Primary cilia function is essential for the development of normal left-right asymmetry, kidneys, heart, central nervous system and many other organs. Current data indicate that the cilium functions as a signal transduction organelle, compartmentalizing receptor-, adaptor- and downstream transduction mechanisms for multiple signaling pathways. However, the identity of essential cilia signals and how signals are transduced by the cilium remains unclear. A complex of four ciliary proteins, inversin, NEK8, ANKS6 and nephrocystin-3 constitutes the so-called ciliary inversin compartment (IC), localized in the proximal portion of the ciliary shaft. We and others found that the IC is required for cilium function in vertebrates, with mutations in INVS, NEK8, ANKS6 and NPHP3 all leading to ciliopathy syndromes that include L-/R-asymmetry perturbation, congenital heart defects and polycystic kidneys. These defects phenocopy the full knockout of PKD2, one of the two major autosomal-dominant polycystic kidney disease genes, suggesting that signaling downstream of ADPKD- and IC-gene products occurs along the same pathway. We found that IC proteins also control ciliary length and intraflagellar transport (IFT) velocity, processes that require cAMP signaling. Unexpectedly, we find that nephrocystin-3 acts as a novel GTPase, suggesting a causal link between IC- and cAMP-signaling. In this grant proposal, we aim to investigate the functional significance of the nephrocystin-3 GTPase mechanism, defining the catalytic parameters of the enzyme, and interrogating molecular connections among the IC constituents and to upstream- and downstream signaling factors. We hypothesize that nephrocystin-3 acts analogous to a stimulatory heterotrimeric G-protein ?-subunit, switching its activity state off and on, along a GTP-hydrolysis cycle.
In Aim #1, we will investigate nucleotide affinitiy and -turnover of the purified recombinant nephrocystin-3, along with the pcy allele that affects the catalytic site, and that gives rise to polycystic kidney disease in mice; compared to G?S. We will test, whether ciliary G-protein coupled receptors may utilize nephrocystin-3 as a signal adaptor, and whether adenylyl cyclases may be stimulated by nephrocystin-3, to generate cAMP.
In Aim #2, we investigate the molecular details of the IC assembly hierarchy. We seek to identify what protein domains are necessary to recruit nephrocystin-3 to its proper localization, to maintain the integrity of the whole complex, and if disease-causing mutations destabilize the structure. We will ultimately measure ciliary cAMP levels, as a function of IC manipulations, and test whether deletion or mutation of individual IC proteins change ciliary cAMP levels. These studies connect ciliopathy gene function to cAMP signaling, thereby addressing outstanding issues in ciliary biology with important implications for cystic kidney disease pathogenesis. The results will help identifying druggable enzymatic targets within the IC that may lead to the design of novel therapies.
/Relevance The disease-causing genes for autosomal-dominant polycystic kidney disease and many other genetic/cystic kidney diseases have been identified, but it remains largely unknown, how gene products perform their signaling functions in health and in disease pathogenesis. Many disease gene products localize to the antenna-like primary cilium of cells, where a specific complex of four proteins, the so-called inversin compartment, seems to act like a switch that can be turned into an ?active? and an ?inactive? signaling position. This study investigates the biochemical properties of this switch-like mechanism, the identity of upstream- and downstream actors in the signaling chain, and the cellular consequences when the signaling pathway is active, thereby exploring novel therapeutic approaches to genetic kidney disease.