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

doi:10.1074/jbc.m804936200

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.

Highlights

Heterotrimeric G-protein G␣ 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
An integral part of the cellular machinery underlying accurate spindle positioning is the involvement of heterotrimeric G-protein G␣ and G␤␥ subunits in a manner considered independent of 7TMR activation and instead involving RIC-8, GoLoco motif 3 proteins, and GTPase-accelerating proteins (“GAPs”; i.e. RGS proteins) (6 –13)
In our working model of C. elegans asymmetric cell division controlled by the G␣ subunits GOA-1 and GPA-16 [18, 19], it is the G␣1⁄7GDP/GPR-1/2 complex that activates the generation of astral microtubule (MT) force on mitotic spindle poles, whereas in a competing model [3, 12, 20], the G␣1⁄7GDP/GoLoco motif complex is required for the nucleotide exchange (“GEF”) activity for RIC-8, thereby generating G␣1⁄7GTP as the presumed active form of the G-protein [12, 21, 22]

Summary

EXPERIMENTAL PROCEDURES

Materials—All peptides were synthesized using Fmoc (N-(9fluorenyl)methoxycarbonyl) group protection, high pressure liquid chromatography-purified, and validated by mass spectrometry at the Tufts University Core Facility (Medford, MA). A pFLAG expression construct encoding the adenosine A2A receptor fused to venus-enhanced YFP is described in Ref. 33. BODIPYFL-GTP␥S binding assays were used to quantify GoLoco motif-promoted G␣i1 GDI activity, as described previously [38]. Electrophysiolgical Studies—The method of recording whole cell Ca2ϩ currents from rat SCG neurons with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA) was described in detail previously [27, 45]. GPSM3 Membrane Recruitment Experiments—Similar to the transmembrane domain-anchored G␣ subunits described in Ref. 48, a pcDNA3.1-based mammalian expression vector was generated to encode CFP-TM-G␣oi subunits consisting of (starting at the N terminus) a signal peptide, enhanced CFP [49], the N-terminal 103 amino acids of the rat ␮-opioid receptor, the N-terminal amino acids of human G␣oA, and amino acids –354 of human G␣i1. Amino acid interaction data were derived using SPACE(CMA) [51] and plotted using MATLAB (The MathWorks, Natick, MA)

RESULTS AND DISCUSSION

Angle change during spindle rocking

Point Mutation to EVIDENCE FOR