Cloning, sequencing, purification, and Gq-dependent activation of phospholipase C-beta 3.
Six mammalian phospholipase C isozymes (PLC-beta 1, PLC-beta 2, PLC-gamma 1, PLC-gamma 2, PLC-delta 1, and PLC-delta 2) have been identified at both protein and DNA levels. Here, cDNAs corresponding to a previously unidentified PLC isozyme were isolated from a rat thyroid cell FRTL cDNA library. Comparison of the predicted amino acid sequence of this new PLC with other known PLC isozymes revealed a high degree of overall similarity with PLC-beta 1 and PLC-beta 2. Thus, the new PLC was named PLC-beta 3. Comparison with PLC-beta 1 and PLC-beta 2 also revealed that the deduced amino-terminal sequence of PLC-beta 3 was incomplete by 10-20 amino acids. With the use of antibodies raised against synthetic peptides corresponding to PLC-beta 3-specific amino acid sequences, we purified PLC-beta 3 from a rat brain particulate fraction. The purified enzyme exhibited an apparent molecular mass of 152 kDa on SDS-polyacrylamide gels, as compared with 150 and 140 kDa for PLC-beta 1 and PLC-beta 2, respectively. Studies of the activation of PLC-beta isozymes by three alpha subunits of Gq class G proteins, alpha q, alpha 11, and alpha 16 in the presence of guanosine 5-O-(3-thiotriphosphate) (GTP gamma S) revealed that the extent of activation decreased in the order of PLC-beta 1 > or = PLC-beta 3 >> PLC-beta 2 for all three alpha subunits, suggesting a certain degree of specificity in the interaction of Gq alpha subunits with different PLC-beta isozymes.
Jhon
D Y
DY
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Lee
H H
HH
Park
D
D
Lee
C W
CW
Lee
K H
KH
Yoo
O J
OJ
Rhee
S G
SG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D017386:Sequence Homology, Amino Acid; D010738:Type C Phospholipases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D007527:Isoenzymes; D004247:DNA; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins