Interaction between Pleckstrin homology domains and G protein betagamma-subunits: analyses of kinetic parameters by a biosensor-based method.
Pleckstrin homology (PH) domains, comprised of rather weakly conserved sequences of about 100 amino acid residues, are a protein motif found in many signaling and cytoskeletal proteins. PH domains have been shown to bind to the betagamma subunits of heterotrimeric GTP-binding proteins (Gbetagamma), but the affinity of PH domains for Gbetagamma has not been quantitatively estimated in detail. To characterize the nature of the interaction between PH domains and Gbetagamma its kinetic parameters were analyzed using a BIAcore instrument. All PH domains tested (PH domains of ras-specific guanine nucleotide exchange factor (ras-GRF), phospholipase (PLC) gamma1, and Son of sevenless protein (Sos)) appeared to bind to Gbeta1gamma2 with affinity constants K(D) of 0.108, 0.318, and 0.208 microM, respectively. The binding of PH domains to Gbetagamma was inhibited by preincubation of Gbetagamma with the GDP-bound but not the GTP-bound form of Gialpha. This study showed a high affinity interaction between PH domains and Gbetagamma, and suggests a potential role of PH domains in Gbetagamma-mediated signal transduction in intact cells.
Sawai
T
T
Eisai Tsukuba Research Laboratories, Ibaraki, Japan.
Hirakawa
T
T
Yamada
K
K
Nishizawa
Y
Y
D015374:Biosensing Techniques; D001792:Blood Platelets / Q000737:chemistry*; D001798:Blood Proteins / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000737:chemistry*; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D007700:Kinetics; D008832:Microchemistry; D010446:Peptide Fragments / Q000737:chemistry*; D010750:Phosphoproteins; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D001798:Blood Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C039077:platelet protein P47; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins