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

FEBS Letters

Maundrell, Kinsey¹  Peitsch, Manuel¹  Arkinstall, Steve¹  Chabert, Christian¹ 

[1] Glaxo Institute for Molecular Biology, 14 Chemin des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland

关键词: G-protein;    Gαq;    PLCβ;    Phosphoinositide hydrolysis;    Synthetic peptide;    Protein modelling;    Effector interaction;   

DOI  :  10.1016/0014-5793(95)00351-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The heterotrimeric G-protein α-chain G_αp 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 (G_αt and G_α1 as well as high regional amino acid conservation between members of the G-protein α-chain family, the precise molecular domains of G_αp 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 G_αq 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). G_αq regions described by inhibitory peptides are conserved selectively in other G-protein a-chains linked to PLCβ1 activation (G_α11, G_α4) and correspond spatially to sites of effector interaction identified in G_αs by scanning mutagenesis and in transducin using site-specific antibodies and peptides. Computer homology modelling of G_αq based on the crystal structure of transducin indicates that regions interacting with PLCβ1 form two parallel α-helices lying at the surface of the G_αq structure. These observations provide the first description of two regions within G_αq critically important for activating PLCβ1, and moreover, indicate that effector binding domains identified in transducin and G_αs are also conserved spatially in G_αq.

【 授权许可】

Unknown