A Cytosolic, Gαq- and βγ-insensitive Splice Variant of Phospholipase C-β4

Abstract

Phospholipase C (PLC)-␤4 has been considered to be a mammalian homolog of the NorpA PLC, which is responsible for visual signal transduction in Drosophila. We reported previously the cloning of a cDNA encoding rat phospholipase C-␤4 (PLC-␤4) (Kim, M. J., Bahk, Y. Y., Min, D. S., Lee, S. J., Ryu, S. H., and Suh, P.-G. (1993) Biochem. Biophys. Res. Commun. 194, 706 -712). We report now the isolation and characterization of a splice variant (PLC-␤4b). PLC-␤4b is identical to the 130-kDa PLC-␤4 (PLC-␤4a) except that the carboxyl-terminal 162 amino acids of PLC-␤4a are replaced by 10 distinct amino acids. The existence of PLC-␤4b transcripts in the rat brain was demonstrated by reverse transcriptionpolymerase chain reaction analysis. Immunological analysis using polyclonal antibody specific for PLC-␤4b revealed that this splice variant exists in rat brain cytosol. To investigate functional differences between the two forms of PLC-␤4, transient expression studies in COS-7 cells were conducted. We found that PLC-␤4a was localized mainly in the particulate fraction of the cell, and it could be activated by G␣ q , whereas PLC-␤4b was localized exclusively in the soluble fraction, and it could not be activated by G␣ q . In addition, both PLC-␤4a and PLC-␤4b were not activated by G-protein ␤␥-subunits purified from rat brain. These results suggest that PLC␤4b may be regulated by a mechanism different from that of PLC-␤4a, and therefore it may play a distinct role in PLC-mediated signal transduction. Phosphoinositide-specific phospholipase C (PLC)1 plays a pivotal role in transmembrane signaling. In response to various extracellular stimuli such as numerous hormones, growth factors, and neurotransmitters, this enzyme catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and thereby generates two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (IP 3 ) (1, 2). Diacylglycerol is a direct activator of protein kinase C, whereas IP 3 induces transient release of calcium from the endoplasmic reticulum into the cytoplasm (3).Multiple PLC isozymes have been purified from a variety of mammalian tissues, and several PLC genes have been cloned (4, 5). As predicted from the cDNAs, the PLC isozymes vary in size, with molecular masses ranging from 85 to 150 kDa. Despite low overall homology among the predicted amino acid sequences, significant sequence similarity exists in two domains that are designated as the X-and the Y-domains. These domains appear to constitute regions important for catalytic activities such as the specific recognition of the substrate and the hydrolysis of its phosphodiester bond. On the basis of the relative locations of the X-and Y-domains in the primary structure, PLC isozymes are classified into three types: ␤, ␥, and ␦. All PLC-␤ types have a carboxyl-terminal 400-amino acid domain that contains an unusually high number of charged residues. On the other hand, the ␥ type has a long stretch of sequence between the X-and Y-domains, and the ␦ type contains neither of the two ...

Signaling properties and expression in normal and tumor tissues of two phospholipase C epsilon splice variants

“…Number of cycles is indicated for PLCe common region and PLCe1a (top panels) while the data for PLCe1b (bottom panels) show amplification after 34 cycles. HEK293T sample (293T) and no templates (/) were included as controls; GAPDH was amplified to verify loading signaling and localization (Kim et al, 1998). Differences in tissue distribution have been reported for most splice variant-subtypes (Rebecchi and Pentyala, 2000).…”

Multiple roles of phosphoinositide-specific phospholipase C isozymes

“…Several alterative splicing variants of PLC-β4 have been reported. We found and identified two alternative splicing variants from rat and bovine brain (24,41,44). The third splicing variant of rat PLC-β4 has an additional 37 nucleotide exon at the C-terminal region (23).…”

The mechanism of phospholipase C-γ1 regulation

“…The existence of multiple forms of PLC enzymes suggests that each isozyme may differ in some respect, in tissue distribution, intracellular localization, regulatory mechanisms, and other downstream functions. Emerging evidence suggests that an additional level of diversity may exist beyond the above subgroup classification, because individual genes may yield multiple PLC products due to alternative splicing of the mRNA (Bahk et al, 1994;Kim et al, 1998). The diversity among the PLC enzymes point to selective coupling of individual PLCs to different receptors and signaling pathways.…”

“…Emerging evidence suggests that an additional level of diversity may exist beyond the above subgroup classification, because individual genes may yield multiple PLC products due to alternative splicing of the mRNA (Bahk et al, 1994;Kim et al, 1998). The diversity among the PLC enzymes point to selective coupling of individual PLCs to different receptors and signaling pathways.…”