PITUITARY KALLIKREIN & PROHORMONE PROCESSING

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Tissue (glandular) kallikrein (TK) is a trypsin-like serine protease which can generate biologically active peptides from inactive precursors with high specificity. I have recently discovered TK in the rat pituitary, and have found that TK exhibits patterns of tissue specific expression and physiological regulation closely paralleling MSH and beta-endorphin in the intermediate lobe, and prolactin (PRL) in the anterior pituitary lobe (AP). The proposed research will examine TK's role in the posttranslational processing of PRL in the AP, and Pro-opiomelanocortin (POMC; the precursor to MSH and beta-endorphin) in the intermediate lobe. Rat pituitary TK will be purified and its physiochemical and enzymatic properties compared to TK from other tissues. The ability of TK to cleave PRL and POMC will be examined using gel-electrophoresis and HPLC, and cleavage sites identified by microsequencing. PRL processing by TK may result in novel hormones with important functions in reproductive biology and mammary growth. Therefore, PRL cleavage products will be bioassayed for classic PRL actions (induction of milk-producing enzymes and stimulation of Nb2 lymphoma cell growth), as well as novel mitogenic actions on rat mammary glands in vivo. The cellular and subcellular localization of TK and cleaved PRL in the rat pituitary will be examined using immunohistochemistry and subcellular fractionation to see if TK is localized in sites of prohormone processing. Changes in TK, PRL and POMC levels, biosynthesis, and secretion will be compared following various physiological, hormonal and pharmacological in vivo and in vivo and in vitro to determine how tightly their regulation is interlinked. Alternative functions for rat pituitary TK will be tested (e.g., kinin generation and kinin modulation of pituitary hormone release). The results will determine whether TK functions to process peptides other than kinins, may elucidate an important mechanism of hormone biosynthesis, and reveal a novel role for PRL folding intermediates. Demonstration of an estrogen- induced pathway for generating PRL fragments with novel mitogenic activity on the mammary gland may be of considerable importance to current theories of the hormonal control of mammary growth and tumorigenesis in both rats and man.