Intramolecular interaction in LGN, an adaptor protein that regulates mitotic spindle orientation

Proper mitotic spindle orientation requires that astral microtubules are connected to the cell cortex by the microtubule-binding protein NuMA, which is recruited from the cytoplasm. Cortical recruitment of NuMA is at least partially mediated via direct binding to the adaptor protein LGN. LGN normally adopts a closed conformation via an intramolecular interaction between its N-terminal NuMA-binding domain and its C-terminal region that contains four GoLoco (GL) motifs, each capable of binding to the membrane-anchored G?(i) subunit of heterotrimeric G protein. Here we show that the intramolecular association with the N-terminal domain in LGN involves GL3, GL4, and a region between GL2 and GL3, whereas GL1 and GL2 do not play a major role. This conformation renders GL1 but not the other GL motifs in a state easily accessible to G?(i). To interact with full-length LGN in a closed state, NuMA requires the presence of G?(i); both NuMA and G?(i) are essential for cortical recruitment of LGN in mitotic cells. In contrast, mInsc, a protein that competes with NuMA for binding to LGN and regulates mitotic spindle orientation in asymmetric cell division, efficiently binds to full-length LGN without G?(i) and induces its conformational change, enhancing its association with G?(i). In nonpolarized symmetrically dividing HeLa cells, disruption of the LGN?NuMA interaction by ectopic expression of mInsc results in a loss of cortical localization of NuMA during metaphase and anaphase and promotes mitotic spindle misorientation and a delayed anaphase progression. These findings highlight a specific role for LGN-mediated cell cortex recruitment of NuMA.