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There is a genetic contribution to fetal alcohol spectrum disorders (FASD), but the identification of candidate genes has been elusive. Ethanol may cause FASD in part by decreasing the adhesion of the developmentally critical L1 cell adhesion molecule through interactions with an alcohol binding pocket on the extracellular domain. Pharmacologic inhibition or genetic knockdown of ERK2 did not alter L1 adhesion, but markedly decreased ethanol inhibition of L1 adhesion in NIH/3T3 cells and NG108-15 cells. Likewise. leucine replacement of S1248, an ERK2 substrate on the L1 cytoplasmic domain. did not decrease L1 adhesion, but abolished ethanol inhibition of L1 adhesion. Stable transfection of NIH/3T3 cells with human L1 resulted in clonal cell lines in which L1 adhesion was consistently sensitive or insensitive to ethanol for more than a decade. ERK2 activity and S1248 phosphorylation were greater in ethanol-sensitive NIH/3T3 clonal cell lines than in their ethanol-insensitive counterparts. Ethanol-insensitive cells became ethanol sensitive after increasing ERK2 activity by transfection with a constitutively active MAP kinase kinase 1. Finally, embryos from two substrains of C57BL mice that differ in susceptibility to ethanol teratogenesis showed corresponding differences in MAPK activity. Our data suggest that ERK2 phosphorylation of S1248 modulates ethanol inhibition of L1 adhesion by inside-out signaling and that differential regulation of ERK2 signaling might contribute to genetic susceptibility to FASD. Moreover, identification of a specific locus that regulates ethanol sensitivity, but not L1 function, might facilitate the rational design of drugs that block ethanol neurotoxicity. [ABSTRACT FROM AUTHOR]

Prenatal ethanol exposure causes fetal alcohol spectrum disorders (FASD) in part by disrupting the neural cell adhesion molecule L1. L1 gene mutations cause neuropathological abnormalities similar to those of FASD. Ethanol and 1-butanol inhibit L1-mediated cell-cell adhesion (L1 adhesion), whereas 1-octanol antagonizes this action. To test the hypothesis that there are alcohol binding sites on L1, we used 3-azibutanol and 3-azioctanol, the photoactivatable analogs of 1-butanol and 1-octanol, to photolabel the purified Ig1-4 domain of human L1 (hL1 Ig1-4). 3-Azibutanol (11 mM), like ethanol, inhibited L1 adhesion in NIH/3T3 cells stably transfected with hL1, whereas subanesthetic concentrations of 3-azioctanol (14 μM) antagonized ethanol inhibition of L1 adhesion. 3-Azibutanol (100-1,000 μM) and 3-azioctanol (10-100 μM) photoincorporated into Tyr-418 on Ig4 and into two adjacent regions in the N terminus, Glu-33 and Glu-24 to Glu-27. A homology model of hL1 Ig1-4 (residues 33-422), based on the structure of the Ig1-4 domains of axonin-1, suggests that Glu-33 and Tyr-418 hydrogen-bond at the interface of Ig1 and Ig4 to stabilize a horseshoe conformation of L1 that favors homophilic binding. Furthermore, this alcohol binding pocket lies within 7 A of Leu-120 and Gly-121, residues in which missense mutations cause neurological disorders similar to FASD. These data suggest that ethanol or selected mutations produce neuropathological abnormalities by disrupting the domain interface between Ig1 and Ig4. Characterization of alcohol agonist and antagonist binding sites on L1 will aid in understanding the molecular basis for FASD and might accelerate the development of ethanol antagonists. [ABSTRACT FROM AUTHOR]