Spatial-temporal patterning of metabotropic glutamate receptor-mediated inositol 1,4,5-triphosphate, calcium, and protein kinase C oscillations: protein kinase C-dependent receptor phosphorylation is not required.

John P. Robarts Research Institute, P. O. Box 5015, 100 Perth Drive, London, Ontario N6A 5K8, Canada.
Journal of Biological Chemistry (Impact Factor: 4.65). 10/2001; 276(38):35900-8. DOI: 10.1074/jbc.M103847200
Source: PubMed

ABSTRACT The metabotropic glutamate receptors (mGluR), mGluR1a and mGluR5a, are G protein-coupled receptors that couple via G(q) to the hydrolysis of phosphoinositides, the release of Ca(2+) from intracellular stores, and the activation of protein kinase C (PKC). We show here that mGluR1/5 activation results in oscillatory G protein coupling to phospholipase C thereby stimulating oscillations in both inositol 1,4,5-triphosphate formation and intracellular Ca(2+) concentrations. The mGluR1/5-stimulated Ca(2+) oscillations are translated into the synchronized repetitive redistribution of PKCbetaII between the cytosol and plasma membrane. The frequency at which mGluR1a and mGluR5a subtypes stimulate inositol 1,4,5-triphosphate, Ca(2+), and PKCbetaII oscillations is regulated by the charge of a single amino acid residue localized within their G protein-coupling domains. However, oscillatory mGluR signaling does not involve the repetitive feedback phosphorylation and desensitization of mGluR activity, since mutation of the putative PKC consensus sites within the first and second intracellular loops as well as the carboxyl-terminal tail does not prevent mGluR1a-stimulated PKCbetaII oscillations. Furthermore, oscillations in Ca(2+) continued in the presence of PKC inhibitors, which blocked PKCbetaII redistribution from the plasma membrane back into the cytosol. We conclude that oscillatory mGluR signaling represents an intrinsic receptor/G protein coupling property that does not involve PKC feedback phosphorylation.

  • [Show abstract] [Hide abstract]
    ABSTRACT: The kisspeptin receptor (KISS1R) is a Gα(q/11)-coupled seven-transmembrane receptor activated by a group of peptides referred to as kisspeptins (Kps). The Kp/KISS1R signaling system is a powerful regulator of GnRH secretion, and inactivating mutations in this system are associated with hypogonadotropic hypogonadism. A recent study revealed that Kp triggers prolonged signaling; not from the inability of the receptor to undergo rapid desensitization, but instead from the maintenance of a dynamic and active pool of KISS1R at the cell surface. To investigate this further, we hypothesized that if a dynamic pool of receptor is maintained at the cell surface for a protracted period, chronic Kp-10 treatment would trigger the sustained activation of Gα(q/11) as evidenced through the prolonged activation of phospholipase C, protein kinase C, and prolonged mobilization of intracellular Ca(2+). Through single-cell analyses, we tested our hypothesis in human embryonic kidney (HEK) 293 cells and found that was indeed the case. We subsequently determined that prolonged KISS1R signaling was not a phenomenon specific to HEK 293 cells but is likely a conserved property of KISS1R-expressing cells because evidence of sustained KISS1R signaling was also observed in the GT1-7 GnRH neuronal and Chinese hamster ovary cell lines. While exploring the regulation of prolonged KISS1R signaling, we identified a critical role for extracellular Ca(2+). We found that although free intracellular Ca(2+), primarily derived from intracellular stores, was sufficient to trigger the acute activation of a major KISS1R secondary effector, protein kinase C, it was insufficient to sustain chronic KISS1R signaling; instead extracellular Ca(2+) was absolutely required for this.
    Endocrinology 10/2012; · 4.72 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cyclical phosphorylation and dephosphorylation of a key residue within the C-terminal domain of the activated type 5 metabotropic glutamate (mGlu₅) receptor is believed to cause the synchronous, oscillatory changes in inositol 1,4,5-trisphosphate and Ca²⁺ levels observed in a variety of cell types. Here, we have attempted to better define the kinase and phosphatase enzymes involved in this modulation. Ca²⁺ and [³H]inositol phosphate ([³H]IP(x) ) measurements in astrocyte preparations have been used to evaluate the effects of pharmacological inhibition of protein kinase C (PKC) and protein phosphatase activities and small interfering RNA-mediated specific PKC isoenzymic knock-down on mGlu₅ receptor signalling. Ca²⁺ oscillation frequency or [³H]IP(x) accumulation in astrocytes stimulated by mGlu₅ receptors, was concentration-dependently decreased by protein phosphatase-1/2A inhibition or by PKC activation. PKC inhibition also increased [³H]IP(x) accumulation two- to threefold and changed the Ca²⁺ response into a peak-plateau response. However, selective inhibition of conventional PKC isoenzymes or preventing changes in [Ca²⁺](i) concentration by BAPTA-AM loading was without effect on mGlu₅ receptor-stimulated [³H]IP(x) accumulation. Selective knock-down of PKCδ was without effect on glutamate-stimulated Ca²⁺ responses; however, selective PKCε knock-down in astrocytes changed Ca²⁺ responses from oscillatory into peak-plateau type. These data confirm the acute regulation of mGlu₅ receptor signalling by protein kinases and protein phosphatases and provide novel data pinpointing the isoenzymic dependence of this regulation in the native mGlu₅ receptor-expressing rat cortical astrocyte. These data also highlight a potential alternative mechanism by which mGlu₅ receptor signalling might be therapeutically manipulated.
    British Journal of Pharmacology 04/2011; 164(2b):755-71. · 5.07 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The calcium-sensing receptor (CaR) elicits oscillatory Ca(2+)(i) mobilization associated with dynamic, inhibitory protein kinase C-mediated phosphorylation of CaR(T888). While modest CaR stimulation elicits Ca(2+)(i) oscillations, greater stimulation either increases oscillation frequency or elicits sustained responses by an unknown mechanism. Here, moderate CaR stimulation (2.5 mm Ca(2+)(o), 10 min) increased CaR(T888) phosphorylation (160-kDa mature receptor) 5-fold in CaR stably transfected HEK-293 cells, whereas 3-5 mm Ca(2+)(o) treatments were without apparent effect. Treatment with 2 mm Ca(2+)(o) caused sustained CaR(T888) phosphorylation (> or = 20 min) and oscillatory Ca(2+)(i) mobilization. However, 5 mm Ca(2+)(o) increased CaR(T888) phosphorylation only briefly while eliciting sustained Ca(2+)(i) mobilization, suggesting that greater CaR activation induces rapid CaR(T888) dephosphorylation, thus permitting sustained Ca(2+)(i) responses. Indeed, 5 mm Ca(2+)(o) stimulated protein phosphatase 2A activity and induced CaR(T888) dephosphorylation following acute phorbol ester pretreatment, the latter effect being mimicked by CaR-positive allosteric modulators (NPS-R467 and l-Phe). Finally, the phosphatase inhibitor calyculin-A reversed CaR-induced inhibition of parathyroid hormone secretion from bovine parathyroid slices and normal human parathyroid cells, demonstrating the physiological importance of phosphorylation status on parathyroid function. Therefore, high Ca(2+)(o)-stimulated protein kinase C acts in concert with high Ca(2+)(o)-induced phosphatase activity to generate and maintain CaR-induced Ca(2+)(i) oscillations via the dynamic phosphorylation and dephosphorylation of CaR(T888).
    Journal of Biological Chemistry 03/2010; 285(19):14170-7. · 4.65 Impact Factor