BIOSYNTHESIS, PROCESSING & SECRETION OF NEUROPEPTIDES & PITUITARY PEPTIDE HORMONE

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Pituitary secretory vesicle enzymes involved in the processing of pro-opiomelanocortin (POMC, pro-ACTH/endorphin) and pro-vasopressin were studied. A 70,000 mol. wt. paired basic residue specific prohormone converting enzyme (PCE), has recently been characterized as a unique calcium activated aspartic protease. Cloning of this enzyme is in progress. Studies were conducted to uncover mechanisms underlying the differential processing of POMC in the anterior and intermediate pituitary and brain. In vitro experiments show that calcium can regulate the extent of cleavage of POMC to smaller products. Thus, intravesicular calcium concentrations may regulate the quantitative differences in processing of beta-LPH to beta-endorphin in the anterior versus the intermediate lobe. Another finding was that 'O'glycosylation at Thr45 of N-POMC inhibited PCE from cleaving the Arg-Lys5O bond to yield N-POMC-49 and Lys5Ogamma3MSH. Since the non-'O'glycosylated form of N-POMC is found in intermediate but not anterior lobe, this post-translational modification of POMC may account for the presence of N-POMC1-49 and Lys5Ogamma3MSH only in intermediate lobe. The signal involved in the targeting of POMC into the regulated secretory pathway was also investigated. Truncated POMC/CAT constructs were transfected into the endocrine AtT-20 cells. Analysis of the intracellular localization of the expressed POMC-CAT fusion protein analyzed by immunofluorescence histochemistry using anti-CAT, and secretion studies with forskolin stimulation indicate that the N-terminal 25 amino acids of the prohormone is sufficient to target POMC into the regulated pathway. Binding studies suggest the presence of a receptor in secretory vesicle membranes which preferentially binds N-POMC1-77 at an acidic DH and may be involved in the sorting process. Our previous work showed that during salt-loading, anterior pituitary POMC mRNA and hypothalamic AVP mRNA levels were increased. Recently, a dramatic increase during salt-loading, of a polyA truncated form of AVP mRNA in the neural pituitary was discovered. This increase persisted with hypothalamic stalk transection suggesting that the AVP mRNA is locally synthesized, most likely in the pituicytes.