Mutational analysis of phospholipase C-beta 2. Identification of regions required for membrane association and stimulation by guanine-nucleotide-binding protein beta gamma subunits.
Members of the beta isozyme subfamily of the phosphoinositide-specific phospholipases C (PLC beta) have recently been shown to be stimulated by both guanine-nucleotide-binding protein alpha and beta gamma subunits. The alpha subunits of the Gq class activate PLC beta isozymes in the order of PLC beta 1 > or = PLC beta 3 >> PLC beta 2, which is different from the order of PLC beta 3 > PLC beta 2 > PLC beta 1 for beta gamma subunit stimulation. The C-terminal region of PLC beta 1, in particular the sequence between Thr903 and Leu1142, has been shown to be involved in interacting with activated alpha q subunits and to contain a region required for efficient membrane association of PLC beta 1 [Park, D., Jhon, D.-Y., Lee, C.-W., Ryu, S. H. & Rhee, S. G. (1993) J. Biol. Chem. 268, 3710-3714, and Wu, D., Jiang, H., Katz, A. & Simon, M. I. (1993) J. Biol. Chem. 268, 3704-3709]. To examine the structure-function relationships of a PLC beta isozyme highly sensitive to beta gamma subunit stimulation, we have altered the cDNA of PLC beta 2 by site-directed mutagenesis and have examined the effects of these structural alterations on the functional properties of the mutant polypeptides. The results show that the C-terminal region of PLC beta 2 downstream of Phe818, which corresponds to Tyr816 of PLC beta 1, contains a region essential for membrane association, but is required neither for the interaction of PLC beta 2 with Ca2+ and the phospholipid substrate, nor for beta gamma subunit stimulation of PLC beta 2. These data suggest that PLC beta isozymes are activated by alpha q and beta gamma subunits via distinct domains.
Schnabel
P
P
Molecular Pharmacology Division, German Cancer Research Center, Heidelberg.
Camps
M
M
Carozzi
A
A
Parker
P J
PJ
Gierschik
P
P
D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017384:Sequence Deletion; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D007527:Isoenzymes; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium