Identification of a structural element in Phospholipase C β2 that interacts with G protein βγ

Abstract

To delineate the specific regions of phospholipase C β2 (PLC β2) involved in binding and activation by G protein βγ subunits, we synthesized peptides corresponding to segments of PLC β2. Two overlapping peptides corresponding to Asn-564-Lys-583 (N20K) and Glu-574-Lys-593 (E20K) inhibited the activation of PLC β2 by βγ subunits (IC50 50 and 150 μM, respectively), whereas two control peptides did not. N20K and E20K, but not the control peptides, inhibited βγ-dependent ADP-ribosylation of Gα11 by pertusis toxin and βγ-dependent activation of phosphoinositide 3-kinase. To demonstrate direct binding of the peptides to βγ subunits, the peptides were chemically cross-linked to purified β1γ2. N20K and E20K cross- linked to both β1 and γ2 subunits, whereas the control peptides did not. Crosslinking to β and γ was inhibited by incubation with excess PLC β2 or PLC β3, whereas cross-linking to γ but not β was inhibited by r-myr- α11. These data together demonstrate specificity of N20K and E20K for G βγ binding and inhibition of effector activation by βγ subunits. The results suggest that an overlapping region of the two active peptides, Glu- 574-Lys-583, mimics a region of PLC β2 that is involved in binding to βγ subunits. Changing a tyrosine to a glutamine in this overlapping region of the peptides inhibited binding of the peptide to βγ subunits. Alignment of these peptides with the three-dimensional structure from PLC δ1 identifies a putative α helical region on the surface of the catalytic domain of PLC β2 that could interact with βγ subunits.