Abstract
Complementary DNAs encoding a previously unidentified phosphoinositide-specific phospholipase C (PLC) isozyme were cloned from a rat brain cDNA library by the polymerase chain reaction with degenerate oligonucleotide primers based on sequences common to three known delta-type PLC isozymes. The encoded polypeptide contains 772 amino acids (calculated molecular mass, 88,966 daltons) and is similar in primary structure to delta-type PLC isozymes, with overall sequence identities of 45% to PLC-delta 1, 72% to PLC-delta 2, and 47% to PLC-delta 3. Thus, the new PLC isozyme was named PLC-delta 4. Recombinant PLC-delta 4 was purified from extracts of HeLa cells that had been infected with vaccinia virus containing the corresponding cDNA. The purified protein exhibited an apparent molecular mass of 90 kDa on SDS-polyacrylamide gels. The specific activity of PLC-delta 4 and its dependence on Ca2+ were similar to those of PLC-delta 1. The distribution of PLC-delta 4 in 16 different rat tissues was studied by immunoblot analysis with PLC-delta 4-specific antibodies of fractions obtained after an enzyme-enrichment procedure. The 90-kDa immunoreactive protein was detected unambiguously in only eight tissues and was present at concentrations that were low compared to those of other major PLC isozymes. A 93-kDa immunoreactive protein was also prominent in testis but was not detected in the other seven positive tissues. The 93-kDa enzyme appears to be derived from a splice variant of the mRNA that encodes the 90-kDa PLC-delta 4 and contains an additional 32 amino acids between the X and Y catalytic domains. Splice variants have not previously been detected for delta-type PLC isozymes.
Highlights
Phosphatidylinositol-specific phospholipase C (PLC)1 plays an important role in receptor-mediated signal transduction by generating two second messenger molecules, inositol 1,4,5trisphosphate (IP3) and diacylglycerol, from phosphatidylinositol 4,5-bisphosphate (PIP2) [1]
As part of our continuing effort to detect previously unidentified PLC isoforms and gather clues pointing to a regulatory mechanism for ␦-type PLC enzymes, we screened a rat brain cDNA library by the polymerase chain reaction (PCR) with oligonucleotide primers based on the amino acid sequences conserved in the X and Y domains of ␦-type isozymes
Restriction enzyme analysis and sequencing of these two fragments revealed that an additional 96 and 42 nt, respectively, were inserted immediately before the Y region of the PLC-␦4 open reading frame. To determine whether these two additional fragments were amplified from alternatively spliced forms of PLC-␦4 mRNA in testis, the entire coding region of PLC-␦4 cDNA was amplified with two sets of primers, all of which corresponded to the 5Ј- or 3Ј-untranslated regions of the cDNA
Summary
Vol 271, No 1, Issue of January 5, pp. 25–31, 1996 Printed in U.S.A. Molecular Cloning, Splice Variants, Expression, and Purification of Phospholipase C-␦4*. The 93-kDa enzyme appears to be derived from a splice variant of the mRNA that encodes the 90-kDa PLC-␦4 and contains an additional 32 amino acids between the X and Y catalytic domains. The amino acid sequence similarity in the X and Y domains is ϳ60 and 40%, respectively, among the nine mammalian enzymes [3, 4]; the similarity is greater when members of the same type of PLC are compared. As part of our continuing effort to detect previously unidentified PLC isoforms and gather clues pointing to a regulatory mechanism for ␦-type PLC enzymes, we screened a rat brain cDNA library by the polymerase chain reaction (PCR) with oligonucleotide primers based on the amino acid sequences conserved in the X and Y domains of ␦-type isozymes.
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