title: Guanine nucleotide dissociation inhibitor activity of the triple GoLoco motif protein G18: alanine-to-aspartate mutation restores function to an inactive second GoLoco motif.
abstract: GoLoco ('Galpha(i/o)-Loco' interaction) motif proteins have recently been identified as novel GDIs (guanine nucleotide dissociation inhibitors) for heterotrimeric G-protein alpha 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 Galpha 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 Galpha(i1) subunit. In binding assays with the non-hydrolysable GTP analogue guanosine 5'-[gamma-thio]triphosphate, G18 exhibits GDI activity, slowing the exchange of GDP for GTP by Galpha(i1). Only the first and third GoLoco motifs within G18 are capable of interacting with Galpha subunits, and these bind with low micromolar affinity only to Galpha(i1) in the GDP-bound form, and not to Galpha(o), Galpha(q), Galpha(s) or Galpha12. 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 Galpha and its bound nucleotide [Kimple, Kimple, Betts, Sondek and Siderovski (2002) Nature (London) 416, 878-881], results in gain-of-function with respect to Galpha binding and GDI activity.
authors.affiliation: Department of Pharmacology, Lineberger Comprehensive Cancer Center, and UNC Neuroscience Center, The University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Randall J
authors.initials: RJ
authors.lastname: Kimple
authors.forename: Francis S
authors.initials: FS
authors.lastname: Willard
authors.forename: Melinda D
authors.initials: MD
authors.lastname: Hains
authors.forename: Miller B
authors.initials: MB
authors.lastname: Jones
authors.forename: Gift K
authors.initials: GK
authors.lastname: Nweke
authors.forename: David P
authors.initials: DP
authors.lastname: Siderovski
mesh_terms: D000409:Alanine / Q000235:genetics; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation
chemical_list: C487218:GPSM3 protein, human; D044385:GTP-Binding Protein alpha Subunits; D020730:Guanine Nucleotide Dissociation Inhibitors; D006153:Guanosine Diphosphate; D001224:Aspartic Acid; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000409:Alanine