PDZ 단백질과의 결합을 통한 GPCR 신호 전달 과정에서의 PLC-β 의 subtype 특이적인 역할에 관한 연구
- PDZ 단백질과의 결합을 통한 GPCR 신호 전달 과정에서의 PLC-β 의 subtype 특이적인 역할에 관한 연구
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- Among phospholipase C (PLC) isozymes (b, g, d, e, z and h), PLC-b plays a key role in G-protein coupled receptor (GPCR) mediated signaling. Despite possessing similar structure and identical enzymatic activity, each PLC-b subtype (b1-b4) displays a unique tissue expression pattern and knock-out phenotype. Thus, it would be interesting to determine whether the PLC-b isozyme has a subtype-specific role. To elucidate the subtype-specific role of PLC-b in GPCR signaling, I induced a subtype-specific knock-down of PLC-b and measured PLC activity and intracellular Ca2+ mobilization upon bradykinin (BK) and lysophosphatidic acid (LPA) treatment in HeLa cells. As a result, I found that cells in which PLC-b1 had been knocked-down showed reduced PLC activity and intracellular Ca2+ mobilization upon BK treatment compared to control and PLC-b3 knock-down cells, whereas upon treatment with LPA, PLC-b3 knock-down cells showed reduced activity than with the other cell type. I also demonstrated that the coupling of GPCRs to PLC-β subtype occurred by physical interaction through PDZ domains, protein-interaction domains that are often found in multi-domain scaffolding proteins. The PDZ scaffold proteins, PAR-3 and NHERF2, physically linked bradykinin B2 receptor (B2R) and Lysophosphatidic acid-2 receptor (LPA2) to their respective coupled PLC-β. Moreover, I showed that LPA-induced cellular proliferation was reduced only in PLC-b3 knock-down cells. Taken together, these data demonstrate that each subtype of PLC-b has a specific role in GPCR-mediated signaling, even within a single cell type. This study strongly suggests that multiple PLC-b subtypes are required in a cell for specific regulation of GPCR signaling.Lysophosphatidic acid (LPA) stimulates Na+/H+ exchanger 3 (NHE3) activity in opossum kidney proximal tubule (OK) cells by increasing the apical membrane amount of NHE3. This occurs by stimulation of exocytic trafficking of NHE3 to the apical plasma membrane by an NHERF2-dependent mechanism. HoIver, it is still unclear how NHERF2 leads to the LPA-induced exocytosis of NHE3. In the current study, I demonstrate that stable expression of exogenous NHERF2 increases LPA-induced phospholipase C (PLC) activation and subsequent elevation of intracellular Ca2+ in opossum kidney proximal tubule (OK) cells. Pretreatment with U73122, a PLC inhibitor, prevented the LPA-induced NHE3 activation and the exocytic trafficking of NHE3. To understand how the elevation of intracellular Ca2+ leads to the stimulation of NHE3, I pretreated OK cells with BAPTA-AM, an intracellular Ca2+ chelator. BAPTA-AM completely blocked the LPA-induced increase of NHE3 activity and surface NHE3 amount by decreasing the LPA-induced exocytic trafficking of NHE3. Pretreatment with GF109203X, a PKC inhibitor, did not affect the percent of LPA-induced NHE3 activation and increase of surface NHE3 amount. From these results, I suggest that NHERF2 plays a necessary role in LPA-induced PLC activation, and that PLC-dependent elevation of intracellular Ca2+ but not PKC activation is necessary for the LPA-induced increase of NHE3 exocytosis.
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