Guanine nuoleotide dissociation inhibitor activity of the triple GoLoco motif protein G18: Alanine-to-aspartate mutation restores function to an inactive second GoLoco motif

Randall J. Kimple, Francis S. Willard, Melinda D. Hains, Miller B. Jones, Gift K. Nweke, David P. Siderovski

Research output: Contribution to journalArticle

49 Scopus citations

Abstract

GoLoco ('Gαi/o-Loco' interaction) motif proteins have recently been identified as novel GDIs (guanine nucleotide dissociation inhibitors) for heterotrimeric G-protein α subunits. G18 is a member of the mammalian GoLoco-motif gene family and was uncovered by analyses of human and mouse genomes for anonymous open-reading frames. The encoded G18 polypeptide is predicted to contain three 19-amino-acid GoLoco motifs, which have been shown in other proteins to bind Ga subunits and inhibit spontaneous nucleotide release. However, the G18 protein has thus far not been characterized biochemically. Here, we have cloned and expressed the G18 protein and assessed its ability to act as a GDI. G18 is capable of simultaneously binding more than one Gαil subunit. In binding assays with the non-hydrolysable GTP analogue guanosine 5′-[γ-thio]triphosphate, G18 exhibits GDI activity, slowing the exchange of GDP for GTP by Gαil. Only the first and third GoLoco motifs within G18 are capable of interacting with Ga subunits, and these bind with low micromolar affinity only to Gαil in the GDP-bound form, and not to Gαo, Gαq, Gαs or Gαl2. Mutation of Ala-121 to aspartate in the inactive second GoLoco motif of G18, to restore the signature acidic-glutamine-arginine tripeptide that forms critical contacts with Ga and its bound nucleotide [Kimple, Kimple, Betts, Sondek and Siderovski (2002) Nature (London) 416, 878-881], results in gain-of-function with respect to Ga binding and GDI activity.

Original languageEnglish
Pages (from-to)801-808
Number of pages8
JournalBiochemical Journal
Volume378
Issue number3
DOIs
StatePublished - 15 Mar 2004

Keywords

  • Fluorescence spectroscopy
  • G-protein regulatory motif (GPR motif)
  • GoLoco motif
  • Guanine nucleotide dissociation inhibitor
  • Heterotrimeric G-protein
  • Surface plasmon resonance

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