Aim 1a: Identify which rhomboid proteases are expressed in glioma cell lines
The mechanism by which extracellular PTPμ fragments are generated remains incompletely understood. Biochemical characterization of PTPμ proteolysis demonstrate that ADAM/MMP inhibition does not completely abrogate cleavage and implicate a serine protease to contribute to shedding of extracellular fragments28 (Figure 2). Rhomboid proteases are the only serine proteases located in the membrane and are predicted to generate fragments of the correct molecular weights.
To test whether rhomboid proteases could be contributing to shedding of PTPμ, we will first determine which rhomboids are expressed in non-invasive and ...view middle of the document...
Aim 1b: Determine which rhomboid proteases cleave and release PTPμ
There are currently four known mammalian rhomboid proteases: RHBDL1, RHBDL2, RHBDL3, and RHBDL4. RHBDL1 expression is restricted to the Golgi apparatus and is only weakly detected at the plasma membrane40. RHBDL2 is the best understood rhomboid protease and is located in the plasma membrane39. RHBDL3 is also known as presenilin-associated rhomboid-like and is localized to the inner mitochondrial membrane40. RHBDL4 is located in endoplasmic reticulum and is involved in endoplasmic reticulum–associated degradation41. Due to their localization within cells, RHBDL3 and RHBDL4 would not be expected to contribute to PTPμ shedding. Therefore, we hypothesize that rhomboid proteases, RHBDL1 or RHBDL2, cleave and release PTPμ ectodomains.
Genetic knockdown of a protein of interest is a powerful tool to study the function of a protein. Using a lentiviral strategy, RHBDL1, RHBDL2, RHBDL3, and RHBDL4 will be knocked down by shRNA. For this aim, investigating the role of rhomboid proteases in the proteolytic processing of PTPμ in glioma cells, the LN-229 invasive glioma cell line will be used. LN-229 cells are the appropriate cell line choice because they endogenously proteolyze PTPμ and generate the same fragments detected in human glioblastoma specimens. To increase the amount of PTPμ material to work with and accurately analyze, LN-229 will be infected with lentiviral particles to express exogenous full-length PTPμ the day after plating.
LN-229 cells will be maintained in complete media and will be plated in 100mm dishes. The next day the cells will be infected with lentiviral particles containing shRNA targeting RHBDL1, RHBDL2, RHBDL3, or RHBDL4. Controls cells will be infected with lentiviral particles containing scrambled shRNA. Protein knockdown using shRNA requires two to three days. Following this period, the cells will be washed and the media will be replaced with serum-free media. The next day cells will then be treated with an ADAM/MMP inhibitor overnight. The following day, cells will be untreated or treated with ionomycin for 30 minutes to stimulate ectodomain shedding. (Ectodomain shedding occurs inefficiently in vitro probably due to the absence of tumor microenvironment specific factors. The calcium ionophore, ionomycin, mimics growth factor stimulation to induce shedding in vitro.) The culture supernatant will be collected. Shed PTPμ fragments will be precipitated from the culture supernatant by TCA. Cells will be harvested for lysates which will contain the cell-associated PTPμ proteins. Shed PTPμ fragments and cell-associated PTPμ proteins will be resolved by SDS-PAGE gels and immunoblotted with the BK2 antibody directed to the MAM domain of PTPμ.
Predictions, alternative approaches, and potential pitfalls:
We predict that RHBDL2 mediates cleavage of PTPμ based on the observation that both proteins are present in filopodial like structures 39,42. Additionally,...