PLC∊ (phospholipase C∊) is a novel PLC that has a CDC25 guanine nucleotide exchange factor domain and two RA (Ras-association) domains of which the second (RA2) is critical for Ras activation of the enzyme. In the present studies, we examined hormonal stimulation to elucidate receptor-mediated pathways that functionally regulate PLC∊. We demonstrate that EGF (epidermal growth factor), a receptor tyrosine kinase agonist, and LPA (lysophosphatidic acid), S1P (sphingosine 1-phosphate) and thrombin, GPCR (G-protein-coupled receptor) agonists, stimulate PLC∊ overexpressed in COS-7 cells. EGF stimulated PLC∊ in an RA2-dependent manner through Ras and Rap. In contrast, LPA, S1P and thrombin stimulated PLC∊ by both RA2-independent and -dependent mechanisms. To determine the G-proteins that mediate the effects of these GPCR agonists, we co-expressed constitutively active G-proteins with PLC∊ and found that Gα12, Gα13, Rho, Rac and Ral stimulate PLC∊ in an RA2-independent manner; whereas TC21, Rap1A, Rap2A and Rap2B stimulate PLC∊ in an RA2-dependent manner similar to H-Ras. Of these G-proteins, we show that Gα12/Gα13 and Rap partly mediate the effects of LPA, S1P and thrombin to stimulate PLC∊. In addition, the stimulation by LPA and S1P is also partly sensitive to pertussis toxin. These studies demonstrate diverse hormonal regulation of PLC∊ by distinct and overlapping pathways.
- epidermal growth factor (EGF)
- lysophosphatic acid (LPA)
- phospholipase Cε
- sphingosine 1-phosphate (S1P)
Abbreviations used: GEF, guanine nucleotide exchange factor; PLC, phospholipase C; GPCR, G-protein-coupled receptor; RA, Ras-association; EGF, epidermal growth factor; EGFR, EGF receptor; RTK, receptor tyrosine kinase; LPA, lysophosphatidic acid; S1P, sphingosine 1-phosphate; GST, glutathione S-transferase; FBS, fetal bovine serum; RGS, regulators of G-protein signalling.
- The Biochemical Society, London ©2004