An investigation into the functional role of the RhoGAP protein SH3BP1/ARHGAP43 in Autosomal Dominant Polycystic Kidney Disease

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a potentially life changing and life-threatening disease that affects up to 1:1000 people. The disease causes fluid filled cysts to grow in the kidneys where they continue to grow to huge sizes through a patient's life. These cysts slowly reduce the function of both kidneys until patients reach end stage kidney failure, most commonly by the ages of 50 to 60. When patients reach end stage kidney failure, they will need dialysis or kidney transplant to survive. ADPKD is caused by a problem in a patient's genetic code which leads to defects in the proteins Polycystin 1 (PC1) or Polycystin 2 (PC2). Parents with one of these genetic problems have a 50:50 chance of passing this down to their children and there is currently no cure. There is only one treatment that can delay developing end stage kidney failure, but it has significant side effects and only slows the disease a small amount.
Even though we have known what damaged proteins cause ADPKD for a number of years, how damage to these proteins causes cysts to grow remains a mystery. By looking at the cells lining these these cysts we can see they grow and divide faster but also move slower and are less adherent to each other than normal cells. However, how PC1 or PC2 damage leads to these changes in cells is not currently know.
This project will aim to understand what the functions of PC1 and PC2 do when not defective and how defects in these proteins lead to the known changes in cell behaviour and therefore cyst growth. We have newly discovered that PC1 interacts with another protein, SH3BP1. This protein has been shown to be important for cell movement and cell to cell adhesion. Further, it is also important in the uptake into the cells of certain receptors that stimulate cell growth after being activated. This process has only recently been discovered and its potential role in ADPKD has not yet been explored. SH3BP1 provides an exciting potential explanation for why defective PC1 leads to the changes in cells we see under the microscope and therefore how cysts develop.
I will use lab-based techniques to establish what parts of PC1 and SH3BP1 interact specifically and if this interaction changes how either protein works. I will also be able to screen for any other proteins that may also be involved in this interaction using mass spectrometry. Using high powered microscopy, I will look at how specific growth receptors are taken up by cells in real time and evaluate if this is different in cells with damaged PC1 or SH3BP1. Finally, to confirm the role of the interaction of PC1 and SH3BP1 in cyst growth, I will see if increasing or decreasing SH3BP1 in cells with damaged PC1 will reduce the growth of cysts in 3D. By completing this project, we will have a better understanding of how cysts grow in ADPKD and this may lead to better treatments for the disease which are desperately needed.

Autosomal Dominant Polycystic Kidney disease (ADPKD) is the most common monogenic kidney disease in man and a leading cause of end stage renal failure. It most commonly results from mutations in PKD1 or PKD2, encoding Polycystin 1 (PC1) and Polycystin 2 (PC2). Treatment for ADPKD is limited and hampered by side effects and reduced efficacy. The mechanisms behind cyst development in ADPKD are unclear but cellular hyperproliferation, reduced cell motility and impaired cell-cell junction formation.
This project aims to evaluate the functional relevance of a recently identified interaction between PC1 and a Rho-GAP protein, SH3BP1. Through its GAP activity towards Cdc42 or Rac1, SH3BP1 regulates cell-cell adhesion, cell migration and actin cytoskeletal organisation. It has also been recently identified as a key regulator or a novel mechanism of endocytosis (Fast Endophilin Mediated Endocytosis, FEME). Important growth factor receptors such as EGFR can be internalised via FEME so thus the process may play a role in cellular proliferation. The SH3BP1-PC1 interaction may provide a mechanistic explanation to the cellular phenotypes observed in cystic cells.
I will use a combination of co-IP, pull downs and mass spectrometry, we will evaluate the exact binding determinants between the PC1-SH3BP1 interaction. PC1 can dephosphorylate interacting proteins (such as PC2) and SH3BP1 is highly phosphorylated. Using phosphoproteomics, I will identify any specific phosphorylation sites on SH3BP1 before evaluating them for functional relevance in cystic cells and cyst development in vitro. Through live cell imaging I will evaluate the internalisation of specific growth factor receptors shown to drive hyperproliferation in ADPKD cells such as EGFR. Using markers of FEME and other forms of endocytosis, I will specify if any forms of endocytosis are deranged in ADPKD. Finally, the role of SH3BP1 in cyst development will be evaluated in vitro through use of 3D cystic cell models.