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 Gαi/o-Loco (GoLoco) motifs. GPSM2 has guanine nucleotide dissociation inhibitor (GDI) activity towards both Gαo- and Gαi-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 Gαi1, Gαi2, and Gαi3 and Gαo. 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 Gαi1-, Gαo-, and Gαs-subunits was assessed by surface plasmon resonance; all of the motifs bound Gαi1, but exhibited low affinity towards Gαo. GDI activity was assessed by a fluorescence-based nucleotide-binding assay, revealing that all four GoLoco motifs are functional as GDIs for Gαi1, Gαi2, and Gαi3. Consistent with our binding studies, the GDI activity of GPSM2 GoLoco motifs on Gαo was significantly lower than that toward Gαi1, suggesting that the in vivo targets of GPSM2 are most likely to be Gαi-subunits.
|Number of pages||11|
|Journal||Biochimica et Biophysica Acta - Molecular Cell Research|
|State||Published - 10 Sep 2005|