Abstract
The metabotropic glutamate receptor type 7 (mGluR7) is the predominant group III mGluR in the presynaptic active zone, where it serves as an autoreceptor to inhibit neurotransmitter release. Our previous studies show that PKC phosphorylation of mGluR7 on Ser-862 is a key mechanism controlling constitutive and activity-dependent surface expression of mGluR7 by regulating a competitive interaction of calmodulin and protein interacting with C kinase (PICK1). As receptor phosphorylation and dephosphorylation are tightly coordinated through the precise action of protein kinases and phosphatases, dephosphorylation by phosphatases is likely to play an active role in governing the activity-dependent or agonist-induced changes in mGluR7 receptor surface expression. In the present study, we find that the serine/threonine protein phosphatase 1 (PP1) has a crucial role in the constitutive and agonist-induced dephosphorylation of Ser-862 on mGluR7. Treatment of neurons with PP1 inhibitors leads to a robust increase in Ser-862 phosphorylation and increased surface expression of mGluR7. In addition, Ser-862 phosphorylation of both mGluR7a and mGluR7b is a target of PP1. Interestingly, agonist-induced dephosphorylation of mGluR7 is regulated by PP1, whereas NMDA-mediated activity-induced dephosphorylation is not, illustrating there are multiple signaling pathways that affect receptor phosphorylation and trafficking. Importantly, PP1γ1 regulates agonist-dependent Ser-862 dephosphorylation and surface expression of mGluR7.
Highlights
MGluR7 is a presynaptic autoreceptor in the CNS, which is regulated by receptor phosphorylation
Our previous studies show that PKC phosphorylation of metabotropic glutamate receptor type 7 (mGluR7) on Ser-862 is a key mechanism controlling constitutive and activity-dependent surface expression of mGluR7 by regulating a competitive interaction of calmodulin and protein interacting with C kinase (PICK1)
As receptor phosphorylation and dephosphorylation are tightly coordinated through the precise action of protein kinases and phosphatases, dephosphorylation by phosphatases is likely to play an active role in governing the activity-dependent or agonist-induced changes in mGluR7 receptor surface expression
Summary
Cells and Antibodies—HEK cells were maintained in DMEM containing 10% fetal bovine serum and 1% L-glutamine. Precleared supernatants were incubated with antibody-bound protein A or G beads (Sigma) for 4 h at 4 °C and washed four times with lysis buffer. Primary cultured cortical neurons (days in vitro 14) were treated with 50 nM okadaic acid or dimethyl sulfoxide for 45 min at 37 °C, and washed three times with ice-cold PBS containing 1 mM MgCl2 and 0.1 mM CaCl2 (PBSϩϩ). The neurons were washed, permeabilized with 0.2% Triton X-100 for 5 min, and blocked with 10% normal goat serum for 1 h, and internalized receptors were visualized by staining with Alexa Fluor 488-conjugated secondary antibody (green). To detect FLAG expression, rabbit anti-FLAG antibody (1:500) was incubated after blocking with normal goat serum, followed by co-staining with Alexa Fluor 648-conjugated secondary antibody. The amount of internalization was quantified by measuring the integrated intensity of green and red signals using MetaMorph software (version 7.0, Universal Imaging Corp.)
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