Identification of a region at the N-terminus of phospholipase C-beta 3 that interacts with G protein beta gamma subunits.
Members of the phospholipase C-beta (PLC-beta) family of proteins are activated either by G alpha or G beta gamma subunits of heterotrimeric G proteins. To define specific regions of PLC-beta 3 that are involved in binding and activation by G beta gamma, a series of fragments of PLC-beta 3 as glutathione-S-transferase (GST) fusion proteins were produced. A fragment encompassing the N-terminal pleckstrin homology (PH) domain and downstream sequence (GST-N) bound to G protein beta 1 gamma 2 in an in vitro binding assay, and binding was inhibited by G protein alpha subunit, G alpha i1. This PLC-beta 3 fragment also inhibited G beta gamma-stimulated PLC-beta activity in a reconstitution system, while having no significant effect on G alpha q-stimulated PLC-beta 3 activity. The N-terminal G beta gamma binding region was delineated further to the first 180 amino acids, and the sequence Asn150-Ser180, just distal to the PH domain, was found to be required for the interaction. Mutation of basic residues 154Arg, 155Lys, 159Lys, and 161Lys to Glu within this region reduced G beta gamma binding affinity and specifically reduced the EC50 for G beta gamma-dependent activation of the mutant enzyme 3-fold. Basal activity and G alpha q-dependent activation of the enzyme were unaffected by the mutations. While these basic residues may not directly mediate the interaction with G beta gamma, the data provide evidence for an N-terminal G beta gamma binding region of PLC-beta 3 that is involved in activation of the enzyme.
Barr
A J
AJ
Departments of Medicine and Immunology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Ali
H
H
Haribabu
B
B
Snyderman
R
R
Smrcka
A V
AV
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D019204:GTP-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D005982:Glutathione Transferase / Q000235:genetics; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D054799:Phospholipase C beta; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D007527:Isoenzymes; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D010738:Type C Phospholipases; C518033:PLCB1 protein, human; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins; C498015:GNAI2 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins