Investigating the role of GRP170 in ENaC biogenesis

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 DESCRIPTION (provided by applicant): The major focus of the study proposed here is to characterize the role of the ER lumenal chaperone, GRP170, on the quality control and trafficking of the Epithelial Sodium Channel (ENaC). ENaC is a heterotrimeric protein composed of α, β, and γ subunits. ENaC is responsible for salt reabsorption across the epithelia of the kidney and lung, and plays a critical role in controlling blood pressure and airway fluid volume. Defects in ENaC degradation are associated with Liddle's Syndrome and pseudohypoaldosteronism type I that result in hyper- and hypotension, respectively. ENaC is monitored by the ER quality control machinery and targeted for ER associated degradation (ERAD). ER associated degradation (ERAD) is the process whereby proteins entering the secretory pathway are subject to degradation when they fail to attain a mature conformation. In fact, even a large percentage of WT ENaC is targeted for the ERAD machinery. Many other disease relevant proteins can also become ERAD substrates, including CFTR (cystic fibrosis), AQP2 (nephrogenic diabetes insipidus), and Pael-R (Parkinson's disease). The focus of this proposal on the role of GRP170 in ENaC quality control and trafficking arises from the following considerations. First, GRP170 is abundant and is a member of the high molecular weight Hsp70-like family of proteins. Second, GRP170 possesses two unique activities: GRP170 acts as a nucleotide exchange factor (NEF) for the ER lumenal Hsp70, and it acts as a "holdase" in vitro and binds to misfolded proteins within the secretory pathway. Third, GRP170 targets the α ENaC subunit for ERAD, but has no effect on the β and γENaC subunits. Fourth, in contrast to its effect on individual subunits, preliminary data suggest that GRP170 promotes the assembly and surface expression of the ENaC channel (αβγ) in Xenopus oocytes. A Fisher Rat Thyroid (FRT) cell system will be used to investigate the differential effect of GRP170 on the monomeric versus assembled ENaC channel. FRT cells form polarized monolayers in culture, allowing for measurement of ENaC subunit stability, surface expression, proteolytic processing, and ENaC function, as measured by amiloride-sensitive Na+ short circuit current. These outputs will be quantified in response to knockdown or overexpression of GRP170. To further characterize the mechanism of GRP170 action on ENaC a series of GRP170 constructs with domain deletions will also be assayed. In addition to its role in regulating ENaC and therefore blood pressure, GRP170 function has been tied to other relevant processes, including insulin secretion, secretion of vascular endothelial growth factor (VEGF), tumor survival, and programmed cell death. Moreover, GRP170 plays a cytoprotective role during ischemia and atherosclerosis. Together, understanding GRP170 mechanism of action will provide novel insights into ENaC function and associated disease states.