Identification of a region at the N-terminus of phospholipase C-beta 3 that interacts with G protein beta gamma subunits.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Barr, AJ; Ali, H; Haribabu, B; Snyderman, R; Smrcka, AV

Published Date

  • February 22, 2000

Published In

Volume / Issue

  • 39 / 7

Start / End Page

  • 1800 - 1806

PubMed ID

  • 10677230

Pubmed Central ID

  • 10677230

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi992021f

Language

  • eng

Conference Location

  • United States