Elsevier

Cellular Signalling

Volume 22, Issue 7, July 2010, Pages 1153-1161
Cellular Signalling

Subtype-specific role of phospholipase C-β in bradykinin and LPA signaling through differential binding of different PDZ scaffold proteins

https://doi.org/10.1016/j.cellsig.2010.03.010Get rights and content

Abstract

Among phospholipase C (PLC) isozymes (β, γ, δ, ε, ζ and η), PLC-β plays a key role in G-protein coupled receptor (GPCR)-mediated signaling. PLC-β subtypes are often overlapped in their distribution, but have unique knock-out phenotypes in organism, suggesting that each subtype may have the different role even within the same type of cells. In this study, we examined the possibility of the differential coupling of each PLC-β subtype to GPCRs, and explored the molecular mechanism underlying the specificity. Firstly, we found that PLC-β1 and PLC-β3 are activated by bradykinin (BK) or lysophosphatidic acid (LPA), respectively. BK-triggered phosphoinositides hydrolysis and subsequent Ca2+ mobilization were abolished specifically by PLC-β1 silencing, whereas LPA-triggered events were by PLC-β3 silencing. Secondly, we showed the evidence that PDZ scaffold proteins is a key mediator for the selective coupling between PLC-β subtype and GPCR. We found PAR-3 mediates physical interaction between PLC-β1 and BK receptor, while NHERF2 does between PLC-β3 and LPA2 receptor. Consistently, the silencing of PAR-3 or NHERF2 blunted PLC signaling induced by BK or LPA respectively. Taken together, these data suggest that each subtype of PLC-β is selectively coupled to GPCR via PDZ scaffold proteins in given cell types and plays differential role in the signaling of various GPCRs.

Introduction

The signaling pathways of numerous extracellular signaling molecules such as lipids, peptides, nucleotides and hormones are transduced via plasma membrane-bound GPCRs. Upon ligand binding, GPCRs sequencially activate heterotrimeric G protein and PLC-β. Activated PLC-β hydrolyzes phosphatidyl inositol 4,5-bisphosphate to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), in turn, IP3 and DAG evoke intracellular Ca2+ mobilization and PKC activation. It is well known that intracellular Ca2+ mobilization and PKC activation are key events involved in GPCR-mediated cellular responses such as migration and proliferation and as well as many others [1], [2].

There are four subtypes of PLC-β in mammals, each of which exhibits different expression patterns. PLC-β1 is the most widely expressed, with highest levels occurring in specific regions of the brain such as the hippocampus. PLC-β3 is also widely expressed, particularly in the brain, liver and parotid gland. PLC-β2 and PLC-β4 are expressed mainly in hematopoietic cells and in the cerebellum respectively [2], [3]. The varying expression patterns of each PLC-β subtype further reflect the defects of PLC-β subtype-specific knock-out mice. For example, the absence of PLC-β1 causes sudden death due to epileptic-like seizures [4]. In these mice, decreased in PLC-β activation by a muscarinic agonist is observed in the temporal lobe, cerebellum and hippocampus. Mice lacking PLC-β2 show defects in chemoattractant-mediated signal transduction. For example, fMLP-induced chemotaxis and superoxide generation are decreased in leukocytes derived from PLC-β2 knock-out mice [5]. Studies on the PLC-β3-specific knock-out mice are more controversial. There is a report that homozygous inactivation of PLC-β3 is lethal at embryonic day 2.5 [6]. However, another knock-out study suggested that PLC-β3 is involved in negative modulation of mu opioid responses [7]. PLC-β4 knock-out mice have a number of defects, especially in motor coordination. Because the mice do not show muscle weakness and bone deformity, it is reasonable to speculate that resultant hypokinetics and waddling gait might be due to a defect in the cerebellum [4].

Although knock-out mice of the different PLC-β subtypes possess distinct phenotypes, it is still unclear whether each PLC-β subtype has a specific role in the cell. The defect in muscarinic receptor signaling in the hippocampus of PLC-β1 knock-out mice, for instant, could be caused not only by the loss of a specific function of PLC-β1 but also by the loss of the major subtype of PLC-β in the hippocampus. As previously described, PLC-β1 and PLC-β3 are ubiquitously expressed. It is well known that a number of cells express multiple subtypes of PLC-β. Vascular smooth muscle cells (VSMC), for example, express all of the PLC-β subtypes [8]. Thus it will broaden our understanding of PLC-β-mediated signaling to address the question of whether each PLC-β subtypes in a cell has a specific role.

Some GPCRs can form a molecular complex by binding with scaffolding protein such as A-kinase anchoring proteins (AKAPs) and postsynaptic density disc-large ZO-1 (PDZ) proteins [9]. It is also known that PLC-β can interact with some of these scaffolding proteins [10], [11]. In a previous report, we showed that the Na+-H+ exchanger regulatory factor 2 (NHERF2), PDZ protein, physically links LPA2 to PLC-β3 in a subtype-specific manner and that Shank2 also regulates the m-GluR-mediated Ca2+ signal [12], [13]. Thus it is possible that a particular PLC-β subtype has a specific role in GPCR-mediated signaling by forming a signaling complex with scaffolding proteins and GPCRs.

To address this possibility, we used siRNA to suppress the expression of the ubiquitously expressed PLC-β1 and PLC-β3 subtypes and assessed the effects on different GPCR-mediated signaling pathways in human cervical carcinoma cells (HeLa) We showed that BK-induced PLC activation and intracellular Ca2+ mobilization is affected specifically by PLC-β1 knock-down. In contrast, LPA-induced PLC activation and intracellular Ca2+ mobilizations are affected by knock-down of PLC-β3 not of PLC-β1. Moreover, we showed that through binding with specific PDZ protein such as PAR-3 and NHERF2, B2R and LPA2 physically interact with PLC-β1 and PLC-β3 respectively. And it is also shown that LPA-induced cell proliferation and cell migration are also affected by PLC-β3 knock-down. Here, we demonstrated that each PLC-β has a specific function on GPCR-mediated signaling even in the same type of cells.

Section snippets

Materials

Lysophosphatidic acid (LPA; 1-oleoyl-2-hydroxy-sn-glycerol-3-phosphate) was purchased from Biomol (Plymouth Meeting, PA). Bradykinin and U73122 were obtained from Calbiochem (La Jolla, CA). Fura-2 penta-acetoxymethylester (Fura 2-AM) was obtained from Molecular Probes (Eugene, OR). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and other cell culture reagents were purchased from BioWhiteker (Walkersville, MD). The monoclonal antibody K-92-3 is specific for PLC-β1, as

Knock-down of PLC-β1 or PLC-β3 reduces BK or LPA-induced PLC activity and intracellular Ca2+ mobilization respectively

To examine the subtype-specific roles of PLC-β in GPCR-mediated signaling, we first examined which PLC-β subtypes were expressed in HeLa cells. Western blot analysis revealed that β1 and β3 subtypes of PLC-β are expressed in HeLa cells. No expression of PLC-β2 and PLC-β4 in this cell line was further confirmed by RT-PCR analysis (Fig. 1A). And to find out ligands which potently activate PLC-β, we measured PLC activity upon treatment of several GPCR ligands such as ATP, sphingosine 1-phosphate,

Discussion

All subtypes of PLC-β have the same primary structure, consisting linearly of a PH domain, an EF hand motif, the PLC catalytic domains X and Y, and then a C2 domain. Additionally, they are all known to be activated by the GTP-bound Gαq and βγ subunits dissociated from the heterotrimeric Gi protein upon GPCR activation [1]. Despite the fact that they possess the same primary structure and activation mechanism, multiple subtypes of PLC-β are expressed by most of cells. What the function of these

Acknowledgments

This work was supported by the grant (M10600000281-06J0000-28110) from the National Research Lab of the Korea Science & Engineering Foundation and by the National Research Foundation of Korea Grant funded by the Korean Government (KRF-2007-341-C00027).

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