Mapping regions of Gαq interacting with PLCβ1 using multiple overlapping synthetic peptides

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Abstract
The heterotrimeric G-protein α-chain Gαp plays a critical role mediating receptor-linked activation of the β 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 α-chains (Gαt and Gα1 as well as high regional amino acid conservation between members of the G-protein α-chain family, the precise molecular domains of Gαp mediating activation of PLCβ1 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 αq sequence. These were tested for their ability to inhibit G αq-dependent activation of recombinant PLCJβ1 using an in vitro reconstitution assay. Peptides from two regions of Gαq mediated up to 100% inhibition of GTPγS-stimulated PLCβ1 activity, and representative peptides from each of these regions were half-maximally effective at 69.3 ± 27.4 μM (n = 4) (G αq: 251–265) and 110.0 ± 41.9 μM (n = 4) (G αq: 306–319). Gαq regions described by inhibitory peptides are conserved selectively in other G-protein a-chains linked to PLCβ1 activation (Gα11, Gα4) and correspond spatially to sites of effector interaction identified in Gαs by scanning mutagenesis and in transducin using site-specific antibodies and peptides. Computer homology modelling of Gαq based on the crystal structure of transducin indicates that regions interacting with PLCβ1 form two parallel α-helices lying at the surface of the Gαq structure. These observations provide the first description of two regions within Gαq critically important for activating PLCβ1, and moreover, indicate that effector binding domains identified in transducin and Gαs are also conserved spatially in Gαq.