Abstract
The beta- but not the gamma- and delta-type isozymes of inositol phospholipid-specific phospholipase C (PLC) are activated by G protein alpha q and beta gamma subunits. 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-beta 2 that participate in the interaction of the enzyme with alpha q and beta gamma subunits, we introduced specific truncations and substitutions in the PLC-beta 2 cDNA at positions corresponding to the carboxyl-terminal and acidic amino acid-rich regions, respectively. After transient expression of these cDNA clones in CV-1 cells, the mutant enzymes were partially purified and their capacity to be activated by alpha q and beta gamma subunits determined. Substitution of glutamine residues for three or all seven of a stretch of consecutive glutamic acids in the acidic domain of PLC-beta 2 affected neither alpha q- 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 alpha q. This result suggests that the carboxyl-terminal region of PLC-beta 2 is required for activation by alpha q, and that beta gamma subunits interact with a different region of the enzyme. Thus, alpha q and beta gamma subunits may independently modulate a single PLC-beta 2 molecule concurrently.
Highlights
The 13- but not the y- and &typeisozymes of inositol conserved domains, the so-called X domain of -160 amino phospholipid-specific phospholipase C (PLC) are acti- acids and the Y domain of -260 amino acids, that appear to vated by G proteina, and By subunits
We investigated this possibility by measuring the amino acid sequence identity among different PLC isozymes aq-and py-dependent activation of various PLC-p2 mutants is low, and theirsizes differ markedly, ranging from 85 to 150 that were generated by altering the two PLC-@-specificrekDa
Activation by aqand j3-y subunits of G proteins is specific to the @type PLC isozymes, the structural characteristics of which include the acidic amino acid-rich region between the X and Y domains and thelong carboxyl-terminal region that followsthe Y domain (Fig. 1).To investigate the roles of these two PLC-j3-specific regions in G protein-dependent activation, we constructed four PLC-82 mutants by substituting glutamines for the first three of seven consecutive glutamic acid residues or for all seven glutamic acids, and by eliminating residues carboxyl-terminal to Gly-934 or Ala-867.The wild-type and mutantenzymeswere transiently expressed by transfecting CV-1 cells with the mammalian expression vector pTMl containing PLC-82 cDNA
Summary
Vol 268,No 34,Issue ofDecember 5,pp. 25952-25957,1993 Printed in U.S.A. Sang Bong Lee$, Seok Hwan Shin$, John R. The distinct structural featuresof the different PLC types residue Gly-934 or to residue Ala-867 resulted in en- appear to correlate with differences in their mechanisms of zymes that were activated by By but not by a,. Two y-type isozymes, PLC-y1 and PLC-72, are result suggests that the carboxyl-terminal region of PLC-82 is required for activation by a,, and that By subunits interact witahdifferent regionof the enzyme. We investigated this possibility by measuring the amino acid sequence identity among different PLC isozymes aq-and py-dependent activation of various PLC-p2 mutants is low, and theirsizes differ markedly, ranging from 85 to 150 that were generated by altering the two PLC-@-specificrekDa. all known PLC isozymes contain two gions: the carboxyl-terminal region downstream of the Y domain and the acidic amino acid-rich region between the X. the payment of page charges. Activation by aqwas achieved by first incubating purified a, for 1 h at 30 "C with 1 mM guanosine GTPrS; a, was stored on ice before being added to the PLC assay mixture
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