Expression (tried Freestyle CHO, ExpiCHO and Freestyle HEK cells) and purification of FL SorCS members (1, 2 and 3) to obtain cryo-EM structure of SorCS members in protein complex (w NMDAR (N1 and N2 subunit)), as no structure is available with the SorCS members in protein complex. Project was terminated after some months, as functional studies showed that we could not use truncated NMDAR for the complex (was initial plan as purified FL NMDAR is difficult to work with).
Is the function of NMDAR SorCS dependent?
- Measure agonist affinity (Gly and Glu) to NMDAR by TEVC in oocytes with/without SorCS1-3 expression. Affinity independent of SorCS expression.
- Measure open probability (Po) of NMDAR by MTSEA assay in oocytes with/without SorCS1-3 expression. N2A have higher Po than NMDAR with N2B subunit and Po is independent of SorCS expression.
- Measure NMDAR current by TEVC in oocytes. NMDAR current is positive dependent of SorCS1 and 2 co-expression, but not SorCS3. We tested different truncated constructs and found that SorCS effect is dependent of the C-terminal tail of N2 subunit. 2/3 of the C-terminal N2 tail is required for a positive SorCS1 and 2 effect.
Is cell surface expression of NMDAR dependent of SorCS expression?
- TEVC suggests, as we report a higher NMDAR current for oocytes co-expressed with SorCS1 and 2 (not 3)
- Measure cell surface expression of NMDAR by biotinylation assay and western blot for quantification (assay not successful, as we obtain unspecific bands in WB for the negative control around the similar size of N2B (we tried different antibodies, but similar effect, however none of the antibodies are quality tested for use in oocytes)
- Change cell systems to quantify cell surface expression. We decided to use HEK cells, as cell system previosly have been used for NMDAR assays and easy to work with (pros). Cons, NMDAR will be in an inactive conformation, as inhibitors need to be applied otherwise HEK cells will die upon over-expression of NMDAR and the observation from TEVC is with an active NMDAR. We don’t know if SorCS:NMDAR complex behave different with active/inactive NMDAR. We use N2A and N2B subunit with ß lactamase tag, so we quantify cell surface expression by absorbance as nitrocefin will change color from yellow to red upon reaction with ß lactamase. As control the assay were tested in oocytes to test the assay with an active NMDAR, but we choose to do the quantification with data from HEK cells as system easier / more robust than oocytes. Cell surface expression experiment showed that SorCS1 and 2, but not SorCS3, increase cell surface expression of NMDAR. It suggests that NMDAR cell surface expression is dependent of SorCS1 and 2 expressions.
Compare protein protein interactions of SorCS1, 2 and 3
SorCS1 and 3 have highes sequence similarity, but we show that SorCS1 and 2 seems to have more similar function regarding their interaction with NMDAR, thus we speculate how is it for the rest of SorCS interaction partners in neurons.
- Learn microscopy and rat dissection of hippocampus to make neuronal cultures and quantify expression by GFP signal in microscope.
- Learn to make Lipid nano particles that encapsules RNA that code for SorCS constructs with TurboID tag (will biotinyle all protein in proximity to SorCS upon addition of biotin, thus the proteins can be collected by lysis and quantified by MS)
- Dissection of hippocampus is time-limit (faster, higher chance for a culture to survive), when I final start to manage the dissection after some try, I had to terminate the project as the project approach its end.
- I quantified neuron samples co-expressed with and without SorCS1, 2 and 3 by Western Blot and we report a higher total expression of N2B for all SorCS members in comparison to control. It suggests that NMDAR expression is SorCS dependent, but further studies are required to tell if SorCS3 can participate in trafficking of NMDAR to the cell surface as SorCS1 and 2. Few results in the literature have suggested that SorCS3 may can participating in trafficking of proteins by forming bigger complexes with PSD proteins (if that is the case, it will explain why we observe no effect of. SorCS3 in experiment performed in HEK and oocytes as we only co-expressed NMDAR and SorCS (PSD proteins not present in these cellular systems)).
