Gary L. Waldo

Kinetic Scaffolding Mediated by a Phospholipase C–β and Gq Signaling Complex

Reciprocal Regulation An essential step in many signaling cascades is inositol lipid hydrolysis catalyzed by phospholipase C–β. The latter is activated by the α subunit of the heterotrimeric G protein Gq, and it in turn inactivates Gαq, thus sharpening the signal. Waldo et al. (p. 974, published online 21 October) report structural and biochemical data that explain the basis of this reciprocal regulation. One domain of phospholipase C–β binds to activated Gαq. Though the phospholipase C–β active site remains occluded in the structure, the plug is probably removed upon G protein–dependent orientation of the lipase at the membrane. A second domain of phospholipase C–β accelerates guanosine triphosphate hydrolysis by Gαq causing the signaling complex to dissociate. A crystal structure shows how the two components of a central signaling complex regulate each other. Transmembrane signals initiated by a broad range of extracellular stimuli converge on nodes that regulate phospholipase C (PLC)–dependent inositol lipid hydrolysis for signal propagation. We describe how heterotrimeric guanine nucleotide–binding proteins (G proteins) activate PLC-βs and in turn are deactivated by these downstream effectors. The 2.7-angstrom structure of PLC-β3 bound to activated Gαq reveals a conserved module found within PLC-βs and other effectors optimized for rapid engagement of activated G proteins. The active site of PLC-β3 in the complex is occluded by an intramolecular plug that is likely removed upon G protein–dependent anchoring and orientation of the lipase at membrane surfaces. A second domain of PLC-β3 subsequently accelerates guanosine triphosphate hydrolysis by Gαq, causing the complex to dissociate and terminate signal propagation. Mutations within this domain dramatically delay signal termination in vitro and in vivo. Consequently, this work suggests a dynamic catch-and-release mechanism used to sharpen spatiotemporal signals mediated by diverse sensory inputs.

A unique fold of phospholipase C-beta mediates dimerization and interaction with G alpha q.

GTP-bound subunits of the Gq family of Gα subunits directly activate phospholipase C-β (PLC-β) isozymes to produce the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. PLC-βs are GTPase activating proteins (GAPs) that also promote the formation of GDP-bound, inactive Gβ subunits. Both phospholipase activation by Gα–GTP subunits and GAP activity require a C-terminal region unique to PLC-β isozymes. The crystal structure of the C-terminal region from an avian PLC-β, determined at 2.4 Å resolution, reveals a novel fold composed almost entirely of three long helices forming a coiled-coil that dimerizes along its long axis in an antiparallel orientation. The dimer interface is extensive (∼3,200 Å2), and, based on gel exclusion chromatography, full length PLC-βs are dimeric, indicating that PLC-βs likely function as dimers. Sequence conservation, mutational data and molecular modeling show that an electrostatically positive surface of the dimer contains the major determinants for binding Gβq. Effector dimerization, as highlighted by PLC-βs, provides a viable mechanism for regulating signaling cascades linked to heterotrimeric G proteins.

G Protein–Coupled Receptor–Mediated Activation of p110β by Gβγ Is Required for Cellular Transformation and Invasiveness

Blocking the activation of p110β by G proteins inhibits tumor cell proliferation. Distinguishing Between PI3Kβ Activators The p110β member of the class IA phosphoinositide 3-kinases (PI3Ks) is unusual in that it is activated by receptor tyrosine kinases (RTKs) and by G protein–coupled receptors (GPCRs), in the latter case through Gβγ dimers. Tumor cells deficient in the phosphatase PTEN depend on the activity of p110β for proliferation; however, it has not been possible to assess the relative contributions of RTKs and GPCRs to p110β activity in this context. Dbouk et al. identified and characterized the Gβγ-binding site of p110β and developed an inhibitor peptide that blocked Gβγ-dependent activation of p110β in vitro but left RTK-mediated activation intact. A cell-permeable version of this peptide inhibited the proliferation and invasiveness of human PTEN-deficient tumor cell lines, suggesting that specifically targeting Gβγ-mediated activation of p110β may provide an effective therapy in the treatment of certain cancers. Synergistic activation by heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) and receptor tyrosine kinases distinguishes p110β from other class IA phosphoinositide 3-kinases (PI3Ks). Activation of p110β is specifically implicated in various physiological and pathophysiological processes, such as the growth of tumors deficient in phosphatase and tensin homolog deleted from chromosome 10 (PTEN). To determine the specific contribution of GPCR signaling to p110β-dependent functions, we identified the site in p110β that binds to the Gβγ subunit of G proteins. Mutation of this site eliminated Gβγ-dependent activation of PI3Kβ (a dimer of p110β and the p85 regulatory subunit) in vitro and in cells, without affecting basal activity or phosphotyrosine peptide–mediated activation. Disrupting the p110β-Gβγ interaction by mutation or with a cell-permeable peptide inhibitor blocked the transforming capacity of PI3Kβ in fibroblasts and reduced the proliferation, chemotaxis, and invasiveness of PTEN-null tumor cells in culture. Our data suggest that specifically targeting GPCR signaling to PI3Kβ could provide a therapeutic approach for tumors that depend on p110β for growth and metastasis.

Mechanism of Activation and Inactivation of Gq/Phospholipase C-β Signaling Nodes

The phospholipase C (PLC) isozymes catalyze conversion of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) into the Ca2+-mobilizing second messenger inositol 1,4,5-trisphosphate (inositol (1,4,5)P3) and the protein kinase-activating second messenger diacylglycerol (DAG).1 Many PLC-dependent cellular responses occur in addition to those mediated by these classical second messengers since the activities of a broad range of membrane, cytosolic, and cytoskeletal proteins are regulated by PtdIns(4,5)P2 binding.2–4 Mammals express 13 different PLCs (Figure 1), which differ markedly in their modes of upstream regulation and physiological functions.5 Many growth factors, antigens, and other extracellular stimuli signal through tyrosine phosphorylation of the PLC-γ isozymes, whereas an even larger group of hormones, neurotransmitters, chemoattractant and chemosensory molecules, and other extracellular stimuli promote physiological effects through heterotrimeric G protein-dependent activation of the PLC-β isozymes. These and other forms of receptor-promoted signaling pathways also communicate to Ras superfamily GTPases, which in turn directly activate certain PLC isoyzmes. Thus, PLC-dependent signal transduction provides one of the major fabrics for communication of cells, and delineation of its function at all levels from the intact animal to the atomic resolution of mechanism is fundamental to understanding mammalian biology. Figure 1 Conserved domain structure of the mammalian PLC isozymes. The 13 functional human PLC isozymes were aligned on the basis of the conservation of the protein sequence, and a dendrogram was constructed to cluster similar sequences into shared branches. The ... Many excellent reviews on PLC-dependent signaling are available that focus, for example, on early aspects of the discovery and function of receptor-promoted formation of Ins(1,4,5)P3/diacylglycerol and mobilization of Ca2+,1,6,7 classification and regulation of the PLC isozymes,8–10 tyrosine phosphorylation-dependent activation,11,12 activation through Ras superfamily GTPases,5,13,14 physiology,15,16 and structure/function.17–19 We have limited this review to a general introduction of the domain and structural features of the PLC isozymes and then consider in detail recent advances made in understanding the mechanisms through which Gα-subunits of the Gq family bind to and activate PLC-β isozymes and how the PLC-β isozymes in turn promote inactivation of this signaling complex by stimulating GTP hydrolysis by the GTP-activated G protein.

Membrane-induced Allosteric Control of Phospholipase C-β Isozymes

Background: Phospholipase C-β (PLC-β) isozymes hydrolyze phosphatidylinositol 4,5-bisphosphate to propagate signals for several physiological responses. Results: Membranes are essential for the allosteric release of autoinhibition of PLC-β isozymes. Conclusion: Activators of PLC-β release autoinhibition by orientating the isozymes at the membrane. Significance: The model described provides a better understanding of PLC-β regulation and potential mechanisms to inhibit their activation. All peripheral membrane proteins must negotiate unique constraints intrinsic to the biological interface of lipid bilayers and the cytosol. Phospholipase C-β (PLC-β) isozymes hydrolyze the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to propagate diverse intracellular responses that underlie the physiological action of many hormones, neurotransmitters, and growth factors. PLC-β isozymes are autoinhibited, and several proteins, including Gαq, Gβγ, and Rac1, directly engage distinct regions of these phospholipases to release autoinhibition. To understand this process, we used a novel, soluble analog of PIP2 that increases in fluorescence upon cleavage to monitor phospholipase activity in real time in the absence of membranes or detergents. High concentrations of Gαq or Gβ1γ2 did not activate purified PLC-β3 under these conditions despite their robust capacity to activate PLC-β3 at membranes. In addition, mutants of PLC-β3 with crippled autoinhibition dramatically accelerated the hydrolysis of PIP2 in membranes without an equivalent acceleration in the hydrolysis of the soluble analog. Our results illustrate that membranes are integral for the activation of PLC-β isozymes by diverse modulators, and we propose a model describing membrane-mediated allosterism within PLC-β isozymes.

Potent and Selective Peptide-based Inhibition of the G Protein Gαq

In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gαq binds its two major classes of effectors, the phospholipase C (PLC)-β isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gαq within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gαq in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gαq. A representative peptide was specific for active Gαq because it did not bind inactive Gαq or other classes of active Gα subunits and did not inhibit the activation of PLC-β3 by Gβ1γ2. In contrast, the peptide robustly prevented activation of PLC-β3 or p63RhoGEF by Gαq; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gαq in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gαq-dependent activation of PLC-β3 at least as effectively as a dominant-negative form of full-length PLC-β3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gαq in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gαq in cells.

Regulation of Phospholipase C-β Isoenzymes

A broad range of hormones, neurotransmitters, growth factors, and chemoattractants produce their physiological effects through phospholipase C (PLC)-catalyzed initiation of the inositol lipid signaling cascade (Berridge and Irvine, 1987). Although tyrosine phosphorylation of the SH2- and SH3-domain-containing PLC-γ isoenzymes accounts for interface of a number of growth factors with this signaling pathway, the majority of extracellular stimuli act through seven-transmembrane-spanning receptors that activate G-proteins that in turn activate isoenzymes of the PLC-β family (Rhee and Choi, 1992). The focus of this chapter is on this latter group of signaling proteins.

Agonist and Guanine Nucleotide Regulation of P 2Y Purinergic Receptor-Linked Phospholipase C

Extracellular adenine nucleotides interact with cell surface receptors to produce a myriad of physiological effects in the central nervous system and peripheral tissues (Burnstock, 1978; Gordon, 1986; Burnstock and Kennedy, 1986; Fleetwood and Gordon, 1987). Burnstock proposed in 1978 that these responses are mediated by two major receptor types: those physiologically activated by adenosine, called P1-purinergic receptors, and exhibiting a potency order of adenosine > AMP > ATP, and those activated by ATP or ADP, called P2-purinergic receptors, and exhibiting the potency order of ATP > ADP > AMP > adenosine. Subsequent studies proved that at least two subtypes of P1-purinergic receptors exist (A1- and A2- purinergic receptors) (Van Calker et al., 1979; Stiles, 1986; Williams, 1987). Subclassification of P2-purinergic receptors proved more difficult, since in contrast to the receptors for adenosine, no good antagonists of P2-purinergic receptors are available, and cell surface hydrolases readily metabolize ATP and ADP to adenosine, which can then produce physiological effects through P1-purinergic receptors. However, the development of relatively non-hydrolyzable analogs of ATP helped resolve this issue, and the observation of differential effects of a large number of ATP and ADP analogs led Burnstock and Kennedy to propose in 1985 that subypes of P2-purinergic receptors exist. P2X-purinergic receptors exhibit the potency order of Ap(CH2)pp > App(CH2)p > ADP > 2-methylthio ATP (2MeSATP) and P2Y-purinergic receptors exhibit the potency order of 2MeSATP > ATP > Ap(CH2)pp = App(CH2)p.