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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Endocrinology Study Section (END)
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Haft, Carol R
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University of Connecticut
Schools of Medicine
United States
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Mains, Richard E; Blaby-Haas, Crysten; Rheaume, Bruce A et al. (2018) Changes in Corticotrope Gene Expression Upon Increased Expression of Peptidylglycine ?-Amidating Monooxygenase. Endocrinology 159:2621-2639
Miller, Megan B; Yan, Yan; Wu, Yi et al. (2017) Alternate promoter usage generates two subpopulations of the neuronal RhoGEF Kalirin-7. J Neurochem 140:889-902
Miller, Megan B; Yan, Yan; Machida, Kazuya et al. (2017) Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity. ACS Chem Neurosci 8:1554-1569
Katrancha, Sara M; Wu, Yi; Zhu, Minsheng et al. (2017) Neurodevelopmental disease-associated de novo mutations and rare sequence variants affect TRIO GDP/GTP exchange factor activity. Hum Mol Genet 26:4728-4740
Kumar, Dhivya; Mains, Richard E; Eipper, Betty A (2016) 60 YEARS OF POMC: From POMC and ?-MSH to PAM, molecular oxygen, copper, and vitamin C. J Mol Endocrinol 56:T63-76
Yan, Yan; Eipper, Betty A; Mains, Richard E (2016) Kalirin is required for BDNF-TrkB stimulated neurite outgrowth and branching. Neuropharmacology 107:227-238
Lu, Jianning; Luo, Ceng; Bali, Kiran Kumar et al. (2015) A role for Kalirin-7 in nociceptive sensitization via activity-dependent modulation of spinal synapses. Nat Commun 6:6820
Puigdellívol, Mar; Cherubini, Marta; Brito, Verónica et al. (2015) A role for Kalirin-7 in corticostriatal synaptic dysfunction in Huntington's disease. Hum Mol Genet 24:7265-85
Miller, Megan B; Vishwanatha, Kurutihalli S; Mains, Richard E et al. (2015) An N-terminal Amphipathic Helix Binds Phosphoinositides and Enhances Kalirin Sec14 Domain-mediated Membrane Interactions. J Biol Chem 290:13541-55
Yan, Yan; Eipper, Betty A; Mains, Richard E (2015) Kalirin-9 and Kalirin-12 Play Essential Roles in Dendritic Outgrowth and Branching. Cereb Cortex 25:3487-501

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