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ORIGINAL ARTICLE

Functional Characterization of Go Signaling through G Protein-Regulated Inducer of Neurite Outgrowth 1

Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois

Abstract

G protein-regulated inducer of neurite outgrowth 1 (GRIN1) was initially identified as a binding protein for guanosine 5'-3-O-(thio)triphosphate–bound Gz. GRIN1 is specifically expressed in brain and interacts selectively with activated subunits of the Gi subfamily. GRIN1 colocalizes with Go at the growth cone of neuronal cells and promotes neurite extension in Neuro2a cells when coexpressed 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 with its targeting to the plasma membrane. Using pull-down assays with glutathione S-transferase–fused GRIN1 deletion mutants, Go binding regions were localized to amino acid residues 716 to 746 and 797 to 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. Coexpression of GRIN1 with GoQ205L or GRIN1(717–827), which lacks Go 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 GoQ205L stimulated the formation of GTP-bound Cdc42 in Swiss 3T3 cells. These results suggest that the binding of activated Go to GRIN1 induces activation of Cdc42, which leads to morphological changes in neuronal cells.

Address correspondence to: Dr. Tohru Kozasa, Department of Pharmacology (M/C 868), University of Illinois at Chicago, 835 South Wolcott Avenue, Chicago, IL 60612. E-mail: tkozas{at}uic.edu

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