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

• Published in: Biochimica et biophysica acta 2005-09, Vol.1745 (2), p.254-264
• Main Authors: McCudden, Christopher R., Willard, Francis S., Kimple, Randall J., Johnston, Christopher A., Hains, Melinda D., Jones, Miller B., Siderovski, David P.
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Animals; Carrier Proteins - genetics; Carrier Proteins - physiology; Cattle; G-protein; GoLoco; GPSM2; GTP-Binding Protein alpha Subunits, Gi-Go - antagonists & inhibitors; GTP-Binding Protein alpha Subunits, Gi-Go - physiology; Humans; Intracellular Signaling Peptides and Proteins; LGN; Molecular Sequence Data; Protein Structure, Tertiary; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism
• ISSN: 0167-4889; 0006-3002; 1879-2596
• DOI: 10.1016/j.bbamcr.2005.05.002

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.