Cyclic AMP-dependent protein kinase phosphorylates group III metabotropic glutamate receptors and inhibits their function as presynaptic receptors.

Recent evidence suggests that the functions of presynaptic metabotropic glutamate receptors (mGluRs) are tightly regulated by protein kinases. We previously reported that cAMP-dependent protein kinase (PKA) directly phosphorylates mGluR2 at a single serine residue (Ser843) on the C-terminal tail region of the receptor, and that phosphorylation of this site inhibits coupling of mGluR2 to GTP-binding proteins. This may be the mechanism by which the adenylyl cyclase activator forskolin inhibits presynaptic mGluR2 function at the medial perforant path-dentate gyrus synapse. We now report that PKA also directly phosphorylates several group III mGluRs (mGluR4a, mGluR7a, and mGluR8a), as well as mGluR3 at single conserved serine residues on their C-terminal tails. Furthermore, activation of PKA by forskolin inhibits group III mGluR-mediated responses at glutamatergic synapses in the hippocampus. Interestingly, beta-adrenergic receptor activation was found to mimic the inhibitory effect of forskolin on both group II and III mGluRs. These data suggest that a common PKA-dependent mechanism may be involved in regulating the function of multiple presynaptic group II and group III mGluRs. Such regulation is not limited to the pharmacological activation of adenylyl cyclase but can also be elicited by the stimulation of endogenous G(s)-coupled receptors, such as beta-adrenergic receptors.

Coordinate regulation of metabotropic glutamate receptors.

Recent studies aimed at identifying the mechanisms that regulate the signaling of metabotropic glutamate receptors (mGluRs) have revealed that both protein kinase and protein phosphatase activity are important in directly modulating mGluR function. The inter-relationship between phosphorylation and dephosphorylation of mGluRs seems to be an important determinant in regulating mGluR function and the subsequent neuromodulatory events elicited by activation of mGluRs.

A pleckstrin homology domain specific for phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P2) and fused to green fluorescent protein identifies plasma membrane PtdIns-4,5-P2 as being important in exocytosis.

Kinetically distinct steps can be distinguished in the secretory response from neuroendocrine cells with slow ATP-dependent priming steps preceding the triggering of exocytosis by Ca(2+). One of these priming steps involves the maintenance of phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) through lipid kinases and is responsible for at least 70% of the ATP-dependent secretion observed in digitonin-permeabilized chromaffin cells. PtdIns-4,5-P(2) is usually thought to reside on the plasma membrane. However, because phosphatidylinositol 4-kinase is an integral chromaffin granule membrane protein, PtdIns-4,5-P(2) important in exocytosis may reside on the chromaffin granule membrane. In the present study we have investigated the localization of PtdIns-4,5-P(2) that is involved in exocytosis by transiently expressing in chromaffin cells a pleckstrin homology (PH) domain that specifically binds PtdIns-4, 5-P(2) and is fused to green fluorescent protein (GFP). The PH-GFP protein predominantly associated with the plasma membrane in chromaffin cells without any detectable association with chromaffin granules. Rhodamine-neomycin, which also binds to PtdIns-4,5-P(2), showed a similar subcellular localization. The transiently expressed PH-GFP inhibited exocytosis as measured by both biochemical and electrophysiological techniques. The results indicate that the inhibition was at a step after Ca(2+) entry and suggest that plasma membrane PtdIns-4,5-P(2) is important for exocytosis. Expression of PH-GFP also reduced calcium currents, raising the possibility that PtdIns-4,5-P(2) in some manner alters calcium channel function in chromaffin cells.

Attenuation of focal adhesion kinase signaling following depletion of agonist-sensitive pools of phosphatidylinositol 4,5-bisphosphate.

The effect of phosphoinositide depletion on focal adhesion kinase (FAK) signaling was investigated in two neuronal cell lines. Treatment of either SH-SY5Y neuroblastoma cells or PC12 cells with wortmannin, at a concentration that inhibits phosphatidylinositol 4-kinase activity, led to a selective depletion of phosphatidylinositol 4-phosphate without significantly altering phosphatidylinositol 4,5-bisphosphate (PIP2) content. An enhanced tyrosine phosphorylation of FAK elicited by agonist occupancy of phospholipase C-coupled receptors (muscarinic cholinergic in SH-SY5Y neuroblastoma or bradykinin in PC12 cells) was blocked completely by wortmannin. Under the above conditions, phosphoinositide resynthesis was prevented, and as a consequence, receptor stimulation led to a marked depletion of PIP2. In contrast, the increased tyrosine phosphorylation of FAK elicited by agents that do not activate phospholipase C (phenylarsine oxide, lysophosphatidic acid, or phorbol ester) persisted in the presence of wortmannin. However, the ability of these agents to elicit an increase in FAK phosphorylation was also prevented if PIP2 was depleted by activation of a phospholipase C-coupled receptor in the presence of wortmannin. The results suggest that agonist-sensitive pools of PIP2 must be maintained for FAK signaling to occur in response to a mechanistically diverse range of stimuli.

Inhibition of beta(2)-adrenergic and muscarinic cholinergic receptor endocytosis after depletion of phosphatidylinositol bisphosphate.

Recent evidence supporting a role for phosphoinositides in the endocytosis of phospholipase C-coupled receptors has prompted an investigation of whether there exists a similar requirement for the internalization of adenylyl cyclase-linked receptors. When 1321N1 astrocytoma cells, which possess both muscarinic cholinergic receptors (mAChRs) that couple to phospholipase C and beta-adrenergic receptors (beta(2)-ARs) linked to adenylyl cyclase, were pretreated with wortmannin (WT) at a concentration known to inhibit phosphatidylinositol 4-kinase activity, the labeling of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP(2)) was reduced. Stimulation of phosphoinositide breakdown by activation of mAChRs in WT-pretreated cells led to a further depletion of PIP(2). As previously demonstrated for SH-SY5Y neuroblastoma, inclusion of WT inhibited the endocytosis of mAChRs in 1321N1 cells by >85%. In contrast, the internalization of beta(2)-ARs was only partially ( approximately 30%) prevented. However, when the concentration of PIP(2) was further reduced by exposure of WT-pretreated 1321N1 cells to a muscarinic agonist, the endocytosis of beta(2)-ARs was substantially inhibited (>70%). Lower concentrations of WT (100 nM) that were sufficient to fully inhibit phosphatidylinositol 3-kinase activity had no effect on either phosphoinositide synthesis or receptor endocytosis. The results indicate that the agonist-induced endocytosis of an adenylyl cyclase-linked receptor such as the beta(2)-AR, like that of the phospholipase C-coupled mAChR, is dependent on the synthesis of phosphoinositides and, in particular, that of PIP(2).

Distinct mechanisms for the endocytosis of muscarinic receptors and Gq/11.

To determine whether the agonist-mediated endocytosis of muscarinic receptors and Gq/11 are mechanistically related events, the internalization of Gq/11 was monitored under conditions established to prevent muscarinic receptor endocytosis. Incubation of SH-SY5Y neuroblastoma cells with oxotremorine-M resulted in the translocation of both muscarinic receptors and Gq/11 into a 'light vesicle' membrane fraction. Although muscarinic receptor translocation was prevented by either the depletion of phosphoinositides or by disruption of clathrin assembly, the endocytosis of Gq/11 was unaffected. These results demonstrate that the agonist-induced internalization of muscarinic receptors and Gq/11 proceed via distinct mechanisms.

A role for a wortmannin-sensitive phosphatidylinositol-4-kinase in the endocytosis of muscarinic cholinergic receptors.

A role for phosphoinositides in the endocytosis of muscarinic cholinergic receptors (mAChRs) has been investigated via inhibition of the activity of phosphatidylinositol-4-kinase (PI4K). Pretreatment of SH-SY5Y neuroblastoma cells with micromolar concentrations of wortmannin (WT), LY-294002, or phenylarsine oxide (PAO), three chemically distinct agents known to inhibit PI4K, resulted in both an inhibition of agonist-induced endocytosis of mAChRs and a selective reduction in the 32P-labeling of phosphatidylinositol-4-phosphate. PAO-mediated inhibition of both receptor endocytosis and phosphoinositide synthesis could be fully reversed by inclusion of the bifunctional thiol 2, 3-dimercaptopropanol. The requirement for phosphoinositide synthesis in mAChR endocytosis was independent of a role for these lipids in the maintenance of the cytoskeleton because disruption of the latter with cytochalasin D, ML-7, or colchicine failed to inhibit receptor internalization. Determination of PI4K activity in subcellular fractions of SH-SY5Y cells indicated that enzyme activity in fractions enriched in endocytic vesicles and cytosol was preferentially inhibited by WT, LY-294002, and PAO, a profile consistent with the subcellular distribution of the 110-kDa beta isoform of PI4K, as determined by Western blot analysis. Activity of PI4Kbeta present in immunoprecipitated cell lysates was inhibited >75% by inclusion of each of the three inhibitors. These results indicate that ongoing synthesis of phosphoinositides is necessary for mAChR endocytosis and that the activity of a WT-sensitive form of PI4K, such as PI4Kbeta, is required.

Down-regulation of phospholipase C-beta1 following chronic muscarinic receptor activation.

To determine whether prolonged activation of a phospholipase C-coupled receptor can lead to a down-regulation of its effector enzyme, SH-SY5Y neuroblastoma cells were incubated for 24 h with the muscarinic receptor agonist, oxotremorine-M. Under these conditions, significant reductions (46-53%) in muscarinic cholinergic receptor density, G(alphaq/11) and phospholipase C-beta1 (but not the beta3-or gamma1 isoforms) were observed. These results suggest that a selective down-regulation of phospholipase C-beta1 may play a role in adaptation to chronic muscarinic receptor activation.

Cytoskeletal and phosphoinositide requirements for muscarinic receptor signaling to focal adhesion kinase and paxillin.

The mechanism whereby agonist occupancy of muscarinic cholinergic receptors elicits an increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin has been examined. Addition of oxotremorine-M to SH-SY5Y neuroblastoma cells resulted in rapid increases in the phosphorylation of FAK (t(1/2) = 2 min) and paxillin that were independent of integrin-extracellular matrix interactions, cell attachment, and the production of phosphoinositide-derived second messengers. In contrast, the increased tyrosine phosphorylations of FAK and paxillin were inhibited by inclusion of either cytochalasin D or mevastatin, agents that disrupt the cytoskeleton. Furthermore, phosphorylation of FAK and paxillin could be prevented by addition of either wortmannin or LY-294002, under conditions in which the synthesis of phosphatidylinositol 4-phosphate was markedly attenuated. These results indicate that muscarinic receptor-mediated increases in the tyrosine phosphorylation of FAK and paxillin in SH-SY5Y neuroblastoma cells depend on both the maintenance of an actin cytoskeleton and the ability of these cells to synthesize phosphoinositides.

Agonist-induced endocytosis of muscarinic cholinergic receptors: relationship to stimulated phosphoinositide turnover.

The ability of muscarinic cholinergic receptors to activate phosphoinositide turnover following agonist-induced internalization has been investigated. Incubation of SH-SY5Y neuroblastoma cells with oxotremorine-M resulted in a time-dependent endocytosis of both muscarinic receptors and alpha subunits of G0 and G11, but not of isoforms of phosphoinositide-specific phospholipase C, into a subfraction of smooth endoplasmic reticulum (V1). Agonist-induced increases in diacylglycerol mass and in 32P-phosphatidate labeling, much of which was of the tetraenoic species, were also observed in the V1 fraction, but these increases persisted when the agonist-induced translocation of receptors into the V1 fraction was blocked. All enzymes of the phosphoinositide cycle were detectable in the V1 fraction. However, with the exception of phosphatidylinositol 4-kinase, none was enriched when compared with cell lysates. Both 32P-labeling studies and enzyme assays point to a very limited capacity of this fraction to synthesize phosphatidylinositol 4,5-bisphosphate, whereas the synthesis of phosphatidylinositol 4-phosphate is robust. These results indicate that endocytosed receptors do not appear to retain their ability to activate phosphoinositide turnover. The availability of the substrate for phospholipase C, phosphatidylinositol 4,5-bisphosphate, may be one factor that limits the activity of muscarinic receptors in this subcellular compartment.