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 Gα 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 Gαi1 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 Gαi1. Only the first and third GoLoco motifs within G18 are capable of interacting with Gα subunits, and these bind with low micromolar affinity only to Gαi1 in the GDP-bound form, and not to Gαo, Gαq, Gαs or Gα12. 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 Gα and its bound nucleotide [Kimple, Kimple, Betts, Sondek and Siderovski (2002) Nature (London) 416, 878–881], results in gain-of-function with respect to Gα binding and GDI activity.
Abbreviations used: BODIPY®, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; EST, expressed sequence tag; Gαi1-ΔN, N-terminal 30-amino-acid truncation mutant of Gαi1; G18-GL1 (etc.), protein containing only the first GoLoco motif of G18 (etc.); G18-GL123, protein containing all three GoLoco motifs of G18; GDI, guanine nucleotide dissociation inhibitor; GoLoco, Gαi/o–Loco interaction; GPCR, G-protein-coupled receptor; GPR, G-protein regulatory; GST, glutathione S-transferase; GTP[S], guanosine 5´-[γ-thio]triphosphate; ORF, open-reading frame; RFU, relative fluorescence units; RGS, regulator of G-protein signalling; SPR, surface plasmon resonance; TEV, tobacco etch virus.