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

Barr, Alastair J. and Ali, Hydar and Haribabu, Bodduluri and Snyderman, Ralph and Smrcka, Alan V. (2000) Identification of a region at the N-terminus of phospholipase C-beta 3 that interacts with G protein beta gamma subunits. Biochemistry, 39 (7). pp. 1800-1806. ISSN 0006-2960

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Official URL: http://dx.doi.org/10.1021/bi992021f

Abstract

Members of the phospholipase C-? (PLC-?) family of proteins are activated either by G? or G?? subunits of heterotrimeric G proteins. To define specific regions of PLC-?3 that are involved in binding and activation by G??, a series of fragments of PLC-?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 ?1?2 in an in vitro binding assay, and binding was inhibited by G protein ? subunit, G?i1. This PLC-?3 fragment also inhibited G??-stimulated PLC-? activity in a reconstitution system, while having no significant effect on G?q-stimulated PLC-?3 activity. The N-terminal G?? 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?? binding affinity and specifically reduced the EC50 for G??-dependent activation of the mutant enzyme 3-fold. Basal activity and G?q-dependent activation of the enzyme were unaffected by the mutations. While these basic residues may not directly mediate the interaction with G??, the data provide evidence for an N-terminal G?? binding region of PLC-?3 that is involved in activation of the enzyme.

Item Type: Article
Subjects: University of Westminster > Science and Technology > Life Sciences, School of (No longer in use)
Depositing User: Rachel Wheelhouse
Date Deposited: 20 Jul 2012 13:37
Last Modified: 20 Jul 2012 13:37
URI: http://westminsterresearch.wmin.ac.uk/id/eprint/10867

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