Regulation of Phospholipase C δ1 activity by GTP-binding proteins: RhoA as an inhibitory modulator

Abstract

Phosphatidylinositol4,5-bisphosphate is hydrolysed by phospholipase C (PLC) to form inositol 1,4,5-trisphosphate and diacylglycerol. Each of these products acts as a second messenger in a variety of cellular responses including contraction and cell proliferation [ 1,2]. The PLC family of enzymes consists of three isoforms (P,y,S) which are differentially regulated and expressed. PLCP has been demonstrated to be regulated by heterotrimeric G proteins of the Gq family or by Py subunits [3,4] whereas PLCy is tyrosine phosphorylated by growth factors [5]. The regulation of PLCG isoforms remains obscure although there is increasing evidence to suggest the involvement of GTP-binding proteins [6,71.Partially purified PLC& was isolated from bovine aortic cytosol by sequential elution through Q-sepharose and heparin agarose chromatography columns, using a linear sodium chloride gradient (50-55OmM) and identified immunologically by western blotting. PLC activity was measured using a method based on that described by Waldo et a1 [8] and using [3H]-phosphatidylinositol as a substrate. hmunoabsorption of PLC& from this sample resulted in a reduction in PLC activity of 89+3% (n=3) indicating that this sample is not contaminated with other isoforms of PLC, known or unknown.in the regulation of PLCGI activity samples were preincubated with G T W , a non-hydrolysable analogue of GTP, and PLC activity was measured. The resting PLC activity was consistently and significantly enhanced by the addition of GTPys ( Figure 1). but not by aluminium fluoride, suggesting the involvement of a monomeric GTP-binding protein.In order to determine whether GTP-binding proteins have a role ** I 300 T I T h control GTPVs Figure 1. Effect of GTPys on PLCGI activity. PLCGl samples were pre-incubated for 30 minutes at 37°C with 100lM G T W prior to measurement of PLC activity. Control activity was expressed as 100% and the GTPys stimulated activity was expressed as a % of the control. Data are mean f sem derived from six different aortae. ** p < 0.01To confirm the presence of a GTP-binding protein in the PLC& fraction, GTP-binding of column fractions were measured using [%]-GTPyS. These results demonstrated that the [35S]-GTpuS binding profile generated by the elution of the sample from heparin-agarose correlated exactly with that of PLCGI activity.Clostridium botulinum exoenzyme C3 (exoenzyme C3), which selectively ADP-ribosylates and inactivates rho GTPases also produced a significant increase in PLCGI activity. (Figure 2), *** I Control Exoenzyme C3 Figure 2. Effect of exoenzyme C3 on PLCGI activity. P E G , was preincubated for 1 hour with exoenzyme C3 (20pg/ml) and NAD prior to measuring PLC activity. Control activity is expressed as 100% and exoenzyme C3-stimulated activity is expressed as % of control. Data represents mean f sem from 4 separate aortae. *** p< 0.001.whereas incubation with NAD alone had no effect on PLC activity. Similarly exoenzyme C3 consistently ADP-ribosylated a 21kDa protein in the PLC& samples (n=3, data not shown) and wes...

Critical roles of Gi/oproteins and phospholipase C-δ1 in the activation of receptor-operated TRPC4 channels

“…PLCδ1 is inhibited by RhoA (36,38). Indeed, expression of a constitutively active RhoA mutant (L63) strongly inhibited TRPC4β (SI Appendix, Fig.…”