The beta-type PLC isozymes differ from other isozymes in that they contain a long carboxyl-terminal region downstream of the Y catalytic domain and a region rich in acidic amino acids between the two separated X and Y catalytic domains. To determine the sites on PLC-beta2 that participate in the interaction of the enzyme with Gqalpha and beta/gamma subunits, we introduced specific truncations and substitutions in the PLC-beta2 cDNA at positions corresponding to the carboxyl-terminal and acidic amino acid-rich regions, respectively. The capacity of the mutants to be activated by Gqalpha and beta/gamma subunits was determined. Substitution of glutamine residues for three or all seven of a stretch of consecutive glutamic acids in the acidic domain of PLC- beta2 affected neither Gqalpha- nor beta/gamma-dependent activation significantly. Carboxyl-terminal truncation to residue Gly-934 or to residue Ala-867 resulted in enzymes that were activated by beta/gamma but not by Gqalpha. This result suggests that the carboxyl-terminal region of PLC-~2 is required for activation by Gqalpha and that beta/gamma subunits interact with a different region of the enzyme. Thus, Gqalpha and beta/gamma subunits may independently modulate a single PLC-beta2 molecule concurrently. The fourth member of mammalian beta-type phospholipase C isozymes, PLC- beta4 was shown to differ from the other three mammalian beta-type isozymes (PLC-beta1, -beta2, and -beta3) 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 were not inhibitory. The monophosphate, diphosphate, and triphosphate nucleoside derivatives were all inhibitory, whereas cyclic nucleotides were ineffective. Unlike the other beta-type isozymes, PLC-beta4 contains the GX(4)GKS consensus sequence for the recognition of the phosphoryl group of nucleotides. e regulation of PLC-beta4 by G proteins was also studied. Similar to the other three PLC-beta isozymes, PLC-beta4 was activated by the alpha subunit of Gqalpha but not by the transducin alpha subunit. However, unlike other PLC-beta isozymes, PLC-~4 was not responsive to activation by Gbeta/gamma subunits.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Intramural Research (Z01)
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National Heart, Lung, and Blood Institute
United States
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