Identification of a structural element in phospholipase C beta2 that interacts with G protein betagamma subunits.
To delineate the specific regions of phospholipase C beta2 (PLC beta2) involved in binding and activation by G protein betagamma subunits, we synthesized peptides corresponding to segments of PLC beta2. Two overlapping peptides corresponding to Asn-564-Lys-583 (N20K) and Glu-574-Lys-593 (E20K) inhibited the activation of PLC beta2 by betagamma subunits (IC50 50 and 150 microM, respectively), whereas two control peptides did not. N20K and E20K, but not the control peptides, inhibited betagamma-dependent ADP-ribosylation of Galphai1 by pertussis toxin and betagamma-dependent activation of phosphoinositide 3-kinase. To demonstrate direct binding of the peptides to betagamma subunits, the peptides were chemically cross-linked to purified beta1gamma2. N20K and E20K cross-linked to both beta1 and gamma2 subunits, whereas the control peptides did not. Cross-linking to beta and gamma was inhibited by incubation with excess PLC beta2 or PLC beta3, whereas cross-linking to gamma but not beta was inhibited by r-myr-alphai1. These data together demonstrate specificity of N20K and E20K for G betagamma binding and inhibition of effector activation by betagamma subunits. The results suggest that an overlapping region of the two active peptides, Glu-574-Lys-583, mimics a region of PLC beta2 that is involved in binding to betagamma subunits. Changing a tyrosine to a glutamine in this overlapping region of the peptides inhibited binding of the peptide to betagamma subunits. Alignment of these peptides with the three-dimensional structure from PLC delta1 identifies a putative alpha helical region on the surface of the catalytic domain of PLC beta2 that could interact with betagamma subunits.
Sankaran
B
B
Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Osterhout
J
J
Wu
D
D
Smrcka
A V
AV
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D054799:Phospholipase C beta; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D018411:Spodoptera; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D003432:Cross-Linking Reagents; D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000246:Adenosine Diphosphate Ribose; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins