Gα selectivity and inhibitor function of the multiple GoLoco motif protein GPSM2/LGN

GPSM2 (G-protein signalling modulator 2; also known as LGN or mammalian Pins) is a protein that regulates mitotic spindle organization and cell division. GPSM2 contains seven tetratricopeptide repeats (TPR) and four Gα_i/o-Loco (GoLoco) motifs. GPSM2 has guanine nucleotide dissociation inhibitor (GDI) activity towards both Gα_o- and Gα_i-subunits; however, a systematic analysis of its individual GoLoco motifs has not been described. We analyzed each of the four individual GoLoco motifs from GPSM2, assessing their relative binding affinities and GDI potencies for Gα_i1, Gα_i2, and Gα_i3 and Gα_o. Each of the four GPSM2 GoLoco motifs (36-43 amino acids in length) was expressed in bacteria as a GST-fusion protein and purified to homogeneity. The binding of each of the four GST-GoLoco motifs to Gα_i1-, Gα_o-, and Gα_s-subunits was assessed by surface plasmon resonance; all of the motifs bound Gα_i1, but exhibited low affinity towards Gα_o. GDI activity was assessed by a fluorescence-based nucleotide-binding assay, revealing that all four GoLoco motifs are functional as GDIs for Gα_i1, Gα_i2, and Gα_i3. Consistent with our binding studies, the GDI activity of GPSM2 GoLoco motifs on Gα_o was significantly lower than that toward Gα_i1, suggesting that the in vivo targets of GPSM2 are most likely to be Gα_i-subunits.