, these results are consistent with a role for proSAAS-derived peptides as neuropeptides that influence body weight independently of their function as inhibitors of prohormone convertase 1.

Concentrations of extracellular glycine in the central nervous system are regulated by Na؉ /Cl ؊ -dependent glycine transporters, GLYT1 and GLYT2. N-Arachidonylglycine (NAGly) is an endogenous inhibitor of GLYT2 with little or no effect on GLYT1 and is analgesic in rat models of neuropathic and inflammatory pain. Understanding the molecular basis of NAGly interactions with GLYT2 may allow for the development of novel therapeutics. In this study, chimeric transporters were used to determine the structural basis for differences in NAGly sensitivity between GLYT1 and GLYT2 and also the actions of a series of related N-arachidonyl amino acids. Extracellular loops 2 and 4 of GLYT2 are important in the selective inhibition of GLYT2 by NAGly and by the related compounds N-arachidonyl-␥-aminobutyric acid and N-arachidonyl-D-alanine, whereas only the extracellular loop 4 of GLYT2 is required for N-arachidonyl-L-alanine inhibition of transport. These observations suggest that the structure of the head group of these compounds is important in determining how they interact with extracellular loops 2 and 4 of GLYT2. Site-directed mutagenesis of GLYT2 EL4 residues was used to identify the key residues Arg 531 , Lys 532 , and Ile 545 that contribute to the differences in NAGly sensitivity.