REMOVAL OF THE CARBOXYL-TERMINAL REGION OF PHOSPHOLIPASE-C-BETA-1 BY CALPAIN ABOLISHES ACTIVATION BY G-ALPHA-Q

Abstract

The 150-kDa phospholipase C (PLC)-beta1 and three immunologically related proteins with molecular sizes of 140, 100, and 45 kDa were purified from bovine brain extracts. Determination of the amino-terminal amino acid sequence of the 45-kDa protein and immunoblots of the purified proteins with sequence-specific antibodies to peptides corresponding to three different regions of PLC-beta1 suggest that a single cleavage at the linkage between amino acid residues 880 and 881 of PLC-beta1 generates the 100- and 45-kDa proteins, which correspond to the amino-terminal and carboxyl-terminal portions, respectively, of PLC-beta1. The Ca2+-dependent protease calpain appears to be responsible for the cleavage of PLC-beta1; the PLC-beta1 amino acid sequence contains PEST sequences which are common to proteins susceptible to calpain, and limited proteolysis of purified PLC-beta1 by calpain generated a 100-kDa protein and a 40-kDa protein that contains the same amino-terminal sequence as the 45-kDa protein. The 140-kDa protein lacks the carboxyl-terminal-most region of PLC-beta1, but there is no evidence it is derived from PLC-beta1 by proteolysis. Cleavage of PLC-beta1 by calpain had no significant effect on catalytic activity measured in the absence of the alpha subunit of the Galphaq but completely abrogated the stimulatory effect of Galphaq. On the other hand, Galphaq activated the 140-kDa enzyme. These results suggest that the region between residue 881 and the most carboxyl-terminal 10 kDa of PLC-beta1 contains the Galphaq interaction site.