Activation of PLC-β by G_qα Subunits

Heterotrimeric G proteins consist of α, β, and γ subunits that are stably associated in the inactive, GDP-bound state. Physical interaction between a G protein and an agonist-occupied receptor triggers the exchange of GDP for GTP on the α subunit and the subsequent dissociation of this subunit from the tightly associated βγ dimer. Both the Gα-GTP and Gβγ entities participate in intracellular signaling (Figure 3). The intrinsic GTPase activity of the Gα subunit mediates the rate-limiting hydrolysis of GTP to GDP and results in the reassociation of this subunit with the Gβγ complex and consequent inactivation of signaling. On the basis of their amino acid sequences and effector interactions, G protein α subunits have been divided into four subfamilies: G_sα, G_iα, G_qα, and G₁₂α. The α subunits (α_q, α₁₁, α₁₄, and α₁₆) of all four members of the G_q subfamily activate PLC-β isozymes but not PLC-γ , PLC-δ, or PLC-ε (9, 36, 37). The four mammalian PLC-β isozymes differ in their tissue distribution as well as in their ability to be activated by G proteins. Expression of PLC-β2 is restricted to hematopoietic cells and that of PLC-β4 is limited to the retina and certain neuronal cells, whereas PLC-β1 and PLC-β3 are expressed more widely. G protein—coupled receptors (GPCRs) that activate the G_qα—PLC-β signaling pathway include those for thromboxane A₂, bradykinin, bombesin, angiotensin II, histamine, vasopressin, acetylcholine (muscarinic m1 and m3), α₁-adrenergic agonists, thyroid-stimulating hormone, C-C and C-X-C chemokines, and endothelin-1.

Figure 3

GPCR-mediated activation of PLC-β isozymes. An agonist-occupied GPCR induces the exchange of GDP for GTP on the G protein α subunit and the subsequent dissociation of this subunit from the membrane-associated Gβγ dimer. (more...)

Gα_q and Gα₁₁ subunits activated by the nonhydrolyzable GTP analog guanosine 5′-O-(3′-thiotriphosphate) (GTP-γ -S) stimulate PLC-β isoforms according to the rank order PLC-β1 ≥ PLC-β3 > PLC-β2 (38, 39), which is consistent with the observation that the affinities of GTP-γ -S-activated Gα_q for PLC-β1 and PLC-β3 are similar [dissociation constants (K_d) of 40–60 nM] to each other and substantially higher than that for PLC-β2 (K_d of 400 nM) (40). PLC-β4 is also activated by G_qα subunits; however, because the basal activity of this enzyme is inhibited by ribonucleotides, including GTP-γ -S, accurate determination of the extent of activation is difficult (41). All four members of the G_qα subfamily activate PLC-β1 (42). Furthermore, Gα₁₆, which is expressed only in hematopoietic cells and is distantly related to the more widely expressed Gα_q (amino acid sequence identity of 55%), activates PLC-β1, -β2, and -β3 in a manner essentially indistinguishable from that of Gα_q (43). However, certain receptors discriminate among the α subunits of the G_q subfamily. The receptor for macrophage chemotactic protein-1, for example, couples to Gα₁₄ and Gα₁₆ but not to Gα_q or Gα₁₁ (44).

The activated Gα_q subunit appears to interact with the COOH-terminal region of PLC-β1 downstream of the Y domain; this region contains the C2 domain (residues 663–802) followed by a sequence (residues 803–1216) that is unique to this subfamily of PLC isozymes. The results of deletion analysis suggested that the region downstream of residue 845 is required for binding of and stimulation by Gα_q (45, 46). Secondary structure predictions indicate that this region (residues 846–1216) is composed predominantly of α helices, and it contains a high proportion of basic residues that appear in clusters (47). Basic residues in two such clusters (residues 911–928 and 1055–1075) are important for stimulation by Gα_q (47). The activated Gα_q subunit also interacts with the C2 domains of PLC-β isoforms (34). Unlike the C2 domain of PLC-δ1, which binds Ca²⁺ and serves as a membrane attachment module (33), the C2 domains of PLC-β1 and PLC-β2 do not bind to membranes. However, they each bind GTP-γ -S-activated Gα_q with high affinity ( K_d, 18 nM); they bind the inactive, GDP-bound Gα_q with a much lower affinity ( K_d values of 120 and 60 nM for PLC-β1 and -β2, respectively) and do not associate with the GTP-γ -S-activated Gα_i (34). In contrast, the C2 domain of PLC-δ1 does not exhibit any measurable affinity for GTP-γ -S-activated Gα_q.

Although G protein subunits are not intrinsically hydrophobic, they are bound to the cell membrane as a result, at least in part, of covalent lipid modification. Gγ subunits are prenylated at their COOH termini, a modification that promotes both the association of the βγ dimer with membranes as well as the interaction of βγ with Gα subunits and effectors. Activated, GTP-bound G_qα subunits remain attached to the cell membrane even after separation from the βγ complex; this association is attributable to the fact that their NH₂-terminal regions are hydrophobic and contain two cysteine residues that are palmitoylated (48). The membrane-embedded G_qα subunits are likely recognized by PLC-β isozymes that are loosely associated with the cell membrane as a result of the interaction of their COOH-terminal basic residues with acidic phospholipids (47) (Figure 3).

The membrane localization of PLC-β isozymes is likely further promoted by the presence of PtdIns(3)P, which binds to the PH domain of PLC-β1 (Figure 3). The evidence for a specific interaction of the PH domain of PLC-β1 with PtdIns(3)P has been provided by direct lipid binding studies and by the observation that the lysophosphatidic acid—induced membrane localization of a fusion construct containing green fluorescent protein and the PH domain of PLC-β1 is prevented by a PtdIns 3-kinase inhibitor (22). The amount of PtdIns(3)P is low in mammalian cells and does not change markedly in response to receptor activation. It is therefore possible that PtdIns(3)P contributes to the membrane association of PLC-β1 only under specific circumstances. Nevertheless, treatment of cells with a PtdIns 3-kinase inhibitor results in a rapid reduction in the abundance of PtdIns(3)P, which is indicative both of rapid turnover of this lipid and of a role for PtdIns 3-kinase in its production. PtdIns 3-kinase isoforms containing a p110β or p110γ catalytic subunit are activated by Gβγ (49). Given that Gβγ subunits also bind to the PH domains of PLC-β isozymes, this interaction may contribute to the membrane localization of these isozymes (see below).

PLC-β isozymes also might be targeted to the membrane environment through interaction with adapter proteins known as Na⁺/K⁺ exchanger regulatory factors (NHERFs) (50). These proteins contain two PDZ domains, one of which interacts with receptors such as the β₂-adrenergic receptor and purinergic P₂Y1 receptor. Interaction with a PDZ domain is mediated through a COOH-terminal sequence, (Ser/Thr)-X-(Val/Leu)-COOH, of PDZ binding proteins. All four PLC-β isozymes contain this PDZ binding motif. PLC-β3 binds specifically to NHERF2, and expression of NHERF2 in cells of the COS line potentiated the activation of PLC-β by carbachol (50).

Activation of PLC-β by Gβγ Subunits

With the exception of PLC-β4, PLC-β isozymes are also activated by Gβγ dimers (39, 41, 51, 52) (Figure 3). The relative sensitivity of PLC-β isozymes to Gβγ subunits differs from that to G_qα subunits; PLC-β1 is the least sensitive to Gβγ (39, 52). Direct binding measurements indicate that, although the Gβγ dimer interacts with PLC-β1, -β2, and -β3, it exhibits a high affinity only for PLC-β2 (40). The activation of PLC-β2 by Gβγ in response to ligation of the luteinizing hormone receptor, V2 vasopressin receptor, β₁- and β₂-adrenergic receptors, m2 muscarinic acetylcholine receptor, and the receptors for the chemoattractants interleukin-8, formyl-Met-Leu-Phe, and complement factor 5a has been demonstrated in transfected COS cells (53). Some of these receptors also stimulate PLC-β through G_qα subunits. Although the concentrations of Gβγ required for maximal activation of PLC-β isoforms in vitro are much larger than the effective concentrations of G_qα subunits, the maximal extents of activation are similar. Thus, both G_qα and Gβγ subunits likely contribute to activation of PLC-β in cells. It was suggested that Gβγ is the predominant mediator of PLC-β activation and that the role of G_qα subunits is to specify the receptors that couple to the enzyme (54). However, a variety of physiological stimuli that activate PLC-β isozymes in normal platelets were not able to activate these isozymes in platelets derived from α_q knockout mice; α_q is the only member of the G_qα subfamily expressed in normal platelets (55). Thus, Gα_q appears essential for PLC-β activation and cannot be replaced by Gβγ in this regard.

The region of PLC-β that interacts with G_qα subunits differs from that responsible for interaction with Gβγ . Thus, COOH-terminal truncation of PLC-β2 generated enzymes that were activated by Gβγ but not by Gα_q (56). The PH domain of PLC-β2 exhibits high affinity for Gβγ subunits bound to membranes (23). A chimeric PLC-δ1 molecule in which the PH domain of this isozyme was replaced with that of PLC-β2 was as sensitive to activation by Gβγ as was intact PLC-β2, whereas native PLC-δ1 is not responsive to Gβγ subunits (24). Furthermore, Gβγ induced the membrane translocation of a fusion protein containing green fluorescent protein and the PH domain of PLC-β1 by a mechanism that also required the production of PtdIns(3)P (22), which suggests that Gβγ and PtdIns(3)P bind to different regions of the PH domain. Gβγ subunits appear to interact with more than one region of PLC-β, however, given that the site of interaction of PLC-β2 with Gβγ was also mapped to the sequence that spans residues Glu574 and Lys583, located in the Y region (57). The region of Gβ subunits that interacts with effector molecules such as PLC-β and adenylate cyclase overlaps with that responsible for binding to Gα subunits, which explains why the βγ complex is able to interact with Gα or with effectors but not with both simultaneously (58). PLC-β2 actually contacts multiple sites within Gβ.

Mammalian cDNAs that encode 7 distinct Gβ subunits and 12 Gγ subunits have been isolated. Although some of these subunits are expressed only in specific tissues and selectivity is apparent in the interaction of Gβ and Gγ subunits, the formation of many different Gβγ dimers is possible. Among several such combinations of Gβ and Gγ subunits tested, all except β₁γ₁ activated purified PLC-β3 with similar potencies (59). In cells, however, not all available G_qα subunits and Gβγ combinations appear to function in activation of PLC. The results of experiments with antisense oligonucleotides directed to mRNAs encoding various G protein subunits suggested that the m1 muscarinic acetylcholine receptor interacts only with G protein complexes composed of the subunits α_q, α₁₁, β₁, β₄, and γ₄ in order to activate PLC in RBL-2H3 cells, despite the fact that the subunits α₁₄, β₂, β₃, γ ₂, γ ₃, γ ₅, and γ ₇ are also expressed in these cells (60).

The G protein β₅ subunit differs from the other β subunits in that it shares only 53% amino acid sequence identity with these other highly conserved β isoforms, and in that when complexed with Gγ , it appears to interact selectively with Gα_q- coupled receptors as a result of its specific association with Gα_q (61). Furthermore, the β₅ subunit discriminates among PLC-β isozymes independently of Gα_q. Thus, when recombinant Gβ₅γ and Gβ₁γ dimers were tested for their ability to activate PLC-β1, PLC-β2, and PLC-β3, Gβ₁γ activated all three PLC-β isozymes whereas Gβ₅γ activated PLC-β1 and PLC-β2 but not PLC-β3 (62).

Deactivation of PLC-β Signaling

G protein—initiated signaling is turned off by hydrolysis of the GTP bound to the Gα subunit, a reaction catalyzed by the intrinsic GTPase activity of the α subunit itself, and the subsequent reassociation of the GDP-bound α subunit with the βγ subunits. This deactivation process was studied in detail by reconstituting the m1 muscarinic acetylcholine receptor, G protein, and PLC-β1 in lipid vesicles (63). The muscarinic agonist carbachol induced a 90-fold increase in PLC activity in the presence of the G_qα subfamily (63). Although the intrinsic GTPase activity of purified Gα_q was low (~0.8 min^-1), the presence of PLC-β1 induced a >50-fold increase in this activity; that is, PLC-β1 is a GTPase-activating protein (GAP) for Gα_q. In the reconstituted system, PLC-β1 also increased the rate of GTP hydrolysis by Gα_q by a factor of up to 60 in the presence of carbachol, which alone stimulated GTPase activity by a factor of 6 to 10. More recent kinetic analysis suggests that PLC-β acts directly on Gα_q to stimulate hydrolysis of bound GTP, and that the other components of the reconstituted system (the m1 muscarinic receptor, Gβγ , and phospholipids) promote only GDP-GTP exchange during steady-state hydrolysis (64). A fragment of the COOH-terminal region of PLC-β1 (residues 903–1042) has also been shown to exhibit GAP activity (65). These results indicate that the receptor and PLC-β1 coordinately regulate the amplitude of the PLC signal and the rate of signal termination (Figure 3).

The GTPase activity of Gα_q is also stimulated by a family of regulatory proteins termed RGS (regulators of G protein signaling). Members of this protein family were first identified genetically as negative regulators of G protein signaling in lower eukaryotes, but more than 20 mammalian isoforms have been identified to date (66). Biochemical evidence indicates that RGS proteins block G protein function predominantly by acting as GAPs to limit the lifetime of the active, Gα_q-GTP complex, resulting in inhibition of PLC-β activity (67–69) (Figure 3). RGS proteins exhibit GAP activity toward G_i and G_q class α subunits but not toward G_sα or G₁₂α subunits (67, 68), and the extent of the RGS protein effect depends on the interaction of these proteins with the receptor rather than on their interaction with a specific Gα subunit (69).

PLC-β Signaling in the Nucleus

PLC signaling occurs not only at the plasma membrane but also in the nucleus (70, 71). PLC-β1 is the major PLC isoform in the nucleus of various cells (72, 73), and the COOH-terminal region of the protein is required for nuclear localization (47). The amount of nuclear PLC-β1 protein, which appears to be activated independently of its counterpart at the plasma membrane by an unknown mechanism, increases during cell growth and decreases during differentiation (74). The changes in the abundance of nuclear PLC-β1 correlate with those in the amount of PtdIns(4,5)P₂ hydrolyzed in the nucleus (70). However, the roles of the DAG and Ins(1,4,5)P₃ so generated remain unclear. Translocation of protein kinase C to the nucleus has been demonstrated coincident with the generation of DAG in this organelle. Analysis of cells lacking PLC-β1 as a result of gene ablation revealed that this isozyme is essential for the onset of DNA synthesis in response to insulin-like growth factor I (74).

Activation of PLC-γ by Receptor Protein Tyrosine Kinases

Polypeptide growth factors, such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), vascular endothelial growth factor, glial cell—derived growth factor, and hepatocyte growth factor, induce PtdIns(4,5)P₂ turnover by activating PLC-γ 1 in a wide variety of cells (53). However, insulin and colony-stimulating factor-1, whose receptors are structurally similar to those of PLC-activating growth factors, generally do not activate PLC-γ 1 (75, 76).

The binding of a growth factor to its receptor results in dimerization of receptor subunits, stimulation of the intrinsic tyrosine kinase activity of the receptor, and autophosphorylation of the receptor on specific tyrosine residues. These phosphorylated residues initiate cellular signaling by acting as high-affinity binding sites for the SH2 domains of various effector proteins. The selectivity of the receptor-effector interaction is determined not only by the residues surrounding the phosphorylated tyrosine of the receptor but also by the structure of the SH2 domain of the effector. In the PDGF receptor (β chain), for example, eight auto-phosphorylation sites have been identified that mediate the specific binding of Src family tyrosine kinases (Tyr579 and Tyr581), growth factor receptor—bound 2 (Grb2) (Tyr716), the 85-kDa subunit of PtdIns 3-kinase (Tyr740 and Tyr751), Ras GAP (Tyr771), SH2 domain—containing protein tyrosine phosphatase 2 (SH-PTP2) (Tyr1009), and PLC-γ 1 (Tyr1021) (77). In addition, Tyr766 of the FGF receptor and Tyr785 of the NGF receptor (Trk) specifically bind PLC-γ 1 (78, 79). However, in the case of the EGF receptor, which contains five autophosphorylation sites located in the COOH-terminal region, such individual sites are not strictly required for the recognition of and association with different SH2 domain—containing proteins. Thus, PLC-γ 1 is able to bind to any of these five phosphorylated tyrosine residues (80, 81).

The individual roles of the two SH2 domains of PLC-γ 1 in interaction of the enzyme with the PDGF receptor were investigated by functional inactivation of each domain and expression of the mutant proteins in a PLC-γ 1-deficient fibroblast cell line (82, 83). The mutant protein in which the COOH-terminal SH2 (C-SH2) domain was disabled was bound to and phosphorylated by the PDGF receptor, and it catalyzed the production of inositol phosphates to an extent only slightly less than that observed with the wild-type enzyme. In contrast, the mutant in which the NH₂-terminal SH2 (N-SH2) domain was impaired did not bind to the PDGF receptor and consequently was neither phosphorylated nor activated. These results suggest that the N-SH2 domain, but not the C-SH2 domain, of PLC-γ 1 is required for PDGF-induced enzyme activation.

Phosphorylation of PLC-γ 1 by PDGF, EGF, FGF, and NGF receptors occurs at identical sites: tyrosine residues 771, 783, and 1254 (84). The role of tyrosine phosphorylation of PLC-γ 1 was investigated by substituting phenylalanine for tyrosine at each of these three sites and expressing the mutant enzymes in cells of the NIH 3T3 line (85). Replacement of Tyr783 with Phe prevented the activation of PLC by PDGF; however, like the wild-type enzyme, the mutant PLC-γ 1 associated with the PDGF receptor. Although mutation of specific autophosphorylation sites of various receptors—including Tyr1021 in the PDGF receptor (β chain) (86), Tyr766 in the FGF receptor (87), and Tyr785 in the NGF receptor (88) as well as Tyr1169 in the vascular endothelial growth factor receptor (Flt) (89), Tyr1015 in the rat glial cell—derived growth factor receptor (Ret) (90), and Tyr1356 of the hepatocyte growth factor receptor (Met) (91)—prevented receptor association with PLC-γ 1 and growth factor—induced production of Ins(1,4,5)P₃, the mutant receptors still mediated growth factor—dependent tyrosine phosphorylation of PLC-γ 1. Thus, the growth factor—induced activation of PLC-γ 1 requires not only PLC-γ 1 tyrosine phosphorylation but also the association of the enzyme with the growth factor receptor. Such receptor association is likely required to target the tyrosine-phosphorylated (activated) enzyme to the membrane environment, where its substrate molecules reside.

PDGF-induced generation of PtdIns(3,4,5)P₃ also appears to contribute to the translocation of PLC-γ 1 to the plasma membrane, resulting in more efficient hydrolysis of PtdIns(4,5)P₂ (92–94). Thus, prevention of PtdIns(3,4,5)P₃ generation in PDGF-stimulated NIH 3T3 cells by exposure to a specific inhibitor of PtdIns 3-kinase (wortmannin or LY294002) resulted in an ~40% decrease in both intracellular generation of Ins(1,4,5)P₃ and the release of intracellular Ca²⁺ (93).Given that PtdIns(3,4,5)P₃ binds to both the NH₂-terminal PH domain (92) and the C-SH2 domain of PLC-γ 1 (93, 94), both of these domains likely contribute to the PtdIns(3,4,5)P₃-dependent translocation of the enzyme to the membrane.

A model depicting these various steps in the activation of PLC-γ by growth factors is depicted in Figure 4. Growth factor stimulation thus triggers autophosphorylation of the receptor protein tyrosine kinase (PTK) on tyrosine residues, which provides a docking site for the N-SH2 domain of PLC-γ 1 and thereby allows the receptor PTK to catalyze the phosphorylation of the bound enzyme. In addition, interaction with the receptor promotes the targeting of PLC-γ 1 to its PtdIns(4,5)P₂ substrate molecules in the membrane. Membrane association is also facilitated by interaction of the PH and C-SH2 domains of PLC-γ 1 with PtdIns(3,4,5)P₃ molecules in the membrane that are produced in response to growth factor stimulation. Although Figure 4 shows phosphorylated PLC-γ located in the vicinity of its substrate as a result of simultaneous association with the receptor PTK and PtdIns(3,4,5)P₃, it is also possible that the binding of the N-SH2 domain to the receptor and that of the C-SH2 or PH domain to PtdIns(3,4,5)P₃ constitute distinct means of targeting PLC-γ 1 to the membrane. Little is known of the roles of the EF-hand, SH3, and C2 domains in receptor-mediated activation of PLC-γ . The SH3 domain of PLC-γ 1 has been shown to bind to and stimulate the guanine nucleotide exchange activity of the protein SOS1 (95).

Figure 4

Growth factor receptor—mediated activation of PLC-γ . (Left) Growth factor stimulation triggers autophosphorylation of the receptor PTK on tyrosine residues, which then function as docking sites for many SH2 domain—containing proteins, (more...)

Autophosphorylation of growth factor receptors and subsequent tyrosine phosphorylation of substrate proteins, including PLC-γ 1, both require H₂O₂, whose concentration increases transiently and which appears to function as an intracellular messenger in growth factor—stimulated cells (96). In addition to the activation of a receptor PTK by the binding of a growth factor, concurrent inhibition of protein . tyrosine phosphatases by H₂O₂ may be necessary to increase the steady-state level of protein tyrosine phosphorylation (97).

Antigen Receptor—Induced Activation of PLC-γ by Nonreceptor Protein Tyrosine Kinases

Hematopoietic cells express a variety of antigen and immunoglobulin (Ig) receptors that are able to bind specifically to a wide spectrum of ligands. Such binding results in a rapid activation of signaling events such as the tyrosine phosphorylation and subsequent activation of PLC-γ 1 or PLC-γ 2. These receptors, which include the T cell antigen receptor (TCR), the B cell antigen receptor (BCR), the high-affinity IgE receptor (FcεRI), and the IgG receptors (FcγRs), comprise multiple polypeptide chains but do not possess intrinsic PTK activity; rather, they activate members of distinct families of nonreceptor PTKs such as the Src, Syk, and Tec families. The ligand-binding extracellular portion of these receptors consists of variable chains that confer specificity, and these chains are noncovalently associated with invariant subunits that serve to couple each receptor to intracellular signaling molecules such as nonreceptor PTKs, Ras, and PtdIns 3-kinase. This coupling is mediated by the presence in the invariant chains of the immune receptor tyrosine-based activation motif (ITAM), with the consensus sequence Asp-X₂-Tyr-X₂-Leu-X₇-Tyr-Asp-X-Leu (where X is any amino acid). Phosphorylation of the two tyrosine residues within this motif by members of the Src family of nonreceptor PTKs generates docking sites for SH2 domain—containing molecules that propagate signals from the activated receptors (98).

The extracellular domain of the TCR is formed by α and β (or γ and δ) subunits, which contain the variable domains responsible for recognition of small peptides bound to major histocompatibility complex class I or class II molecules. The noncovalently associated invariant chains of the TCR include the CD3 complex (CD3γ , CD3δ, and two copies of CD3ε) and the ζ chain homo dimer. Each of the CD3 subunits contains one copy of the ITAM sequence, whereas the ζ chain contains three copies of this motif (Figure 5). The binding of antigen by the TCR triggers activation of Lck and Fyn, two members of the Src family of PTKs, by an undefined mechanism. Either one or both of these enzymes phosphorylates the tyrosine residues located within the ITAM sequences of the CD3 and ζ chains. These pairs of phosphorylated tyrosine residues serve as binding sites for the tandem SH2 domains of ZAP-70, a member of the Syk family of PTKs. Lck or Fyn then phosphorylates the bound ZAP-70, resulting in its activation. Together with Lck and Fyn, activated ZAP-70 phosphorylates various downstream substrates, including PLC-γ 1, LAT (linker for activation of T cells), and SLP-76 (SH2 domain—containing leukocyte protein of 76 kDa).

Figure 5

TCR-induced activation of PLC-γ 1. (Top) Ligation of the TCR triggers the activation of Lck and Fyn by unknown mechanisms. Either or both of these Src family PTKs then phosphorylates tyrosine residues within ITAM sequences located in TCRζ (more...)

LAT is a palmitoylated, transmembrane domain—containing protein that is predominantly localized to membranes (99). In response to TCR stimulation, LAT is phosphorylated on at least four tyrosine residues: Tyr132, Tyr171, Tyr191, and Tyr226 (100). Phosphorylated LAT associates in vivo with PLC-γ 1, the adapter protein Gads, and other proteins as a result of interaction of its phosphorylated tyrosine residues with their SH2 domains. As in its interaction with the PDGF receptor, only the N-SH2 domain of PLC-γ 1 is necessary for binding to phosphorylated LAT (101). The Tyr132 residue of LAT mediates binding of PLC-γ 1, whereas Tyr171 and Tyr191 are responsible for binding Gads. Gads is abundant in T cells and, through its COOH-terminal SH3 domain, associates with the adapter protein SLP-76, thus linking SLP-76 to LAT (102). Studies with a LAT-deficient Jurkat T cell line indicate that LAT is essential for signaling through PLC and Ras (103). Engagement of the TCR on the mutant cells resulted in normal tyrosine phosphorylation of the ζ chain and ZAP-70; however, the extent of tyrosine phosphorylation of PLC-γ 1 was markedly reduced and PLC-γ -mediated PtdIns(4,5)P₂ hydrolysis and mobilization of intracellular Ca²⁺ were not apparent. T cells also express two members, Itk and Rlk, of the Tec family of nonreceptor PTKs, which contain an NH₂-terminal PH domain followed by SH3, SH2, and kinase domains. T cells from mice lacking both Itk and Rlk also exhibit a reduced extent of PLC-γ 1 activation in response to TCR stimulation (104).

On the basis of these various observations, it has been proposed that activated ZAP-70 phosphorylates LAT, and that phosphorylated LAT first recruits unphosphorylated PLC-γ 1 and positions it close to activated ZAP-70 and subsequently localizes the phosphorylated PLC-γ 1 in proximity to its substrate (Figure 5). The tyrosine phosphorylation of PLC-γ 1 may also be mediated to a certain extent by Fyn or Lck or by ZAP-70 without the help of LAT. SLP-76 is also required for TCR-induced phosphorylation of PLC-γ 1 and activation of the Ras signaling pathway, but not for the tyrosine phosphorylation of most proteins in response to TCR stimulation (105). Although the mechanism by which SLP-76 contributes to PLC-γ 1 activation is not clear, the recruitment of Itk by phosphorylated SLP-76 might serve to position Itk in close proximity to LAT-associated PLC-γ 1 (106).

The BCR is composed of an IgM molecule (consisting of two heavy chains and two light chains) noncovalently associated with two heterodimers of the invariant Igα and Igβ subunits, each of which contains one copy of the ITAM sequence. Engagement of the BCR results in the activation of Lyn, the most abundant Src family PTK in B cells, by an unknown mechanism that involves autophosphorylation on Tyr416 (located within the catalytic domain of the human protein) (107). Activated Lyn phosphorylates the pair of tyrosine residues in the ITAM sequences of Igα and Igβ, which results in the recruitment of Syk through its tandem SH2 domains. The ITAM-bound Syk is then phosphorylated on Tyr519 (located in the activation loop) by Lyn. This sequential activation of Lyn and Syk is similar to that of Lck-Fyn and ZAP-70 in T cells. However, in Lyn-deficient B cells, Syk is still partially activated and transmits signals to PLC-γ 2 (107), which suggests that Syk activation, unlike that of ZAP-70, is not strictly dependent on members of the Src family.

PLC-γ 1 is tyrosine phosphorylated after recruitment through its SH2 domains to phosphorylated Syk when reconstituted in COS cells (108). However, PLC-γ 2, not PLC-γ 1, is expressed in B cells. Furthermore, PtdIns(3,4,5)P₃ and Btk, a Tec family PTK, have been implicated in BCR-induced PLC-γ signaling (109, 110). BCR engagement activates PtdIns 3-kinase, resulting in the conversion to PtdIns(3,4,5)P₃ of a small fraction of presumably the same pool of PtdIns(4,5)P₂ as that targeted by PLC-γ 2. The resulting PtdIns(3,4,5)P₃ interacts with the PH domain of Btk, thereby promoting its membrane targeting and activation either by autophosphorylation or through its phosphorylation mediated by Lyn and Syk. The importance of Btk PH domain function is evidenced by the fact that spontaneously arising mutations in the region of the Btk gene encoding this domain cause X-linked agammaglobulinemia in humans and mice (111). Activated Btk is thought to contribute to PLC-γ 2 activation, at least in part, by catalyzing its tyrosine phosphorylation. Thus, treatment of B cells with wortmannin inhibits the tyrosine phosphorylation of PLC-γ 2. PtdIns(3,4,5)P₃ may also influence PLC-γ 2 activity directly through its interaction with the PH and SH2 domains of the enzyme, similar to its role in the PDGF-induced activation of PLC-γ 1 (Figure 4). Consistent with this scenario, engagement of both the BCR and the IgG receptor Fcγ IIb, the latter of which recruits the PtdIns(3,4,5)P₃ phosphatase SHIP and thereby prevents PtdIns(3,4,5)P₃ accumulation, blocks the activation of PLC-γ 2 (110).

A protein known as B cell linker (BLNK) or SLP-65 is also an essential component of the PLC-γ2 activation pathway in B cells (112). Phosphorylation of BLNK by Syk induces its translocation to the cell membrane. The phosphorylation of BLNK does not appear to require Btk, given that it occurs normally in Btk-deficient cells. On the basis of these various observations, a model that integrates the roles of PtdIns(3,4,5)P₃, Btk, Syk, and BLNK in PLC-γ 2 activation has been proposed (Figure 6). Phosphorylated, membrane-bound BLNK is thought to recruit PLC-γ 2 to the membrane, where it is phosphorylated and activated by membrane-associated Btk. Given that the SH2 domain of Btk, in addition to its PH domain, is required for full activation of PLC-γ 2 in B cells, this domain likely plays a role in the membrane localization of Btk by binding to tyrosine-phosphorylated BLNK.

Figure 6

BCR-induced activation of PLC-γ 2. (Top) BCR engagement triggers the activation of Lyn by an unknown mechanism. Activated Lyn phosphorylates tyrosine residues within ITAM sequences located in the Igα and Igβ chains. The two phosphorylated (more...)

The products of PtdIns 3-kinase also contribute to PLC-γ activation in response to ligation of Fc receptors, a family of receptors that bind to soluble Ig and immune complexes via the Fc region of Ig. For example, PtdIns 3-kinase is required for the translocation and tyrosine phosphorylation of PLC-γ 1 (but it has no effect on PLC-γ 2 activation) in response to antigen stimulation of FcεRI in RBL2H3 mast cells (113). In platelets, which express a single class of Fcγ R (Fcγ RIIA), inhibition of PtdIns 3-kinase prevents PLC-γ 2 activation induced by cross-linking of this receptor (114). Whereas inhibition of PtdIns 3-kinase does not affect tyrosine phosphorylation of PLC-γ 2, it blocks the stable association of this isozyme with the membrane-cytoskeleton interface that occurs at an early stage of platelet activation. Both SLP-76 and BLNK may also play important roles in Fcγ R-mediated PLC-γ activation (115).

G Protein—Coupled Receptor Activation of PLC-γ via Nonreceptor Protein Tyrosine Kinases

Although G protein—coupled receptors (GPCRs) lack intrinsic PTK activity, tyrosine phosphorylation of PLC-γ occurs in response to ligation of several such receptors, including those for acetylcholine (muscarinic), angiotensin II, thrombin, platelet-activating factor, and ATP (116–119). c-Src appears to be responsible for the phosphorylation of PLC-γ 1 in vascular smooth muscle cells and platelets; the introduction of antibodies to c-Src into these cells by electroporation inhibited the tyrosine phosphorylation of PLC-γ 1 elicited by angiotensin II or platelet-activating factor, respectively.

Activation of many GPCRs also induces tyrosine phosphorylation of receptor PTKs such as those for PDGF, EGF, and insulin-like growth factor I (120). Gβγ subunits have been implicated in the mechanism of GPCR-induced transactivation of receptor PTKs. Overexpression of Gβγ subunits in COS cells was sufficient to increase EGF receptor phosphorylation (120). In cultured cells from vascular smooth muscle, angiotensin II induced association of c-Src with the EGF receptor in the presence of EGF receptor kinase inhibitors, which suggests that activation of c-Src precedes EGF receptor transactivation (121). The activated α subunits of G_i or G_s proteins were recently shown to bind directly to the catalytic domain of c-Src and to increase its kinase activity, whereas neither the α subunits of G_q or G₁₂ nor Gβγ subunits exhibited this effect (122). Transactivation of a receptor PTK results in the formation of a structural scaffold for the assembly of a signaling complex (including PLC-γ ) that resembles that formed in response to interaction of the receptor PTK with its ligand (Figure 7).

Figure 7

GPCR-induced activation of PLC-γ by PTKs. An agonist-occupied GPCR induces the exchange of GDP for GTP on the α subunit of G_s or G_i and the subsequent dissociation of the α subunit from the membrane-associated Gβγ (more...)

In some instances, the GPCR itself contributes to a signaling complex that includes c-Src or Janus kinase (JAK). In HEK-293 and COS cells, the agonist-occupied β₂-adrenergic receptor forms a complex with activated c-Src in a manner that is dependent on receptor desensitization (120). This indirect interaction is mediated by the binding of c-Src to the receptor-bound adapter protein β-arrestin, and it results in the targeting of both c-Src and the receptor to clathrin-coated pits. Thus, the binding of β-arrestin to β₂-adrenergic receptors, which terminates receptor coupling to G proteins, also initiates a second wave of signal transduction in which the desensitized receptor functions as a structural component of a PTK cascade. Whether a similar PTK cascade operates for other GPCRs that induce PLC-γ activation remains unknown.

Stimulation of the angiotensin II receptor (AT₁ receptor) in vascular smooth muscle cells activates JAK2 by inducing its tyrosine phosphorylation and association with the receptor. This association requires a Tyr-Ile-Pro-Pro (YIPP) motif that is located in the intracellular COOH-terminal domain of the receptor and which is identical to the binding site in the PDGF receptor for the SH2 domain of PLC-γ 1 (117). JAK2 does not contain an SH2 domain; rather, its interaction with the AT₁ receptor appears to be mediated by the action of SH-PTP2 as a linker. Stimulation of the AT1 receptor induces phosphorylation of the tyrosine residue in the YIPP motif, and the phosphorylated tyrosine residue serves as the binding site for PLC-γ 1 (123). Both PLC-γ 1 and the SH-PTP2—JAK2 complex thus appear to bind to the same site on the AT₁ receptor.

Activation of PLC-γ by Other Lipid-Derived Messengers

Alternative mechanisms for the activation of PLC-γ that do not rely on tyrosine phosphorylation appear to exist. Phosphatidic acid has been shown to activate both tyrosine-phosphorylated and unphosphorylated forms of PLC-γ 1 to similar extents by increasing their affinity for substrate vesicles (124, 125). Given that phosphatidic acid is an immediate product of phosphatidylcholine hydrolysis by phospholipase D (PLD), activation of PLD in cells may also result in activation of PLC-γ (Figure 8).

Figure 8

Receptor-mediated activation of PLC-γ by phosphatidic acid generated by PLD (left) and by the concerted action of tau (or AHNAK) and AA generated by cytosolic phospholipase A₂. Although PLC-γ is not shown in close proximity to the membrane, (more...)

Unsaturated fatty acids, such as arachidonic acid (AA), also stimulate PLC-γ activity independently of tyrosine phosphorylation in the presence of various splicing variants of the microtubule-associated protein tau (126). Although tau is expressed exclusively in neurons, nonneuronal cells also contain a protein that, together with AA, activates PLC-γ . This activating protein was recently identified as a 680-kDa molecule termed AHNAK (giant in Hebrew) (127). AHNAK contains ~30 repeats of a motif of 128 amino acids. A glutathione S-transferase fusion protein containing one of the repeated motifs activated PLC-γ 1 at nanomolar concentrations in the presence of AA, which suggests that an intact AHNAK molecule contains multiple sites capable of activating PLC-γ .

Activation of PLC-γ by AHNAK appears highly similar to that by tau, even though the only similarity in the primary structures of these two proteins is a high percentage of proline residues. These two activators competed with each other in the activation of PLC-γ , further supporting the notion that they share a common mechanism of activation. Tau interacts with all three of the activation reaction components: PLC-γ , AA, and PtdIns(4,5)P₂. Both tau and AHNAK coimmunoprecipitate with PLC-γ , but not with PLC-β or PLC-γ isozymes.

Although the concentration of AA in resting cells is relatively low, large amounts of this fatty acid are released from phosphatidylcholine by the action of cytosolic PLA₂ in response to cell stimulation. It is therefore likely that certain stimuli that induce activation of cytosolic PLA₂ may also indirectly trigger the activation of PLC-γ in the presence of tau or AHNAK (Figure 8). Activation of cytosolic PLA₂ requires mobilization of intracellular Ca²⁺, and thus may occur secondarily to PLC activation; the subsequent activation of PLC-γ by the AA produced as a result of the action of cytosolic PLA₂ may thus contribute to a positive feedback loop in the hydrolysis of PtdIns(4,5)P₂. On the other hand, PtdIns(4,5)P₂ is a potent activator of cPLA₂ (3), so that hydrolysis of PtdIns(4,5)P₂ by PLC attenuates the activity of cytosolic PLA₂, constituting a negative feedback loop in terms of AA generation.