Received for publication June 15, 2004.
Revised December 4, 2004.
Accepted for publication December 6, 2004.

Functional characterization of Go signaling through GRIN1

Abstract

GRIN1 (G protein-regulated inducer of neurite outgrowth 1) was initially identified as a binding protein for GTPgS-bound Gz. GRIN1 is specifically expressed in brain and interacts selectively with activated subunits of Gi subfamily. GRIN1 colocalizes with Go at the growth cone of neuronal cells and promotes neurite extention in Neuro2a cells when co-expressed with constitutively active mutant GoQ205L. These results suggest that GRIN1 functions as a downstream target for Go. However, GRIN1 does not contain domains that are homologous to known signaling motifs. To understand the mechanisms of Go-GRIN1 pathway, we analyzed functional domains of GRIN1 that are involved in binding with Go or its targeting to the plasma membrane. Using pull down assays with GST-fused GRIN1 deletion mutants, Go binding regions were localized to amino acid residues 716-746 and 797-827 of GRIN1. The Go binding region of GRIN1 did not demonstrate GTPase accelerating activity for Go. 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. Co-expression of GRIN1 with GoQ205L or GRIN1(717-827), which lacks Go binding region, promoted microspike formation in Swiss3T3 cells or neurite extention in Neuro2a cells. The dominant-negative mutant of Cdc42 blocked these morphological changes. Co-expression of GRIN1 and GoQ205L stimulated formation of GTP-bound Cdc42 in Swiss3T3 cells. These results suggest that the binding of activated Go to GRIN1 induces activation of Cdc42, which leads to morphological changes in neuronal cells.

Key words: Gi family, G protein regulation, Cdc42, rho, rac, other small G proteins

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