Phospholipase Cγ activation and phosphoinositide hydrolysis are essential for embryonal development

It is now well established that the metabolism of phosphoinositides plays an important role in the control of cell growth, differentiation, and survival, as well as cell architecture (1, 2). The various isoforms of phospholipase C (PLC) catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] to generate two second messengers, Ins(1,4,5)P₃ and diacylglycerol. It has been shown that Ins(1,4,5)P₃ mediates the release of Ca²⁺ from intracellular stores, while diacylglycerol functions as a intracellular ligand of protein kinase C (PKC).

A great variety of extracellular signals stimulate PLC activity, leading to PtdIns(4,5)P₂ hydrolysis and activation of downstream effector systems. PtdIns(4,5)P₂ hydrolysis is stimulated by activation of G protein-coupled receptors, receptors for hormones, growth factors, cytokines, antigens, and integrins, among others (1, 2). In mammals there are at least 10 genes that encode for PLC isoforms, and additional diversity in structure and function is generated by alternative RNA splicing. The 10 mammalian PLC isoforms can be classified into three groups called PLCβ, PLCγ, and PLCδ (3). There are four PLCβ, two PLCγ, and four PLCδ isoforms. All PLC isoforms contain a conserved catalytic domain, a pleckstrin homology (PH) domain in the amino terminus of the protein and additional regulatory sequences (Fig. 1). It has been shown that the PH domain of PLCδ binds with high affinity and specificity to PtdIns(4,5)P₂ and to the head group Ins(1,4,5)P₃ (4). On the basis of both biochemical and structural studies it was concluded that the PH domain of PLCδ tethers the enzyme to the cell membrane, enabling processive substrate catalysis (4–7). Although the physiological ligands of the PH domains of PLCβ and PLCγ are not known, it is reasonable to assume that these PH domains are also involved in regulated targeting of their host proteins to cell membranes. Analysis of the three-dimensional structure of PLCδ by x-ray crystallography revealed the presence of two additional protein domains (8): a C2 domain that is responsible for Ca²⁺-dependent binding of the catalytic domain to the cell membrane, and an EF domain that functions as a flexible link between the PH domain and the rest of the enzyme. Since all PLC isoforms contain a conserved catalytic domain, as well as C2, EF, and PH domains, it is likely that all isoforms catalyze the hydrolysis of PtdIns(4,5)P₂ by a common mechanism (9).

Figure 1.

Schematic representation of the three isoforms of phospholipase C (PLC). All three isoforms (PLCβ, PLCγ, and PLCδ) contain PH, EF hand, and C2 domains in addition to the conserved catalytic domain that is responsible for PtdIns(4,5)P₂ hydrolysis. PLCβ interacts with G proteins by means of its carboxyl-terminal region. PLCγ is recruited by receptor and nonreceptor protein tyrosine kinases by means of its SH2 domains. In mammals there are four PLCβ, two PLCγ, and four PLCδ isoforms.

Ji et al. (10) explored the biological role of PLCγ1 by generating mice with a disrupted Plcγ1 gene. They demonstrated that disruption of the Plcγ1 gene led to early embryonic lethality at embryonic day 9.0. Histological analysis of PLCγ1 (−/−) mice embryos demonstrated that beyond embryonic day 8.5 the expression of PLCγ1 is essential for normal growth and development. The early embryonal lethality of mice deficient in PLCγ1 revealed the essential role of this enzyme in the development of the embryo. Ji et al. (10) proposed that PLCγ1 is required for the proliferation of an embryonic cell or cells that appear at embryonic day 9.0 or, alternatively, that all the cells of PLCγ1 (−/−) embryo fail to grow at a rate necessary for normal development of the embryo.

PLCγ1 does not appear to be required for the growth of all cell types. Ji et al. (10) were able to prepare embryonic fibroblasts from PLCγ1 (−/−) mice and to demonstrate that these cells respond to serum stimulation, by releasing Ca²⁺ from internal stores. These results are consistent with earlier studies demonstrating that recruitment and activation of PLCγ1 in response to fibroblast growth factor (FGF) or platelet-derived growth factor (PDGF) stimulation is not required for the proliferation of fibroblasts or myoblasts and for neuronal differentiation of PC12 cells (11–14). It therefore appears that certain cells are able to proliferate, survive, and undergo differentiation in the absence and PLCγ1 activation. Yet PLCγ1 activation appears to be crucial for the growth and survival of certain cell types that are essential for normal embryonal development (10). It is possible that the cells deficient in PLCγ1 survive because they express other PLC isoforms or exhibit redundant signaling pathways that compensate for the loss of PLCγ1. It has been demonstrated that the two isoforms of PLCγ, PLCγ1 and PLCγ2, are activated by protein tyrosine kinases (2, 3, 9). These enzymes contain Src homology 2 and 3 (SH2 and SH3) domains; the SH2 domains are responsible for the binding of these enzymes to tyrosine autophosphorylation sites on activated receptor protein tyrosine kinases (15). In addition, a variety of extracellular signals induce tyrosine phosphorylation of PLCγ1, a step which is essential for enzyme activation and for stimulation of PtdIns(4,5)P₂ hydrolysis (16). Because of their similar structures, PLCγ1 and PLCγ2 are probably regulated by similar mechanisms. However, while PLCγ1 is widely expressed, PLCγ2 is expressed mainly in immune cells, and therefore PLCγ2 cannot compensate for the loss of PLCγ1 from most tissues.

PLCβ is activated by G protein-coupled receptors such as the muscarinic acetylcholine receptor m1, bradykinin, and endothelin receptors, among many other receptors (1–3, 9). PLCβ is activated by receptors that activate the G_q family of heterotrimeric G proteins, and the activation is mediated by the α₂, α₁₁, α₁₄, and α₁₆ subunits of G_q. In addition, PLCβ isoforms can be activated by Gβγ proteins in response to stimulation of certain G protein receptors (2, 3, 17). By contrast, not much is known about extracellular signals that regulate the activity of PLCδ. It has been recently demonstrated that PLCδ can be activated by stimulation of the α₁-adrenergic receptor coupled to an unusual G_hα protein (18).

The essential role of PLCγ1 in embryonal development is also consistent with the recent finding that activation of certain G protein-coupled receptors leads to tyrosine phosphorylation and stimulation of PLCγ1 activity (19). Hence, PLCγ1 lies downstream of both receptor tyrosine kinases and G protein-coupled receptors. Most cells express only one form of PLCγ (usually PLCγ1). Therefore, elimination of PLCγ1 could not be compensated for by other PLC isoforms that are subject to regulation by different extracellular signals. On the other hand, disruption of individual PLCβ or PLCδ genes by homologous recombination may not necessarily lead to embryonal lethality, since many cells express several PLCβ or PLCδ isoforms. Moreover, it is possible that some of the biological effects of PLCβ could be mediated by PLCγ, as various G protein-coupled receptors can activate PLCγ, thus providing a potential compensatory mechanism for loss of PLCβ isoforms.

A detailed elucidation of the biological roles of PLC isoforms will be determined by generation of mice deficient in the various PLC isoforms. With these tools in hand, it will be possible to cross between mice with individually disrupted Plc genes and thus generate mice that are deficient in two or more PLC isoforms. These experiments will surely be performed in the near future and provide us with additional insights concerning the many biological roles of these fascinating enzymes.