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 r ole mediating receptor-linked activation of the beta isoforms of PLC w hich 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 am ino acid conservation between members of the G-protein alpha-chain fam ily, the precise molecular domains of G(alpha q) mediating activation of PLC beta 1 are unknown. To map sites responsible for effector inter action 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 o f recombinant PLC beta 1 using an in vitro reconstitution assay. Pepti des from two regions of G(alpha q) mediated up to 100% inhibition of G TP 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 pept ides are conserved selectively in other G-protein alpha-chains linked to PLC beta 1 activation (G(alpha 11), G(alpha 14)) and correspond spa tially to sites of effector interaction identified in G(alpha s), by s canning mutagenesis and in transducin using site-specific antibodies a nd peptides. Computer homology modelling of G(alpha q) based on the cr ystal 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 descriptio n of two regions within G(alpha q) critically important for activating PLC beta 1, and moreover, indicate that effector binding domains iden tified in transducin and G(alpha s) are also conserved spatially in G( alpha q).