In vitro and in vivo approaches for the functional characterization of the scaffold protein, GRASP Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/r494vn28f

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  • Studies using the pluripotent embryonic carcinoma cell line, P19, as a retinoic acid (RA)-responsive model system have been instrumental towards our understanding of the RA-dependent signaling pathways in development and homeostasis. Grp1-associated scaffold protein (GRASP; also known as Tamalin) was first identified by our group, as a gene robustly induced by RA treatment in P19 cells. GRASP was reported to influence the cell-surface expression of interacting membrane receptors in vitro albeit by an unknown mechanism. Furthermore, the in vivo role of GRASP is poorly understood. Mice with a germline deletion of Grasp (GRASP-/-) do not exhibit any gross morphological, behavioral or sexual deficits and exhibit mild, altered sensitivities to morphine and cocaine administration. The goals of our studies herein were to investigate the mechanistic details of GRASP for the role in trafficking and cell-surface, localization of membrane receptors in vitro and the role of GRASP in vivo. Previously, our group had identified a strong interaction of GRASP with Grp1, a guanine nucleotide exchange factor for the small G protein, Arf6. In this study we show GRASP localizes in intracellular recycling endosomal compartment(s), recruits Grp1 to these structures, and facilitates activation of Arf6. Activation of Arf6 regulates key aspects of vesicular trafficking, actin reorganization and cellular migration. Furthermore, we show that GRASP preferentially regulates intracellular membrane trafficking by the Arf6-dependent/clathrin-independent pathway. We have shown that GRASP is expressed in the adult murine skin. Our studies demonstrate a robust induction of GRASP transcripts in murine dermis and epidermis following exposure to ultraviolet (UV) rays. We also report the generation of novel mice with germline deletion of Grasp (GRASP-/-), that exhibit altered proliferative and apoptotic responses to UV exposure. Ongoing investigations suggest that the observed phenotypes of GRASP-/- mice after UV exposure maybe due to the dysregulation of the subcellular localization of the tumor suppressor protein, p53. Taken together, our in vitro and in vivo approaches have provided new insight in the role of GRASP as scaffold protein that regulates intracellular trafficking pathways.
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