................................................................................................................................ i Co-Authorship Statement .................................................................................................. iii Acknowledgments .............................................................................................................. iv Table of

Determining the Molecular Mechanisms of PACS-1-mediated Protein Sorting

SNW1 regulates Notch signaling in neuroblastoma through interacting with RBPJ.

Using Col2.3GFP transgenic mice expressing GFP in maturing osteoblasts, we isolated Col2.3GFP+ enriched osteoblasts from 3 sources. We performed RNA-sequencing, identified 593 overlapping genes and confirmed these genes are highly enriched in osteoblast differentiation and bone mineralization annotation categories. The top 3 annotations are all associated with endoplasmic reticulum and Golgi vesicle transport. We selected 22 trafficking genes that have not been well characterized in bone for functional validation in MC3T3-E1 pre-osteoblasts. Transient siRNA knockdown of trafficking genes including Sec24d, Gosr2, Rab2a, Stx5a, Bet1, Preb, Arf4, Ramp1, Cog6 and Pacs1 significantly increased mineralized nodule formation and expression of osteoblast markers. Increased mineralized nodule formation was suppressed by concurrent knockdown of P4ha1 and/or P4ha2, encoding collagen prolyl 4-hydroxylase isoenzymes. MC3T3-E1 pre-osteoblasts with knockdown of Cog6, Gosr2, Pacs1 or Arf4 formed more and larger ectopic mineralized bone nodules in vivo, which was attenuated by concurrent knockdown P4ha2. Permanent knockdown of Cog6 and Pacs1 by CRISPR/Cas9 gene editing in MC3T3-E1 pre-osteoblasts recapitulated increased mineralized nodule formation and osteoblast differentiation. In summary, we have identified several vesicle trafficking genes with roles in osteoblast function. Our findings provide potential targets for regulating bone formation. 

The role of vesicle trafficking genes in osteoblast differentiation and function

Corticotropic cells of the anterior pituitary gland release adrenocorticotropic hormone (ACTH) in a regulated manner to promote the production of cortisol and androgens. The process of ACTH secretion is partly mediated by the phosphofurin acidic cluster sorting protein 1 (PACS-1); however, the underlying mechanisms behind this regulation remain unclear. Herein, we demonstrated PACS-1 interactions with the short transient receptor potential channel 3 (TRPC3) calcium transporter and the extended synaptotagmin-1 (ESyt1) endoplasmic reticulum–plasma membrane tethering protein. Importantly, PACS-1 promoted interactions between TRPC3 and ESyt1 and regulated their plasma membrane localization. Lastly, we demonstrated that PACS-1 is required for a proper store-operated calcium entry (SOCE) response and that ESyt1 regulates ACTH secretion through an unknown mechanism regulated by PACS-1. Overall, our study provides new insights into the physiological role PACS-1 plays in modulating intracellular calcium levels and regulating ACTH secretion in corticotropic cells.

PACS-1 Interacts with TRPC3 and ESyt1 to Mediate Protein Trafficking while Promoting SOCE and Cooperatively Regulating Hormone Secretion

PACS-1 and adaptor protein-1 mediate ACTH trafficking to the regulated secretory pathway.

The regulated secretory pathway is a specialized form of protein secretion found in endocrine and neuroendocrine cell types. Pro-opiomelanocortin (POMC) is a pro-hormone that utilizes this pathway to be trafficked to dense core secretory granules (DCSGs). Within this organelle, POMC is processed to multiple bioactive hormones that play key roles in cellular physiology. However, the complete set of cellular membrane trafficking proteins that mediate the correct sorting of POMC to DCSGs remain unknown. Here, we report the roles of the phosphofurin acidic cluster sorting protein-1 (PACS-1) and the clathrin adaptor protein 1 (AP-1) in the targeting of POMC to DCSGs. Upon knockdown of PACS-1 and AP-1, POMC is readily secreted into the extracellular milieu and fails to be targeted to DCSGs.

/pdf/pacs-1-and-adaptor-protein-1-mediate-acth-trafficking-to-the-12dp628ewf.pdf

PACS-1 and Adaptor Protein-1 Mediate ACTH Trafficking to the Regulated Secretory Pathway

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PACS-1 and adaptor protein-1 mediate ACTH trafficking to the regulated secretory pathway.

PACS-1 and Adaptor Protein-1 Mediate ACTH Trafficking to the Regulated Secretory Pathway