Rationale: MacMARCKS was identified as an interaction partner of the presynaptic metabotropic glutamate receptor mGluR7 in a Y2H screen. Endogenous mGluR7 can be immunoprecipitated from cultured cerebellar granule cells and MacMARCKs can be co-immunoprecipitated (Fig. 5a). Overexpression of myc-tagged mGluR7 and GFP-tagged MacMARCKS in cultured cerebellar granule cells induces localization of both proteins to the plasma membrane of the soma and dendrites as assessed by ICC (Fig. 5f). Expression of a mGluR7 mutant devoid of the binding domain to MacMARCKS (mGluR7(ΔMacMARCKS) causes a retention of MacMARCKS in the cytoplasm of the cell (Fig. 5g).
mGluR7 is involved in promoting G-protein-mediated inhibition of voltage-sensitive calcium channels (VSCCs). Co-expression of mGluR7A and MacMARCKS in cultured cerebellar granule cells strongly reduces tonic inhibition of VSCCs (Fig. 6b1) indicating an indirect role of MacMARCKS for the regulation of voltage-cated calcium channel activity.
Antibody for WB was generated using purified GST-MacMARCKS fusion protein. Authors specify that the antibody was "not optimal for immunofluorescent labelling", therefore GFP-tagged MacMARCKS was overexpressed in cells for ICC. No KO-control.
25/8/2017 Pim
- The MacMARCKS protein is inferred to exert the function at the presynapse based on (1) the mentioned presynaptic localization of the mGLUR7 and (2) the presence of MacMARCKs/MRP/MARCKSL1 in brain synapses (ticket #833). Experimental description: [...] a new-born rat brain cDNA library subcloned into the pAD vector were sequentially transformed into... To generate a MAcMARCKs expression plasmid, the entire coding sequence of MacMARCKs was amplified by PCR from the isolated pAD clone using...