MAPPING REGIONS OF G(ALPHA-Q) INTERACTING WITH PLC-BETA-1 USING MULTIPLE OVERLAPPING SYNTHETIC PEPTIDES

The heterotrimeric G-protein alpha-chain G(alpha q) plays a critical role mediating receptor-linked activation of the beta isoforms of PLC which hydrolyse membrane inositol-containing phospholipids to generate the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. Despite knowledge of the three-dimensional structure of two G-protein alpha-chains (G(alpha t) and G(alpha i1)) as well as high regional amino acid conservation between members of the G-protein alpha-chain family, the precise molecular domains of G(alpha q) mediating activation of PLC beta 1 are unknown. To map sites responsible for effector interaction we employed 188 peptides each of 15 residues and corresponding to overlapping regions of the complete G(alpha q) sequence. These were tested for their ability to inhibit G(alpha q)-dependent activation of recombinant PLC beta 1 using an in vitro reconstitution assay. Peptides from two regions of G(alpha q) mediated up to 100% inhibition of GTP gamma S-stimulated PLC beta 1 activity, and representative peptides from each of these regions were half-maximally effective at 69.3 +/- 27.4 mu M (n = 4)(G(alpha q): 251-265) and 110.0 +/- 41.9 mu M (n = 4)(G(alpha q): 306-319), G(alpha q) regions described by inhibitory peptides are conserved selectively in other G-protein alpha-chains linked to PLC beta 1 activation (G(alpha 11), G(alpha 14)) and correspond spatially to sites of effector interaction identified in G(alpha s), by scanning mutagenesis and in transducin using site-specific antibodies and peptides. Computer homology modelling of G(alpha q) based on the crystal structure of transducin indicates that regions interacting with PLC beta 1 form two parallel alpha-helices lying at the surface of the G(alpha q) structure. These observations provide the first description of two regions within G(alpha q) critically important for activating PLC beta 1, and moreover, indicate that effector binding domains identified in transducin and G(alpha s) are also conserved spatially in G(alpha q).