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

GoLoco ('Gα_i/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 Gα_il 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 Gα_il. 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 Gα_il in the GDP-bound form, and not to Gα_o, Gα_q, Gα_s or Gα_l2. 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.