Functional characterization of Galphao signaling through G protein-regulated inducer of neurite outgrowth 1.

G protein-regulated inducer of neurite outgrowth 1 (GRIN1) was initially identified as a binding protein for guanosine 5'-3-O-(thio)triphosphate-bound Galphaz. GRIN1 is specifically expressed in brain and interacts selectively with activated alpha subunits of the Gi subfamily. GRIN1 colocalizes with Galphao at the growth cone of neuronal cells and promotes neurite extension in Neuro2a cells when coexpressed with constitutively active mutant GalphaoQ205L. These results suggest that GRIN1 functions as a downstream target for Galphao. However, GRIN1 does not contain domains that are homologous to known signaling motifs. To understand the mechanisms of Galphao-GRIN1 pathway, we analyzed functional domains of GRIN1 that are involved in binding with Galphao or with its targeting to the plasma membrane. Using pull-down assays with glutathione S-transferase-fused GRIN1 deletion mutants, Galphao binding regions were localized to amino acid residues 716 to 746 and 797 to 827 of GRIN1. The Galphao binding region of GRIN1 did not demonstrate GTPase accelerating activity for Galphao. GRIN1 localized in the cell periphery in Neuro2a cells, and two cysteine residues at C-terminal region of GRIN1 (Cys818 and Cys819) were shown to be critical for its membrane targeting. Coexpression of GRIN1 with GalphaoQ205L or GRIN1Delta(717-827), which lacks Galphao binding region, promoted microspike formation in Swiss 3T3 cells or neurite extension in Neuro2a cells. The dominant-negative mutant of Cdc42 blocked these morphological changes. Coexpression of GRIN1 and GalphaoQ205L stimulated the formation of GTP-bound Cdc42 in Swiss 3T3 cells. These results suggest that the binding of activated Galphao to GRIN1 induces activation of Cdc42, which leads to morphological changes in neuronal cells.