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

Figure 6.

Figure 6

Knockdown of endogenous PICK1 does not have any effect on mGluR-mediated signaling and AMPAR endocytosis when both mGluR1 and mGluR5 are activated.A and B, Western blot (A) and quantitation of the western blots (B) showing that in control cells, application of 100 μM R,S-DHPG for 5 min increased the phosphorylation of MAP kinases. The receptors that recycled to the cell surface in 2.5 h also induced phosphorylation of MAP kinases upon application of the agonist (N = 3). C and D, Western blot (C) and quantitation of the western blots (D) suggested that similar to control cells, in PICK1 knockdown cells, initial application of 100 μM R,S-DHPG for 5 min increased the phosphorylation of MAP kinases and application of the agonist after 2.5 h also resulted in the increase in the phosphorylation of MAP kinases (N = 3). E and F, representative images (E) and quantitation of the mGluR-mediated AMPAR endocytosis (F) suggested that in control cells, initial application of 100 μM R,S-DHPG for 5 min led to the endocytosis of AMPARs. The mGluRs that recycled back to the cell surface in 2.5 h were able to induce the endocytosis of GluA1-containing receptors when they were stimulated with 100 μM R,S-DHPG for 5 min. On the other hand, application of 100 μM R,S-DHPG did not cause endocytosis of GluA1-containing receptors when the mGluR recycling was inhibited with okadaic acid and FK-506 (N: control:: untreated = 37; DHPG = 36; 2.5 h untreated = 37; 2.5 h DHPG = 41; 2.5 h (OA + FK-506) untreated = 39; 2.5 h (OA + FK-506) DHPG = 36). G and H, representative images (G) and quantitation of the mGluR-mediated AMPAR endocytosis (H) showed that in shPICK1 transfected cells, initial application of 100 μM R,S-DHPG for 5 min led to the endocytosis of GluA1-containing receptors, and the receptors that recycled back to the cell surface in 2.5 h post agonist application also induced the internalization of GluA1-containing receptors. When the mGluR recycling was inhibited with okadaic acid and FK-506, application of 100 μM R,S-DHPG could not induce endocytosis of GluA1-containing receptors (N: shPICK1:: untreated = 31; DHPG = 31; 2.5 h untreated = 39; 2.5 h DHPG = 37; 2.5 h (OA + FK-506) untreated = 33; 2.5 h (OA + FK-506) DHPG = 35). 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.