IDENTIFICATION OF A STRUCTURAL ELEMENT IN PHOSPHOLIPASE-C BETA-2 THATINTERACTS WITH G-PROTEIN BETA-GAMMA-SUBUNITS

To delineate the specific regions of phospholipase C beta 2 (PLC beta 2) involved in binding and activation by G protein beta gamma subunits , we synthesized peptides corresponding to segments of PLC beta 2, Two overlapping peptides corresponding to Asn-564-Lys-583 (N20K) and Glu- 574-Lys-593 (E20K) inhibited the activation of PLC beta 2 by beta gamm a subunits (IC50 50 and 150 mu M, respectively), whereas two control p eptides did not, N20K and E20K, but not the control peptides, inhibite d beta gamma-dependent ADP-ribosylation of G alpha(i1) by pertussis to xin and beta gamma-dependent activation of phosphoinositide 3-kinase, To demonstrate direct binding of the peptides to beta gamma subunits, the peptides were chemically cross-linked to purified beta(1) gamma(2) . N20K and E20K cross-linked to both beta(1) and gamma(2) subunits, wh ereas the control peptides did not, Cross-linking to beta and gamma wa s inhibited by incubation with excess PLC beta 2 or PLC beta 3, wherea s cross-linking to gamma but not beta was inhibited by r-myr-alpha(i1) . These data together demonstrate specificity of N20K and E20K for G b eta gamma binding and inhibition of effector activation by beta gamma subunits, The results suggest that an overlapping region of the two ac tive peptides, Glu-574-Lys-583, mimics a region of PLC beta 2 that is involved in binding to beta gamma subunits, Changing a tyrosine to a g lutamine in this overlapping region of the peptides inhibited binding of the peptide to beta gamma subunits, Alignment of these peptides wit h the three-dimensional structure from PLC delta 1 identifies a putati ve alpha helical region on the surface of the catalytic domain of PLC beta 2 that could interact with beta gamma subunits.