title: A point mutation to Galphai selectively blocks GoLoco motif binding: direct evidence for Galpha.GoLoco complexes in mitotic spindle dynamics.
abstract: Heterotrimeric G-protein Galpha subunits and GoLoco motif proteins are key members of a conserved set of regulatory proteins that influence invertebrate asymmetric cell division and vertebrate neuroepithelium and epithelial progenitor differentiation. GoLoco motif proteins bind selectively to the inhibitory subclass (Galphai) of Galpha subunits, and thus it is assumed that a Galphai.GoLoco motif protein complex plays a direct functional role in microtubule dynamics underlying spindle orientation and metaphase chromosomal segregation during cell division. To address this hypothesis directly, we rationally identified a point mutation to Galphai subunits that renders a selective loss-of-function for GoLoco motif binding, namely an asparagine-to-isoleucine substitution in the alphaD-alphaE loop of the Galpha helical domain. This GoLoco-insensitivity ("GLi") mutation prevented Galphai1 association with all human GoLoco motif proteins and abrogated interaction between the Caenorhabditis elegans Galpha subunit GOA-1 and the GPR-1 GoLoco motif. In contrast, the GLi mutation did not perturb any other biochemical or signaling properties of Galphai subunits, including nucleotide binding, intrinsic and RGS protein-accelerated GTP hydrolysis, and interactions with Gbetagamma dimers, adenylyl cyclase, and seven transmembrane-domain receptors. GoLoco insensitivity rendered Galphai subunits unable to recruit GoLoco motif proteins such as GPSM2/LGN and GPSM3 to the plasma membrane, and abrogated the exaggerated mitotic spindle rocking normally seen upon ectopic expression of wild type Galphai subunits in kidney epithelial cells. This GLi mutation should prove valuable in establishing the physiological roles of Galphai.GoLoco motif protein complexes in microtubule dynamics and spindle function during cell division as well as to delineate potential roles for GoLoco motifs in receptor-mediated signal transduction.
authors.affiliation: Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA. willardfs@lilly.com
authors.forename: Francis S
authors.initials: FS
authors.lastname: Willard
authors.forename: Zhen
authors.initials: Z
authors.lastname: Zheng
authors.forename: Juan
authors.initials: J
authors.lastname: Guo
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Digby
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Kimple
authors.forename: Jason M
authors.initials: JM
authors.lastname: Conley
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Johnston
authors.forename: Dustin
authors.initials: D
authors.lastname: Bosch
authors.forename: Melinda D
authors.initials: MD
authors.lastname: Willard
authors.forename: Val J
authors.initials: VJ
authors.lastname: Watts
authors.forename: Nevin A
authors.initials: NA
authors.lastname: Lambert
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Ikeda
authors.forename: Quansheng
authors.initials: Q
authors.lastname: Du
authors.forename: David P
authors.initials: DP
authors.lastname: Siderovski
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans; D002462:Cell Membrane / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism*; D006801:Humans; D008870:Microtubules / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D051381:Rats; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D008941:Spindle Apparatus
chemical_list: D044385:GTP-Binding Protein alpha Subunits