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 microM (n = 4) (G alpha q: 251-265) and 110.0 +/- 41.9 microM (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 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.
Arkinstall
S
S
Glaxo Institute for Molecular Biology, Plan-les-Ouates, Geneva, Switzerland.
Chabert
C
C
Maundrell
K
K
Peitsch
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003198:Computer Simulation; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007527:Isoenzymes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology; D054799:Phospholipase C beta; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics; D013329:Structure-Activity Relationship; D015293:Transducin / Q000378:metabolism; D010738:Type C Phospholipases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins; D015293:Transducin