Production of bioactive peptides involves precursors, enzymes, storage in secretory granules, routing of granules to release sites and the machinery for regulated release. Peptidyiglycine alpha-Amidating Monooxygenase (PAM) is one of the few peptide processing enzymes spanning the granule membrane. During the previous grant period we found that expression of PAM affects trafficking of proopiomelanocortin (POMC) and prohormone convertase 1 (PC1) and organization of the cytoskeleton in AtT-20 corticotrope tumor cells. Kalirin, a new member of the Dbl family of GDP/GTP exchange factors for small GTP binding proteins, was identified by its interaction with routing determinants in the cytosolic domain of PAM. Expression of Kalirin in AtT-20 cells alters cytoskeletal organization and POMC trafficking. By exploring interactions of the lumenal domains of PAM with granule content proteins, and the Kalirin-mediated interactions of PAM with cytosolic components, we will develop an understanding of how the status of the lumenal milieu is communicated to relevant cytosolic factors.
Aim 1 is to evaluate the interplay of lumenal and cytosolic interactions in the trafficking of PAM and POMC, and in alterations in the cytoskeleton. We will use AtT-20 cells exhibiting inducible expression of individual domains of PAM to identify domains involved in trafficking and cytoskeletal organization.
Aim 2 is to determine the functional significance of the interaction of Kalirin with PAM and other proteins. A Kalirin homologue in pituitary will be sought. The mechanisms through which Kalirin interacts with the secretory pathway and the cytoskeleton will be examined using intact and permeabilized AtT-20 cells and primary pituitary cultures.
Aim 3 is to identify features of the PAM cytosolic domain governing its interactions with Kalirin. Membrane PAM proteins unable to interact with Kalirin will be used to study the mechanism through which PAM affects regulated secretion.
Aim 4 is to define the functional domains of Kalirin and its homologues. The spectrin-like region essential for the interaction of Kalirin with the cytosolic domain of PAM will be delineated and the activity of the GDP/GTP exchange factor domain will be assessed. Binding interactions of the pleckstrin homology and src homology 3 domains of Kalirin will be identified.
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