Glutamatergic regulation of serine racemase via reversal of PIP2 inhibition

Abstract

D-serine is a physiologic coagonist with glutamate at NMDA-subtype glutamate receptors. As D-serine is localized in glia, synaptically released glutamate presumably stimulates the glia to form and release D-serine, enabling glutamate/D-serine cotransmission. We show that serine racemase (SR), which generates D-serine from L-serine, is physiologically inhibited by phosphatidylinositol (4,5)-bisphosphate (PIP2) presence in membranes where SR is localized. Activation of metabotropic glutamate receptors (mGluR5) on glia leads to phospholipase C-mediated degradation of PIP2, relieving SR inhibition. Thus mutants of SR that cannot bind PIP2 lose their membrane localizations and display a 4-fold enhancement of catalytic activity. Moreover, mGluR5 activation of SR activity is abolished by inhibiting phospholipase C.

Fig. 1.

SR binds to phospholipids. (A) Immunocytochemistry of SR in mouse primary culture glia is consistent with localization to the plasma membrane and small granular structures that may be vesicles. SR is highlighted in green while the glial marker, glial fibrillary acidic protein (GFAP), is shown in red. (B) PIP strip assays with purified His-SR reveal prominent binding to a variety of lipids. Singly phosphorylated PIPs provide the most prominent binding with somewhat lesser binding for various forms of PIP2 and least for PIP3. PtdIns, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PA, phosphatidic acid; and PS, phosphatidylserine. (C) Liposomal assays in vitro with His-SR give similar findings as in B. PI, phosphatidylinositol. (D) PolyPIPosome assays in HEK293 cells transfected with myc-tagged SR show greatest binding to PI(4,5)P2. (E) Similar results as in D are seen in intact mouse brain. Akt, a known interactor of lipids, is used as a positive control. (F) Fluorescence anisotropy assays with pure SR and BODIPY-TMR-labeled PIP2 reveal specific interactions between SR and PIP2 with a K_d of ≈1 μM. Boiled SR does not interact with PIP2. (G) Fluorescence anisotropy of the PH domain of PLCδ1 provides a K_d of ≈0.25 μM, similar to values for SR. Unlabeled PIP2 potently displaces binding with a K_i of 0.1 μM. Bars represent the mean ± SEM of 3 independent experiments each performed in triplicate.

Fig. 2.

PIP2 inhibits SR. (A) Assays of SR in the presence of 50-μM lipids reveal specific inhibition by a mixture of PIPs, while PA moderately inhibits SR. 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) and 1-oleoyl-2-acetyl-sn-glycerol (OAG) are diacylglycerol derivatives. (B) SR is markedly inhibited by 50 μM PIP2 and PIP3. The singly phosphorylated PIs do not significantly impact activity. (C) Inositol phosphate head groups lacking the hydrophobic fatty acid component do not inhibit SR. (D) PI(4,5)P2 concentration dependently inhibits SR with an IC₅₀ of 13 μM. Bars represent the mean ± SEM of 3 independent experiments each performed in triplicate.

Fig. 3.

Mutation of specific SR residues prevents its inhibition via PIP2 binding. (A) GDDA-BLAST analysis reveals a hidden peripheral lipid-binding (PLB) domain within the SR sequence. Specific potential lipid interacting surface residues are also identified. (B) PIP2 PolyPIPosome assay with pure wild-type vs. mutant SR proteins reveals K96, K137, and L168 as potential lipid binding residues, because their mutation to alanine hampers interaction with PIP2. (C) SR activity assays with or without PIP2 show that SR-K96A, K137A, and L168A are resistant to PIP2 inhibition. All 3 mutants are basally as active as the wild-type protein. (D) Mouse primary culture glia transfected with myc-tagged wild-type SR or the L168A mutant demonstrate a requirement of L168 for interaction with PIP2 in glia, as L168A does not coprecipitate with PIP2 PolyPIPosomes. (E) In intact primary glia the nonlipid interacting myc-tagged L168A mutant SR has a diffuse cytosolic distribution, whereas the wild-type protein is highly concentrated at lipid membranes, particularly the plasma membrane (arrows). SR is represented in green, while the nuclear stain, DAPI, is shown in blue. Bars represent the mean ± SEM of 3 independent experiments each performed in triplicate.

Fig. 4.

PIP2 inhibits SR by competing with ATP. (A) Model of mammalian SR showing ATP in yellow, magnesium ion in black, leucine 168 in pink, and lysines 75, 77, 96, and 137 in green. The PIP2 interacting residues are adjacent to the ATP binding site on SR, presumably interfering with the activation of SR by ATP. (B) Fluorescence anisotropy reveals that the binding of SR to PIP2 is competitively reversed by ATP in a concentration-dependent fashion. (C) Liposomal assays also show that ATP reverses the SR–PIP2 interaction in a concentration-dependent manner. (D) Pure His-SR enzyme activity was measured in the presence or absence of PIP2 and increasing concentrations of ATP. PIP2 inhibits SR activity by interfering with ATP, as PIP2 causes a 100-fold increase in the EC₅₀ for ATP with no change in maximal activation. (E) SR enzyme activity measured in the presence or absence of PIP2 and increasing L-serine concentration displays a substantial reduction in V_max with no alteration in K_m. Hence, PIP2 inhibits SR noncompetitively. Bars represent the mean ± SEM of 3 independent experiments each performed in triplicate.

Fig. 5.

Metabotropic glutamate receptor activation stimulates SR by reversing PIP2 inhibition. (A) HEK293 cells transfected with wild-type SR and mGluR5 display a 4-fold increase in SR activity upon treatment with 50 μM of the mGluR agonist DHPG for 30 min. The PLC inhibitor U73122 (10 μM) reverses mGluR5-mediated activation of SR. The non-PIP2 binding mutant L168A is 4 times more active basally than the wild-type enzyme. Additionally, L168A activity does not appear to be altered by either DHPG or U73122, reflecting an absence of mGluR5-mediated regulation. (B) Glial cells endogenously expressing mGluR5 and SR show increased D-serine production with 50 μM DHPG treatment for 30 min. U73122 (10 μM) reverses this effect, demonstrating that glutamate transmission activates SR by degrading phospholipids. Bars represent the mean ± SEM of 3 independent experiments, each performed in triplicate.

Fig. 6.

Model for D-serine signaling in the brain. D-serine is synthesized from L-serine by SR and stored primarily within astrocytes ensheathing neuronal synapses containing NMDA receptors. SR and D-serine may also occur in neurons (10). When the presynaptic neuron releases glutamate, it acts not only on the postsynaptic neuron, but also on the ensheathing astrocyte, resulting in the activation of metabotropic glutamate receptors and consequent degradation of PIP2 by PLC; subsequent activation of SR occurs through ATP binding. In the synaptic cleft, D-serine binds to the glycine/D-serine binding site on the NMDA receptor and, in conjunction with L-glutamate, elicits opening of the receptor channel. This model affords a potential activation mechanism of the D-serine synthesis needed for NMDA neurotransmission.