Secretory granules require, at different stages of maturity, very different granule constituents and membrane components. Instead of regarding these highly specialized organelles as sequestered and protected from the rest of the cell, current data suggest that substantial signaling occurs across secretory granule membranes. Expression of the peptide amidating monooxygenase (PAM), a granule membrane protein, causes changes in cytoskeletal organization. Known PAM cytosolic interactor proteins include Trio and Kalirin, multifunctional Rho GDP/GTP exchange factors (GEFs) capable of affecting cytoskeletal organization and gene expression. The experiments proposed focus on PAM and its interactions with Trio/Kalirin in pituitary endocrine cells.
Aim 1 tests the hypothesis that Trio and Kalirin play different roles by examining regulated secretion and cytoskeletal organization in pituitary cells with reduced levels of either Trio or Kalirin. The capabilities of the individual Rho GEF domains of Trio/Kalirin will be compared to those of the intact molecules.
Aim 2 explores the structure and functional roles of the non-catalytic domains of Trio and Kalirin. Trio/Kalirin-initiated pathways involving Pak, a Rac-activated kinase known to modulate cytoskeletal organization, and Cdk5, a Pro-directed kinase known to affect neurite outgrowth and hormone secretion from beta-cells and anterior pituitary endocrine cells, will be explored. Preliminary data demonstrating a regulatory role for intramolecular interactions of the first SH3 domain of Kalirin and neighboring Pro-rich sequences will be pursued using domain deletions and cell permeant peptide inhibitors. The secretory granule entry signal in the cytosolic domain of PAM will be defined in Aim 3 and complexes of PAM with other proteins will be isolated and characterized. Cytosolic proteins whose binding to secretory granule membranes is dependent on lumenal pH will be sought and a scaffolding role for Trio/Kalirin in organizing signaling pathways will be evaluated. With a mechanistic understanding of some of the ways in which secretory granules communicate to the surrounding cytosol, therapeutic means of controlling hormone secretion can be sought.
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