Determination of the primary structure of PLC-154 demonstrates diversity of phosphoinositide-specific phospholipase C activities

doi:10.1016/0092-8674(88)90549-1

Cell

Volume 54, Issue 2, 15 July 1988, Pages 171-177

https://doi.org/10.1016/0092-8674(88)90549-1 Get rights and content

Abstract

Protein sequence analysis of a bovine brain phosphoinositide-specific phospholipase C (PI-PLC; PLC-154) has permitted the isolation of a cDNA that appears to code for this protein. Transient expression of this cDNA in COS-1 cells demonstrates that the cDNA encodes a functional phospholipase C that migrates at approximately 150,000 daltons. A transcript of approximately 7 kb is observed in RNA derived from bovine brain and a related transcript of the same size is present in certain human cell lines. Southern blot analysis indicates that one or possibly two genes hybridize with a PLC-154 probe. Regions of homology between PLC-154 and the previously described PLC-148 allow the assignment of a putative catalytic domain to the central region of PLC-154.

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Citation Excerpt :
In the late 80 s, three PLC isozymes, namely PLC-γ, -β, and -δ, were isolated from bovine brain cytosol and their cDNA sequences were obtained [29,140,141]. Subsequently, multiple sub-types were shown to exist in each of these three classes from various tissues using protein purification, RT-PCR using specifically-designed primers or a screening method using low stringency hybridization with probes made from the conserved X or Y regions [30,142–150]. Thus 4 PLCβ (1-4) and 2 PLCγ (1, 2) enzymes were found to exist.
Phosphoinositide-specific phospholipase Cs (PLCs) are expressed in all mammalian cells and play critical roles in signal transduction. To obtain a comprehensive understanding of these enzymes in physiology and pathology, a detailed structural, biochemical, cell biological and genetic information is required. In this review, we cover all these aspects to summarize current knowledge of the entire superfamily. The families of PLCs have expanded from 13 enzymes to 16 with the identification of the atypical PLCs in the human genome. Recent structural insights highlight the common themes that cover not only the substrate catalysis but also the mechanisms of activation. This involves the release of autoinhibitory interactions that, in the absence of stimulation, maintain classical PLC enzymes in their inactive forms. Studies of individual PLCs provide a rich repertoire of PLC function in different physiologies. Furthermore, the genetic studies discovered numerous mutated and rare variants of PLC enzymes and their link to human disease development, greatly expanding our understanding of their roles in diverse pathologies. Notably, substantial evidence now supports involvement of different PLC isoforms in the development of specific cancer types, immune disorders and neurodegeneration. These advances will stimulate the generation of new drugs that target PLC enzymes, and will therefore open new possibilities for treatment of a number of diseases where current therapies remain ineffective.
Reflections on the days of phospholipase C
2013, Advances in Biological Regulation
Citation Excerpt :
This nomenclature proved reasonable in 1988, when distinct cDNAs corresponding to the β, γ, and δ forms of PLC (in our laboratory) (Suh et al., 1988b, c), and a cDNA corresponding to the α form of PLC (in the laboratory of Frank Bennett and Stanley Crooke) (Bennett et al., 1988) were cloned. Other laboratories also reported amino acid sequences for PLC-β (Katan et al., 1988) and PLC-γ (Stahl et al., 1988). When the sequences of enzymes β, γ, and δ (all of these sequences were deduced from the nucleotide sequences of rat or bovine brain cDNAs) were compared, despite a low overall homology between the three enzymes, a significant sequence similarity was found in two regions: one of about 150 amino acids and the other of about 120 amino acids.
Upregulation of pancreatic polypeptide-sensitive neuropeptide Y (NPY) receptors in estrogen-induced hypertrophy of the anterior pituitary gland in the Fischer-344 rat
2000, Molecular and Cellular Endocrinology
Implants of diethylstilbestrol inducing anterior pituitary prolactinomas in female Fischer-344 rats produced a considerable elevation of high-affinity binding of either rat or human pancreatic polypeptide (hPP). No comparable upregulation of high-affinity binding sites was noted for the ligand [¹²⁵I](Leu³¹,Pro³⁴)hPYY (LP-PYY) (masked by 2 nM hPP to force selectivity for the Y₁ sites), or of the Y₂-selective ligand [¹²⁵I]hPYY(3–36). The Y₅-like sites displayed the rank order of potency of hPP=rPP, hNPY, LP-PYY>pPYY(1–36)>hNPY(2–36)>hPYY(3–36)≫aPP, similar to the previously described rabbit kidney or hypothalamic Y₅-like receptors. The PP binding in the anterior pituitary was not sensitive to the Y₁-selective non-peptidic antagonist BIBP-3226. The PP binding was highly sensitive to alkali metal cations, and to a N5-substituted amiloride antagonist of sodium transport, but not to a C2-guanido substituted amiloride antagonist of sodium channels. The binding was also sensitive to phospholipase C antagonist U-73122, and to alkylating α-adrenergic agonist chloroethylclonidine. Lactotrophs isolated in Percoll gradients after enzymic dispersion of the anterior pituitary gland retained the high-affinity PP binding. Following removal of estrogen implants, the hPP binding sites decreased to very low levels within 3 days, in parallel to the decrease of plasma prolactin.
Sensitivity of orexin-A binding to phospholipase C inhibitors, neuropeptide Y, and secretin
2000, Biochemical and Biophysical Research Communications
The binding of [¹²⁵I] orexin-A (Ox-A) to particulates from Chinese hamster ovary (CHO) cells expressing the cloned orexin-A receptor, or from rat forebrain areas, was sensitive to blockers of phosphatidylinositol-specific phospholipase C (PtdIns-PLC) U-73122 and ET-18-OCH₃, little affected by phospholipase A₂ inhibitor quinacrine, and not sensitive to D609, a xanthate inhibitor of phosphatidylcholine-selective PLC. Interaction of the receptor with a PtdIns-PLC was further indicated by a large sensitivity of the binding to Ca²⁺. Up to 50% of the binding was sensitive to the G-protein nucleotide site agonist GTP-γ-S. Ligand attachment to the orexin-A receptor thus depends on an association with both PtdIns-PLC and G-protein α-subunits. In all paradigms examined, the binding of [¹²⁵I]orexin-A was competed by human/rat neuropeptide Y (hNPY) and porcine secretin with a potency similar to orexin-A (IC₅₀ range 30–100 nM). The rank order of potency for NPY-related peptides was hNPY > porcine peptide YY (pPYY) > (Leu³¹, Pro³⁴) human PYY > human PYY(3-36) > hNPY free acid > human pancreatic polypeptide. Among secretin-related peptides, the rank order of potency was porcine secretin ≥ orexin-A > human pituitary adenylate cyclase-activating peptide > orexin-B > porcine vasoactive intestinal peptide. Among opioid peptides, rat β-endorphin and camel δ-endorphin were much less active than NPY and secretin, and two enkephalins were inactive at 1 μM. In view of high abundance of NPY in forebrain, the above cross-reactivity could indicate a significant contribution of NPY to signaling via orexin-A receptors.
Enhanced phosphoinositide hydrolysis via overexpression of phospholipase C β1 or δ1 inhibits stimulus-induced insulin release in insulinoma MIN6 cells
1999, Biochemical and Biophysical Research Communications
To study the effects of enhanced phosphoinositide hydrolysis on insulin secretion, phosphoinositide-specific phospholipase Cβ1 (PLCβ1) or PLCδ1 was overexpressed in insulinoma MIN6 cells via adenoviral vectors. Inositol phosphate production stimulated by NaF (with AlCl3) in PLCβ1-overexpressing cells and that stimulated by KCl or glucose in both PLCβ1- and PLCδ1-overexpressing cells were greater than that in control cells. In addition, reduced phosphatidylinositol-4,5-bisphosphate levels were observed in these cells stimulated by NaF or KCl. The greater phosphoinositide hydrolysis was accompanied by 25–45% inhibition of insulin secretion. These data suggest that excessive phosphoinositide hydrolysis inhibits secretagogue-induced insulin release in MIN6 cells.
Catalytic domain of phosphoinositide-specific phospholipase C (PLC): Mutational analysis of residues within the active site and hydrophobic ridge of PLCδ1
1998, Journal of Biological Chemistry
Structural studies of phospholipase C δ1 (PLCδ1) in complexes with the inositol-lipid headgroup and calcium identified residues within the catalytic domain that could be involved in substrate recognition, calcium binding, and catalysis. In addition, the structure of the PLCδ1 catalytic domain revealed a cluster of hydrophobic residues at the rim of the active site opening (hydrophobic ridge). To assess a role of each of these residues, we have expressed, purified, and characterized enzymes with the point mutations of putative active site residues (His³¹¹, Asn³¹², Glu³⁴¹, Asp³⁴³, His³⁵⁶, Glu³⁹⁰, Lys⁴³⁸, Lys⁴⁴⁰, Ser⁵²², Arg⁵⁴⁹, and Tyr⁵⁵¹) and residues from the hydrophobic ridge (Leu³²⁰, Phe³⁶⁰, and Trp⁵⁵⁵). The replacements of most active site residues by alanine resulted in a great reduction (1,000–200,000-fold) of PLC activity analyzed in an inositol lipid/sodium cholate mixed micelle assay. Measurements of the enzyme activity toward phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bis-phosphate (PIP₂) identified Ser⁵²², Lys⁴³⁸, and Arg⁵⁴⁹ as important for preferential hydrolysis of polyphosphoinositides, whereas replacement of Lys⁴⁴⁰selectively affected only hydrolysis of PIP₂. When PLC activity was analyzed at different calcium concentrations, substitutions of Asn³¹², Glu³⁹⁰, Glu³⁴¹, and Asp³⁴³ resulted in a shift toward higher calcium concentrations required for PIP₂ hydrolysis, suggesting that all these residues contribute toward Ca²⁺binding. Mutational analysis also confirmed the importance of His³¹¹ (∼20,000-fold reduction) and His³⁵⁶(∼6,000-fold reduction) for the catalysis. Mutations within the hydrophobic ridge, which had little effect on PIP₂hydrolysis in the mixed-micelles, resulted in an enzyme that was less dependent on the surface pressure when analyzed in a monolayer. This systematic mutational analysis provides further insights into the structural basis for the substrate specificity, requirement for Ca²⁺ ion, catalysis, and surface pressure/activity dependence, with general implications for eukaryotic phosphoinositide-specific PLCs.

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Cell

Determination of the primary structure of PLC-154 demonstrates diversity of phosphoinositide-specific phospholipase C activities

Abstract

J. Biol. Chem.

Trends Biochem. Sci.

J. Biol. Chem.

J. Biol. Chem.

Life Sci.

Gene

J. Biol. Chem.

J. Biol. Chem.

Inositol trisphosphate and diacylglycerol: two interacting second messengers

Annu. Rev. Biochem.

Domain mapping of PLC-148

Protein kinases in cellular signal transduction: tyrosine kinase growth factor receptors and protein kinase C

SV40-transformed simian cells support the replication of early SV40 mutants

Cell

Guanine nucleotide stimulates production of inositol trisphosphate in rat cortical membranes

Biochem. J.

Phosphatidylinositol turnover in receptor mechanism and signal transduction

Annu. Rev. Pharmacol. Toxicol.