Abstract
Heterotrimeric G protein subunits regulate their effectors by protein-protein interactions. The regions involved in these direct interactions have either signal transfer or general binding functions (Buck, E., Li, J., Chen, Y., Weng, G., Scarlata, S., and Iyengar, R. (1999) Science 283, 1332-1335). Although key determinants of signal transfer regions for G protein subunits have been identified, the mechanisms of signal transfer are not fully understood. We have used a combinatorial peptide approach to analyze one Gbeta region, Gbeta86-105, involved in signal transfer to the effector phospholipase C (PLC)-beta2 to gain a more mechanistic understanding of Gbeta/PLC-beta2 signaling. Binding and functional studies with the combinatorial peptides on interaction with and stimulation/inhibition of phospholipase Cbeta2 indicate that binding affinity can be resolved from EC(50) for functional effects, such that peptides that have wild type binding affinities have 15- to 20-fold lower EC(50) values. Although more potent, these peptides display a much lower extent of maximal stimulation. These peptides synergize with Gbetagamma or peptides encoding the second Gbeta42-54 signal transfer region in maximally stimulating phospholipase C-beta2. Other combinatorial peptides from the Gbeta86-105 region that bind to PLC-beta2 by themselves submaximally stimulate and extensively inhibit Gbetagamma stimulation of PLC-beta2. The intrinsic stimulation function can be attributed to Arg-96 and Ser-97, the synergy function to Trp-99, and the binding affinity to Thr-87, Val-90, Pro-94, Arg-96, Ser-97, and Val-100. These results indicate that, even within signal transfer regions, residues involved in binding can be resolved from those involved in signal transfer and that signal transfer is likely to be achieved through dynamic rather than steady-state interactions.
Highlights
Protein-protein interactions represent a major mode by which information is propagated along cell signaling pathways
Screening of the Combinatorial Peptide Library—We screened a combinatorial peptide library based on the sequence derived from the G 86 –105 region for binding to phospholipase C (PLC)-2 using the peptides on plasmids method (11)
The library had greater than 109 individual members, and we completed three rounds of panning on phospholipase C- (PLC-)2 immobilized in microtiter plate wells
Summary
Materials—Library vectors and electrocompetent cells were a gift from Affymax, Palo Alto, CA. Bacterial Strains, Plasmids, and Oligonucleotides for the Construction of the Combinatorial Peptide on Plasmid Library—Escherichia coli ARI814 electrocompetent cells, the pJS142 library vector, and the pELM3 MBP vector were from Peter Schatz at Affymax Corp., Palo Alto, CA The use of these reagents to construct combinatorial libraries has been previously described (11). 0.25 g of purified PLC-2 in HEK buffer (35 mM HEPES, 0.1 mM EDTA, 50 mM KCl, 1 mM DTT) was added to the wells of a 96-well microtiter plate (Dynatech) and allowed to shake gently at 4 °C for 1 h. This allowed PLC-2 to adhere to the wells of the plates.
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