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. 2023 May 18;299(6):104837. doi: 10.1016/j.jbc.2023.104837

Figure 8.

Figure 8

PICK1 is specifically involved in the mGluR1-mediated signaling and mGluR1-mediated AMPAR endocytosis.A and B, Western blot (A) and quantitation of the western blots (B) showing that in control cells that were treated with the mGluR5 antagonist MTEP, application of 100 μM R,S-DHPG for 5 min led to the phosphorylation of MAP kinases. Furthermore, the receptors recycled to the cell surface in 2.5 h and also showed the ability to induce phosphorylation of MAP kinases on application of 100 μM R,S-DHPG for 5 min (N = 3). C and D, Western blot (C) and quantitation of the western blots (D) suggested that although in PICK1 knockdown cells, in presence of MTEP, initial application of 100 μM R,S-DHPG for 5 min led to the phosphorylation of MAP kinases, but application of 100 μM R,S-DHPG after 2.5 h did not increase the phosphorylation of MAP kinases (N = 3). E and F, representative images (E) and quantitation of the endocytosis index (F) suggested that in control cells (treated with MTEP), the receptors that recycled back to the cell surface in 2.5 h were able to induce the internalization of GluA1-containing receptors when they were stimulated with 100 μM R,S-DHPG for 5 min. Application of 100 μM R,S-DHPG did not cause endocytosis of GluA1-containing receptors when the mGluR1 recycling was inhibited with okadaic acid and FK-506 (N: control:: untreated = 30; DHPG = 32; 2.5 h untreated = 30; 2.5 h DHPG = 31; 2.5 h (OA + FK-506) untreated = 29; 2.5 h (OA + FK-506) DHPG = 29). G and H, representative images (G) and quantitation of the mGluR-mediated AMPAR endocytosis (H) showed that in shPICK1 transfected cells that were treated with mGluR5 antagonist MTEP, although initial application of 100 μM R,S-DHPG for 5 min led to the endocytosis of GluA1-containing receptors, application of 100 μM R,S-DHPG after 2.5 h did not induce the internalization of GluA1-containing receptors. Expectedly, in okadaic acid and FK-506–treated cells, 100 μM R,S-DHPG did not cause endocytosis of GluA1-containing receptors (N: shPICK1:: untreated = 31; DHPG = 32; 2.5 h untreated = 34; 2.5 h DHPG = 33; 2.5 h (OA + FK-506) untreated = 33; 2.5 h (OA + FK-506) DHPG = 32). Results are presented as means ± SD. Scale bar represents 10 μm. ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05; n.s, p > 0.05. DHPG, 3, 5-dihydroxyphenylglycine; mGluR, metabotropic glutamate receptor; PICK1, protein interacting with C kinase 1; MTEP, (3-((2-methyl-1,3-thiazol-4-yl) ethynyl) pyridine).