A point mutation to Gαi selectively blocks GoLoco motif binding: Direct evidence for Gα·GoLoco complexes in mitotic spindle dynamics

Francis S. Willard, Zhen Zheng, Juan Guo, Gregory J. Digby, Adam J. Kimple, Jason M. Conley, Christopher A. Johnston, Dustin Bosch, Melinda D. Willard, Val J. Watts, Nevin A Lambert, Stephen R. Ikeda, Quansheng Du, David P. Siderovski

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Heterotrimeric G-protein Gα 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 (Gαi) of Gα subunits, and thus it is assumed that a Gαi·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 Gαi subunits that renders a selective loss-of-function for GoLoco motif binding, namely an asparagine-to-isoleucine substitution in the αD-αE loop of the Gα helical domain. This GoLoco-insensitivity ("GLi") mutation prevented Gαi1 association with all human GoLoco motif proteins and abrogated interaction between the Caenorhabditis elegans Gα subunit GOA-1 and the GPR-1 GoLoco motif. In contrast, the GLi mutation did not perturb any other biochemical or signaling properties of Gαi subunits, including nucleotide binding, intrinsic and RGS protein-accelerated GTP hydrolysis, and interactions with Gβγ dimers, adenylyl cyclase, and seven transmembrane-domain receptors. GoLoco insensitivity rendered Gαi 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 Gαi subunits in kidney epithelial cells. This GLi mutation should prove valuable in establishing the physiological roles of Gαi·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.

Original languageEnglish (US)
Pages (from-to)36698-36710
Number of pages13
JournalJournal of Biological Chemistry
Volume283
Issue number52
DOIs
StatePublished - Dec 26 2008

Fingerprint

Amino Acid Motifs
Spindle Apparatus
Point Mutation
Proteins
Microtubules
Cell Division
Cells
Mutation
RGS Proteins
Asymmetric Cell Division
Protein Interaction Domains and Motifs
Heterotrimeric GTP-Binding Proteins
Isoleucine
Asparagine
Caenorhabditis elegans
Protein Subunits
Ground penetrating radar systems
Invertebrates
Metaphase
Signal transduction

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Willard, F. S., Zheng, Z., Guo, J., Digby, G. J., Kimple, A. J., Conley, J. M., ... Siderovski, D. P. (2008). A point mutation to Gαi selectively blocks GoLoco motif binding: Direct evidence for Gα·GoLoco complexes in mitotic spindle dynamics. Journal of Biological Chemistry, 283(52), 36698-36710. https://doi.org/10.1074/jbc.M804936200

A point mutation to Gαi selectively blocks GoLoco motif binding : Direct evidence for Gα·GoLoco complexes in mitotic spindle dynamics. / Willard, Francis S.; Zheng, Zhen; Guo, Juan; Digby, Gregory J.; Kimple, Adam J.; Conley, Jason M.; Johnston, Christopher A.; Bosch, Dustin; Willard, Melinda D.; Watts, Val J.; Lambert, Nevin A; Ikeda, Stephen R.; Du, Quansheng; Siderovski, David P.

In: Journal of Biological Chemistry, Vol. 283, No. 52, 26.12.2008, p. 36698-36710.

Research output: Contribution to journalArticle

Willard, FS, Zheng, Z, Guo, J, Digby, GJ, Kimple, AJ, Conley, JM, Johnston, CA, Bosch, D, Willard, MD, Watts, VJ, Lambert, NA, Ikeda, SR, Du, Q & Siderovski, DP 2008, 'A point mutation to Gαi selectively blocks GoLoco motif binding: Direct evidence for Gα·GoLoco complexes in mitotic spindle dynamics', Journal of Biological Chemistry, vol. 283, no. 52, pp. 36698-36710. https://doi.org/10.1074/jbc.M804936200
Willard, Francis S. ; Zheng, Zhen ; Guo, Juan ; Digby, Gregory J. ; Kimple, Adam J. ; Conley, Jason M. ; Johnston, Christopher A. ; Bosch, Dustin ; Willard, Melinda D. ; Watts, Val J. ; Lambert, Nevin A ; Ikeda, Stephen R. ; Du, Quansheng ; Siderovski, David P. / A point mutation to Gαi selectively blocks GoLoco motif binding : Direct evidence for Gα·GoLoco complexes in mitotic spindle dynamics. In: Journal of Biological Chemistry. 2008 ; Vol. 283, No. 52. pp. 36698-36710.
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