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Abstract
To delineate the specific regions of phospholipase C beta2 (PLC beta2) involved in binding and activation by G protein betagamma subunits, we synthesized peptides corresponding to segments of PLC beta2. Two overlapping peptides corresponding to Asn-564-Lys-583 (N20K) and Glu-574-Lys-593 (E20K) inhibited the activation of PLC beta2 by betagamma subunits (IC50 50 and 150 microM, respectively), whereas two control peptides did not. N20K and E20K, but not the control peptides, inhibited betagamma-dependent ADP-ribosylation of Galphai1 by pertussis toxin and betagamma-dependent activation of phosphoinositide 3-kinase. To demonstrate direct binding of the peptides to betagamma subunits, the peptides were chemically cross-linked to purified beta1gamma2. N20K and E20K cross-linked to both beta1 and gamma2 subunits, whereas the control peptides did not. Cross-linking to beta and gamma was inhibited by incubation with excess PLC beta2 or PLC beta3, whereas cross-linking to gamma but not beta was inhibited by r-myr-alphai1. These data together demonstrate specificity of N20K and E20K for G betagamma binding and inhibition of effector activation by betagamma subunits. The results suggest that an overlapping region of the two active peptides, Glu-574-Lys-583, mimics a region of PLC beta2 that is involved in binding to betagamma subunits. Changing a tyrosine to a glutamine in this overlapping region of the peptides inhibited binding of the peptide to betagamma subunits. Alignment of these peptides with the three-dimensional structure from PLC delta1 identifies a putative alpha helical region on the surface of the catalytic domain of PLC beta2 that could interact with betagamma subunits.
Highlights
Many transmembrane receptors coupled to heterotrimeric G proteins can initiate the hydrolysis of phosphatidylinositol 4,5bisphosphate (PIP2)1 to produce inositol trisphosphate (IP3) and diacylglycerol
Peptide Design—There is significant homology between phospholipase C 2 (PLC 2) and phospholipase C (PLC) ␦1 in the 116-amino acid region of PLC 2 that was found to bind to ␥ subunits [16]: 33% identity and 47% similarity overall with a 70-amino acid region having 54% identity and 67% similarity (Fig. 1)
We examined the structure of PLC ␦1 to determine which regions of PLC 2 would be likely to be on the surface of the protein and accessible to G protein ␥ subunits
Summary
Many transmembrane receptors coupled to heterotrimeric G proteins can initiate the hydrolysis of phosphatidylinositol 4,5bisphosphate (PIP2) to produce inositol trisphosphate (IP3) and diacylglycerol. The first 100 amino acids are predicted to form a pleckstrin homology domain [5] This domain in PLC ␦1, when expressed in isolation, binds to PIP2 and IP3 and when removed from PLC ␦1, inhibits anchoring to PIP2-containing membranes but does not inhibit catalysis (6 – 8). The structure shows that the N-terminal region between the pleckstrin homology domain and the X domain has a structural fold that is very similar to the EF-hand domain found in Ca2ϩ-binding proteins, including calmodulin. Removal of the C-terminal third of PLC 1 abolishes activation by G␣q, but Ca2ϩ-dependent activity remains intact [11, 12]. Further analysis of this region has served
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