P2Y2 Purinergic and M3 Muscarinic Acetylcholine Receptors Activate Different Phospholipase C-β Isoforms That Are Uniquely Susceptible to Protein Kinase C-dependent Phosphorylation and Inactivation

Abstract

Activation of phospholipase C-beta (PLC-beta) by G protein-coupled receptors typically results in rapid but transient second messenger generation. Although PLC-beta deactivation may contribute to the transient nature of this response, the mechanisms governing PLC-beta deactivation are poorly characterized. We investigated the involvement of protein kinase C (PKC) in the termination of PLC-beta activation induced by endogenous P2Y(2) purinergic receptors and transfected M(3) muscarinic acetylcholine receptors (mAChR) in Chinese hamster ovary cells. Activation of P2Y(2) receptors causes Galpha(q/11) to associate with PLC-beta3, whereas M(3) mAChR activation causes Galpha(q/11) to associate with both PLC-beta1 and PLC-beta3 in these cells. Phosphorylation of PLC-beta3, but not PLC-beta1, is induced by activating either P2Y(2) receptors or M(3) mAChR. We demonstrate that PKC rather than protein kinase A mediates the G protein-coupled receptor-induced phosphorylation of PLC-beta3. The PKC-mediated phosphorylation of PLC-beta3 diminishes the interaction of Galpha(q/11) with PLC-beta3, thereby contributing to the termination PLC-beta3 activity. These findings indicate that the distinct temporal profiles of PLC activation by P2Y(2) receptors and mAChR may arise from the differential activation of PLC-beta1 and PLC-beta3 by the receptors, coupled with a selective PKC-mediated negative feedback mechanism that targets PLC-beta3 but not PLC-beta1.

Calcium Mobilization Evoked by Hepatocellular Swelling Is Linked to Activation of Phospholipase Cγ

“…In the latter two tissues, volume-sensitive PLC activation appears to involve autocrine stimulation of P2Y purinergic receptors. Although it is known that P2Y receptors can activate PLC␤ and PLC␥ (23,24), the identity of PLC isoforms that mediate volume-sensitive purinergic Ca 2ϩ signaling has not been elucidated. As we have shown that swelling-mediated increases in [Ca 2ϩ ] i in HTC cells are independent of purinergic receptor stimulation (4), it is likely that distinct mechanisms are involved in volume-sensitive PLC activation in liver.…”

Loss of Inositol 1,4,5-trisphosphate receptors from bile duct epithelia is a common event in cholestasis

“…In fact, densitometry showed that M3 muscarinic receptor expression actually was increased by 298% ± 9% relative to normal controls (P < 0.0005, Figure 5B), consistent with previous reports. 8 The α subunits of the G q and G 11 G proteins, which are coupled to the M3 muscarinic receptor, 19 were studied in normal and bile duct-ligated biliary epithelia by the same technique. The amounts of the subunits were similar under both conditions ( Figure 5C and D); cells after BDL expressed 93.2% ± 2.1% of the amount of Gα q /α 11 expressed by normal bile duct cells (P > 0.3).…”

“…These steps include binding of ACh to the M3 muscarinic receptor, activation of an associated G protein, and then PLCβ activation. 19 To determine whether any of these upstream signal transduction events also are affected by BDL, we examined expression of the M3 muscarinic ACh receptor, Gα q /α 11 protein, and PLCβ3. Immunoblot analysis showed that M3 muscarinic receptor expression was not decreased after BDL ( Figure 5A).…”

Pemphigus Vulgaris IgG and Methylprednisolone Exhibit Reciprocal Effects on Keratinocytes

“…Quantitative Phosphorylation Assay-Quantitative phosphorylation assay was designed based on the established protocols described elsewhere (51,52,66,67). Briefly, the DJM-1 cells were grown to ϳ75% confluence in 75-cm 2 flasks in KGM containing 0.09 mM Ca 2ϩ without hydrocortisone supplement (Clonetics, Walkerville, MD) at 37°C in a humid 5% CO 2 incubator, and then incubated for additional 48 h in minimal essential medium containing 10% dialyzed fetal bovine serum, 1.8 mM Ca 2ϩ , and 10 ng/ml epidermal growth factor (all from Invitrogen).…”