ET-1, LPA, and thrombin previously have been shown to stimulate cellular PLC activity (25
). Classically, these and other hormones acting through GPCRs have been proposed to activate PLCβ isoforms through Gq
family G-proteins and Gβγ subunits (25
). In addition, recent studies have raised the possibility that these agonists could activate PLCγ isoforms through transactivation of receptor tyrosine kinase receptors (18
). The discovery of PLCε, however, raises the possibility that these agonists might activate this novel PLC family member. PLCε has been shown to be regulated by multiple G-proteins, including Ras (4
), Rho (9
), and G12
) family G proteins and by Gβγ subunits (11
). Since ET-1, LPA, and thrombin receptors couple to Gq
, and G12
family G proteins (25
) and have been shown to activate Ras (18
) and Rho (43
), these agonists could potentially activate PLCβ isoforms and/or PLCε, changing the view of how these GPCR agonists regulate cellular PI hydrolysis. To determine whether these agonists couple to PLCβ isoforms and/or PLCε, we identified Rat-1 fibroblasts as a cell line that expresses both of these PLC family members and examined the effects of knocking down specific isoforms on agonist-stimulated PI hydrolysis.
Our studies show that ET-1, LPA, and thrombin, acting through endogenous GPCRs, couple to endogenous PLCε in Rat-1 fibroblasts. This is the first demonstration that these agonists physiologically couple to PLCε. In addition, and consistent with previous paradigms (1
), we also show that these diverse agonists activate endogenous PLCβ3. Thus, under physiologic conditions, ET-1, LPA, and thrombin dually regulate both PLCε and PLCβ3 in Rat-1 fibroblasts.
GPCR agonist regulation of these PLC isoforms, however, is not a simple, simultaneous activation of PLCε and PLCβ3. Importantly, we demonstrate that the stimulation of these PLC isoforms is temporally distinct. Our studies show that ET-1, LPA, and thrombin activate PLCβ3 acutely (peak at 15 s; duration ~1 min) but not PLCε. In contrast, ET-1, LPA, and thrombin activate PLCε during sustained PI hydrolysis (>30 s to at least 60 min). This distinct temporal activation of PLCε and PLCβ3 is most apparent for LPA, doses ≤1 μm
, and thrombin, which couple to PLCβ3 acutely and almost exclusively to PLCε during sustained stimulation. Consistent with this observation, overexpression of PLCε had no effect on acute but markedly increased sustained agonist-stimulated PI hydrolysis. ET-1 was similarly coupled to PLCβ3 acutely but, in addition to activating PLCε during the sustained phase, also partly activated PLCβ3, indicating an agonist dependence for the temporal activation of these isoforms. Interestingly, a difference in the activation of PLC by these agonists is consistent with an earlier study that noted ET-1 and LPA regulate PLC activity by distinct mechanisms in Rat-1 fibroblasts (44
). Overall, however, PLCβ3 predominantly accounts for acute and PLCε for sustained PLC responses. Interestingly, in vascular smooth muscle cells, angiotensin II has been shown to stimulate PLCβ1 acutely followed by activation of PLCγ1 (20
), suggesting that acute activation of PLCβ isoforms may be a general phenomenon.
Whereas we have demonstrated that ET-1, LPA, and thrombin regulate PLCβ3 and PLCε, our studies also suggest that these agonists regulate other PLC isoforms. This is apparent, because acute stimulation was only partly inhibited by knockdown of PLCβ3 () or the combined knockdown of both PLCβ3 and PLCε (). Similarly, high doses (≥3 μm
) of LPA activate an unknown PLC during sustained stimulation (). It is possible that either PLCδ1 or PLCγ1, which we show are present in this cell line, mediates these responses. In Rat-1 fibroblasts, ET-1 and LPA have been shown to transactivate the epidermal growth factor receptor (18
), and therefore PLCγ1 might be activated. In addition, whereas PLCε and PLCβ3 appear to be regulated independently because of their distinct activation profiles and additive effects on cellular PLC activity in combined knockdown studies, other PLC isoforms may be activated downstream of these enzymes. In this regard, PLCβ2 and PLCδ1 have been suggested to exist as an inactive heterodimer, and upon Gβγ subunit stimulation of PLCβ2, PLCδ1 is released to hydrolyze PIs (21
). Whether PLCδ1 is involved in PLCβ3 or PLCε signaling remains to be determined.
Previously, we demonstrated that the GPCR agonists LPA, S1P, and thrombin stimulate PLCε overexpressed in COS-7 cells (9
). The present studies confirm that LPA and thrombin couple to PLCε and that the interaction is physiological. Interestingly, in COS-7 cells overexpressing PLCε or PLCβ isoforms, these agonists stimulated PLCε to a markedly greater extent than PLCβ1 or PLCβ2, suggesting preferential coupling to PLCε. However, IP accumulation was measured at 60 min, and acute stimulation was not examined. Because the current studies demonstrate that these agonists regulate PLCβ3 acutely and PLCε in a sustained manner, earlier time points may reveal coupling to PLCβ isoforms.
We have also shown previously that LPA and thrombin activate PLCε through Gα12/13
and Rap (9
). It is possible that a similar mechanism mediates activation of PLCε by thrombin and LPA in Rat-1 fibroblasts, and preliminary studies examining the effects of knocking down Gα12/13
are consistent with this hypothesis.3
Thus, differential activation of Gq
or Ras family G-proteins may mediate the temporal stimulation of PLCβ3 and PLCε, respectively. Interestingly, we observed that thrombin stimulated PLCε at least 50-fold more potently than PLCβ3 (). Other investigators have found similar differences in the potency for thrombin activation of Rho through G12
, which is 20-fold greater than activation through Gq
). Riobo et al.
) have suggested that this difference may be determined by the affinity of the receptor for a given G protein, and this phenomenon may help explain potency differences observed in our studies. Furthermore, our studies also indicate that the signaling pathway(s) underlying thrombin or LPA activation of PLCβ3 desensitize acutely within the first 90 s of stimulation (), as opposed to the pathways regulating PLCε, which remain active for extended periods of at least 60 min (). Whether this difference reflects differential G-protein regulation (i.e.
), a functionally different subset of receptors or intrinsic PLC enzymatic regulation remains to be determined.
Consistent with the temporal activation of PLCε and PLCβ3, we demonstrated a direct functional correlation with IP3
R ubiquitination. Certain GPCRs that activate PLC have been shown to induce down-regulation of IP3
), possibly as a physiologic, protective mechanism to prevent overstimulation through the GPCR effector system (36
). This down-regulation is mediated by the ubiquitin-proteasome pathway and probably involves an IP3
- and Ca2+
R structural change that triggers ubiquitination and subsequent proteasome targeting (36
appears to be required, because microinjection of an IP3
analog causes proteasome-mediated down-regulation (48
), and mutations in the region of the IP3
R that bind IP3
inhibit ubiquitination (47
). Indeed, induction of IP3
R ubiquitination and degradation requires sustained activation of PLC (28
). For example, sustained activation of PLC with carbachol in SH-SY5Y neuroblastoma cells (37
), cholecystokinin and bombesin in AR4–2J pancreatoma cells (32
), or angiotensin II in WB rat liver epithelial cells (39
) induces IP3
R ubiquitination and down-regulation. In contrast, agonists that do not activate PLC in a sustained manner, such as carbachol or substance P in AR4–2J cells (32
) or epidermal growth factor, vasopressin, or bradykinin in WB cells (39
), fail to induce ubiquitination or down-regulation. Our data concur with these studies, since inhibition of sustained, but not acute, PI hydrolysis stimulated by thrombin or ET-1 inhibits IP3
R ubiquitination. Thus, knockdown of PLCε, which has no effect on acute PI hydrolysis but inhibits sustained PI hydrolysis, markedly inhibited thrombin and ET-1 induced ubiquitination, an effect dependent on the lipase function of the PLC (). In contrast, knockdown of PLCβ3 had no effect on thrombin-stimulated ubiquitination and only partly inhibited ET-1, consistent with its effects on PI hydrolysis. This is the first demonstration of the coupling of specific PLC isoforms to this important cellular adaptive response.
The physiologic role of the differential activation of PLCβ3 and PLCε is not known. However, the observed temporal and dose-dependent differences in their activation have multiple implications. Acutely, peak IP3
release through the IP3
), and PLCβ3 probably contributes to this response. The potential role for PLCε and sustained PLC activation is less clear. During sustained stimulation, IP3
can be phosphorylated to higher IPs, including inositol 1,3,4,5-tetrakisphosphate and inositol hexakisphosphate, which have been shown to regulate vesicle transport and nuclear transcription factors (52
). Similarly, time-dependent production of different DAG species has been noted, which likely have distinct physiologic roles (53
). It is intriguing to speculate that sustained agonist activation of PLCε may down-regulate the IP3
R to redirect signaling to these alternative pathways. Furthermore, our studies show a dose-dependent stimulation of PLCε and PLCβ3, which suggests that local agonist concentrations may determine which pathway is activated. This is most apparent for thrombin, which activates PLCε with a potency almost 2 orders of magnitude greater than PLCβ3 (). Thus, concentrations of 3–10 pm
would activate signaling pathways involving PLCε but have little effect on those utilizing PLCβ3. A concentration dependence of ET-1, where concentrations less than 1 nm
influx but greater stimulate mobilization of intracellular Ca2+
, has been noted previously in Rat-1 cells (54
). Whether differential regulation of PLCε or PLCβ3 accounts for this or other dose-dependent effects remains to be determined. Elucidating the temporally distinct products generated by isoform-specific PLCs and defining their physiologic role is an important area of future investigation.
In summary, our studies demonstrate that the GPCR agonists ET-1, LPA, and thrombin couple to both PLCε and PLCβ3 in Rat-1 fibroblasts. Whereas there is some agonist-dependent overlap, activation of these isoforms occurs in a temporally distinct manner whereby PLCβ3 is activated acutely and PLCε in a sustained manner. This temporal regulation of sustained, but not acute, activation of PLCε and PLCβ3 functionally correlates with IP3R ubiquitination. In addition, other PLC isoforms, possibly PLCδ1 and/or PLCγ1, are also simultaneously activated. Clearly, these studies demonstrate that GPCR activation of PLC is complex and involves an agonist- and dose-dependent, temporal activation of multiple PLC isoforms, which translates into discrete regulatory functions.