To know whether or not the set of genes involved in the inositol phospholipid signaling pathway already existed in the early evolution of animals, phospholipase Cs (PLCs) were cloned from Ephydatia fluviatilis (freshwater sponge) and Hydra magnipapillata strain 105 (hydra). Two PLC cDNAs, PLC-betaS and PLC-gammaS, were cloned from sponge and three cDNAs, PLC-betaH1, PLC-betaH2, and PLC-deltaH, from hydra. From the domain organization and the divergence pattern in the PLC family tree, the sponge PLC-betaS and PLC-gammaS and the hydra PLC-deltaH are possibly homologous to the vertebrate PLC-beta, PLC-gamma and PLC-delta subtypes, respectively. A detailed phylogenetic analysis suggests that the hydra PLC-betaH1 and PLC-betaH2 are homologs of the vertebrate PLC-beta1/2/3/Drosophila PLC21 and the vertebrate PLC-beta4/Drosophila norpA, respectively. A phylogenetic analysis of the PLC family and the protein kinase C (PKC) family, together with that of the G protein alpha subunit (Galpha) family, reveals that the origin of the set of genes G(alpha)q, PLC, PKC involved in the inositol phospholipid signaling pathway is very old, going back to dates before the parazoan-eumetazoan split, the earliest branching among extant animal phyla (Koyanagi, 1998).

The rpa (receptor potential absent) mutation of the blowfly, Calliphora erythrocephala, reduces the light-evoked responses of photoreceptor cells and renders the fly blind. This phenotype is similar to the phenotype caused by norpA mutations in Drosophila which have been shown to occur within a gene encoding phospholipase C. In Western blots, norpA antiserum stains a protein in homogenates of wild-type Calliphora eye and head that is similar in molecular weight to the NorpA protein. Very little staining of this protein is observed in similar homogenates of rpa mutant. Moreover, NorpA antiserum strongly stains retina in immunohistochemical assays of wild-type adult head, but not in rpa mutant. Furthermore, eyes of rpa mutant have a reduced amount of phospholipase C activity compared to eye of wild-type Calliphora. These data suggest that the rpa mutation occurs in a phospholipase C gene of the blowfly that is homologous to the norpA gene of Drosophila (McKay, 1994a).

Invertebrate visual signal transduction is initiated by rhodopsin activation of a guanine nucleotide binding protein, Gq, which stimulates phospholipase C (PLC) activity. A 140-kDa PLC enzyme has been purified from squid photoreceptors that is regulated by squid Gq. In these studies, an additional PLC enzyme was purified from the cytosol of squid photoreceptors and identified as a 70-kDa protein by SDS-polyacrylamide gel electrophoresis. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by PLC-70 is optimal at pH 5 in the presence of 100 microM Ca2+ with a specific activity of 10.3 micromol min-1 mg-1. A polyclonal antibody raised against purified PLC-70 does not recognize purified PLC-140, and proteolytic digestion of the two purified enzymes with trypsin or Staphylococcus aureaus V8 protease shows distinct patterns of peptide fragments, indicating that PLC-70 is not a fragment of PLC-140. The partial amino acid sequence of the protein shows homology with PLC21 and norpA isozymes cloned from Drosophila, and mammalian PLC beta isozymes. Reconstitution of purified GTPgammaS-bound soluble squid Gq with PLC-70 results in significant enhancement of PIP2 hydrolysis over a range of Ca2+ concentrations and shifts the maximum activation by calcium to 1 microM. These results suggest that cephalopod phototransduction is mediated by Gq activation of more than one cytosolic PLC enzyme (Mitchell, 1998).

Conserved regions of norpA cDNA were used to isolate bovine cDNAs. These encode four alternative forms of phospholipase C of the beta class that are highly homologous to the NorpA protein and expressed preferentially in the retina. Two of the variants are highly unusual in that they lack much of the N-terminal region present in all other known phospholipases C. The sequence conservation between these proteins and the NorpA protein is higher than that between any other known phospholipases C. GTPase sequence motifs found in proteins of the GTPase superfamily are found conserved in all four variants of the bovine retinal protein as well as the NorpA protein but not in other phospholipases C. Results suggest that these proteins together with the NorpA protein constitute a distinctive subfamily of phospholipases C that are closely related in structure, function, and tissue distribution. Mutations in the NorpA gene, in addition to blocking phototransduction, cause light-dependent degeneration of photoreceptors. In view of the strong similarity in structure and tissue distribution, a defect in these proteins may have similar consequences in the mammalian retina (Ferreira, 1993).

Inositol phospholipid-specific phospholipase C (PLC) isozymes in bovine retina have been characterized . Chromatography of a retinal homogenate on a heparin column partially resolves six peaks of PLC activity, which differ in their relative selectivities for the substrates phosphatidyl 4,5-bisphosphate (PIP2) and phosphatidylinositol (PI). Five of the peaks correspond to the known PLC isozymes PLC-beta 1, PLC-beta 3, PLC-gamma 1, PLC-delta 1, and PLC-delta 2. PLC-beta 1, PLC-beta 3, PLC-gamma 1, and PLC-delta 1 in the retinal fractions were identified by immunoblotting with isozyme-specific antibodies, and PLC-delta 2 was identified by direct sequencing of tryptic peptides. PLC-gamma 2 and PLC-beta 2 were not detectable by immunoblot analysis. In addition to five of the seven mammalian PLC isozymes identified to date, bovine retina contained a previously unidentified PLC, which exhibited the highest selectivity for PIP2 over PI. The new PLC was purified from a retinal particulate fraction to yield a preparation that contained a major protein band with an apparent molecular mass of 130 kDa on SDS-polyacrylamide gels. Sequence analysis of 12 tryptic peptides derived from the 130-kDa protein suggested that the primary structure of the new PLC is similar to those members of beta-type PLC isozymes, especially to that of PLC-norpA, which was originally identified in Drosophila eye. The new enzyme was thus named PLC-beta 4. A search of a rat brain cDNA library with the polymerase chain reaction and oligonucleotide primers based on common PLC amino acid sequences resulted in the cloning of a rat brain cDNA corresponding to a previously uncharacterized PLC. The cDNA encodes a putative polypeptide of 1176 amino acids, with a calculated molecular mass of 134,532 daltons, that contains the sequences of all 12 tryptic peptides of PLC-beta 4. Furthermore, the deduced amino acid sequence of the encoded protein is more related to PLC-norpA than to any of the three mammalian PLC-beta isozymes. These results suggest that the brain cDNA encodes PLC-beta 4, which is likely a mammalian homolog of PLC-norpA (Lee, 1993).

Phospholipase C-beta 4(PLC-beta 4), a new member of phospholipase C isozyme, was purified from bovine cerebellum. The cDNA encoding rat PLC-beta 4 has been cloned from a cDNA library prepared from rat brain. The predicted open reading frame encodes a protein of 1,176 amino acids with a calculated molecular weight of 134,552. The deduced amino acid sequence exhibits 39, 36, and 36% identity with the sequences of rat PLC-beta 1, human PLC-beta 2, and rat PLC-beta 3, respectively. The amino acid sequence of PLC-beta 4, especially, shows higher identity (50%) with norpA PLC sequence from Drosophila melanogaster than those of other PLC-beta subtypes, suggesting that the PLC-beta 4 might be a mammalian PLC equivalent of norpA PLC implicated in photosignal transduction in Drosophila (Kim, 1993).

Transient transfection assays were used to determine how the activity of phospholipase C beta 4, which is preferentially expressed in retina, is regulated. An expression vector carrying the full-length cDNA corresponding to phospholipase C beta 4 was constructed and co-transfected into COS-7 cells together with cDNA encoding the alpha subunits of the Gq class and various beta and gamma subunits corresponding to the heterotrimeric GTP-binding proteins. All the alpha subunits of the Gq class, including G alpha q, G alpha 11, G alpha 14, G alpha 15, and G alpha 16 can activate PLC beta 4 and none of the G beta gamma subunits tested, including G beta 1 gamma 1, G beta 1 gamma 2, G beta 1 gamma 3, or G beta 2 gamma 2, activate phospholipase C beta 4. In control experiments, cotransfection with cDNA encoding the alpha subunit of transducin or Gi2 gives no activation of PLC beta 4. These results indicate that phospholipase C beta 4 is activated by G alpha subunits that are members of the Gq class, and, like the phospholipase C beta 1 isoform, it is refractory to activation in the transfection assay by many of the combinations of beta and gamma subunits found in the heterotrimeric G-proteins (Jiang, 1994).

PLC-beta 4 has now been shown to differ from the other three mammalian beta-type isozymes (PLC-beta 1, -beta 2, and -beta 3) in that it is selectively inhibited by ribonucleotides. The inhibition requires the 5'-phosphate and 2'-hydroxyl groups of ribose as well as the base moiety. Thus, deoxyribonucleotides and ribose 5-phosphate are not inhibitory. The monophosphate, diphosphate, and triphosphate nucleoside derivatives are all inhibitory, whereas cyclic nucleotides are ineffective. Purine nucleotides are more potent inhibitors than pyrimidine nucleotides. Unlike the other beta-type isozymes, PLC-beta 4 contains the GX4GKS consensus sequence for the recognition of the phosphoryl group of nucleotides. In the absence of ribonucleotides, the specific activity of PLC-beta 4 toward phosphatidyl-inositol 4,5-bisphosphate is four to five times the average specific activity of PLC-beta 1 and PLC-beta 3. Thus, nucleotide-dependent inhibition may serve to reduce the activity of PLC-beta 4 in the absence of a hormonal signal. The regulation of PLC-beta 4 by G-proteins was also studied. Similar to the other three PLC-beta isozymes, PLC-beta 4 is activated by the alpha subunit of Gq but not by the transducin alpha subunit. However, unlike other PLC-beta isozymes, PLC-beta 4 was not responsive to activation by G beta gamma subunits (C. W. Lee, 1994).

The Drosophila norpA gene encodes a phosphatidylinositol-specific phospholipase C (PI-PLC) expressed predominantly in photoreceptors and involved in phototransduction. However, no direct role for a phospholipase C in vertebrate phototransduction has been identified to date. Bovine cDNAs encoding PI-PLC isoforms expressed predominantly in the retina have been isolated and characterized. These isoforms have higher homology to the NorpA protein than to any other known PI-PLC. Evidence is presented that the NorpA-homologous bovine retinal PI-PLCs, although found in other retinal neurons as well, are found in cones but not in rods. The results suggest that the phototransduction cascade in cones may utilize phospholipase C in addition to phosphodiesterase (Ferreira, 1994).

Inositol phospholipid-specific phospholipase C (PLC) generates two important second messengers, inositol triphosphate and diacylglycerol. Rat PLC beta 4 cDNA is highly homologous to the norpA cDNA of Drosophila. PLC beta 4 gene expression has been mapped in rat brain tissue sections by in situ hybridization. The PLC beta 4 gene is expressed at high abundance in cerebellar Purkinje cells and neurones of the substantia nigra, the median geniculate bodies and the thalamic nuclei. PLC beta 4 transcripts are also detected in the mammillary nuclei, the neocortex, the habenula and the olfactory bulbs. The specific pattern of gene expression should help to clarify the relationships between the PLC beta 4 and various constituents of second-messenger systems involved in transduction mechanisms triggered by the stimulation of seven transmembrane domain receptors. The strong gene expression in Purkinje cells and retinal neurones suggests that PLC beta 4 may be involved in the pathogenesis of mouse and human neurological diseases characterized by ataxia and retinal degeneration (Roustan, 1995).

Defects in the Drosophila norpA gene encoding a phosphoinositide-specific phospholipase C (PLC) block invertebrate phototransduction and lead to retinal degeneration. The mammalian homolog, PLCB4, is expressed in rat brain, bovine cerebellum, and the bovine retina in several splice variants. To determine a possible role of PLCB4 gene defects in human disease, several overlapping cDNA clones were isolated from a human retina library. The composite cDNA sequence predicts a human PLC beta 4 polypeptide of 1022 amino acid residues (MW 117,000). This PLC beta 4 variant lacks a 165-amino-acid N-terminal domain characteristic for the rat brain isoforms, but has a distinct putative exon 1 unique for human and bovine retina isoforms. A PLC beta 4 monospecific antibody detects a major (130 kDa) and a minor (160 kDa) isoform in retina homogenates. Somatic cell hybrids and deletion panels were used to localize the PCLB4 gene to the short arm of chromosome 20. The gene was further sublocalized to 20p12 by fluorescence in situ hybridization (Alvarez, 1995).

Expression of G protein-regulated phospholipase C (PLC) beta 4 in the retina, lateral geniculate nucleus, and superior colliculus implies that PLC beta 4 may play a role in the mammalian visual process. A mouse line that lacks PLC beta 4 was generated and the physiological significance of PLC beta 4 in murine visual function was investigated. Behavioral tests using a shuttle box demonstrated that the mice lacking PLC beta 4 are impaired in their visual processing abilities, whereas they showed no deficit in their auditory abilities. In addition, the PLC beta 4-null mice show 4-fold reduction in the maximal amplitude of the rod a- and b-wave components of their electroretinograms relative to their littermate controls. However, recording from single rod photoreceptors did not reveal any significant differences between the PLC beta 4-null and wild-type littermates, nor were there any apparent differences in retinas examined with light microscopy. While the behavioral and electroretinographic results indicate that PLC beta 4 plays a significant role in mammalian visual signal processing, isolated rod recording shows little or no apparent deficit, suggesting that the effect of PLC beta 4 deficiency on the rod signaling pathway occurs at some stage after the initial phototransduction cascade and may require cell-cell interactions between rods and other retinal cells (Jiang, 1996).

A variety of extracellular signals are transduced across the cell membrane by the enzyme phosphoinositide-specific phospholipase C-beta (PLC-beta) coupled with guanine-nucleotide-binding G proteins. There are four isoenzymes of PLC-beta, beta1-beta4, but their functions in vivo are not known. The roles of PLC-beta1 and PLC-beta4 in the brain were examined by generating null mutations in mice: PLCbeta1-/- mice developed epilepsy and PLCbeta4-/- mice showed ataxia. The molecular basis of these phenotypes were determined. PLC-beta1 is involved in signal transduction in the cerebral cortex and hippocampus by coupling predominantly to the muscarinic acetylcholine receptor, whereas PLC-beta4 works through the metabotropic glutamate receptor in the cerebellum, illustrating how PLC-beta isoenzymes are used to generate different functions in the brain (Kim, 1997).

Phospholipase C (PLC)-beta4 has been considered to be a mammalian homolog of the NorpA PLC, which is responsible for visual signal transduction in Drosophila. A splice variant of PLC-beta4, PLC-beta4b, is identical to the 130-kDa PLC-beta4 (PLC-beta4a) except that the carboxyl-terminal 162 amino acids of PLC-beta4a are replaced by 10 distinct amino acids. The existence of PLC-beta4b transcripts in the rat brain was demonstrated by reverse transcription-polymerase chain reaction analysis. Immunological analysis using polyclonal antibody specific for PLC-beta4b reveals that this splice variant exists in rat brain cytosol. To investigate functional differences between the two forms of PLC-beta4, transient expression studies in COS-7 cells were conducted. PLC-beta4a is localized mainly in the particulate fraction of the cell, and it can be activated by Galphaq, whereas PLC-beta4b is localized exclusively in the soluble fraction, and it can not be activated by Galphaq. In addition, both PLC-beta4a and PLC-beta4b are not activated by G-protein betagamma-subunits purified from rat brain. These results suggest that PLC-beta4b may be regulated by a mechanism different from that of PLC-beta4a, and therefore it may play a distinct role in PLC-mediated signal transduction (Kim, 1998).