Our overall hypothesis is that ethanol-induced inhibition of cell adhesion, one possible cause of damage in the developing neural system, is mediated by alcohol binding sites on the L1 cell adhesion molecule. Different alcohols show remarkable structural specificity for alcohol inhibition of cell adhesion (agonist action). Other alcohols (e.g., 1-octanol) noncompetitively antagonize the effects of ethanol on Ll-mediated cell adhesion and on the development of mouse whole embryo cultures. We hypothesize that these sites are on L1 because the brains of children with L1 mutations resemble those of children with fetal alcohol spectrum disorder (FASD). The novel aspect of this proposal is the use of recently developed photoaffinity alcohol analogs, 3-azibutanol (an agonist) and 3-azioctanol (an antagonist) to photolabel the agonist and antagonist binding sites respectively.
Specific Aim 1 tests the hypothesis that there are alcohol agonist sites on the L1 adhesion molecule by photolabeling them with 3-azibutanol, which acts similarly to ethanol on cell adhesion. Purified L1 photolabeled with [3H]3-azibutanol will be digested and fragments photoincorporating alcohol separated by HPLC. The stoichiometry of photoincorporation will be measured by mass spectrometry. Further, digestion of target fragments will produce fragments suitable for sequencing by mass spectrometry. To assess pharmacological relevance, the apparent dissociation constant and pharmacology of each photoincorporation site will be determined and compared to those obtained from parallel cell adhesion experiments.
Specific Aim 2 similarly tests the hypothesis that there are separate alcohol antagonist sites on L1 using the photoaffinity label, 3-azioctanol, which inhibits ethanol-induced inhibition of cell adhesion at micromolar concentrations. We expect to define at least one amino acid in the binding pocket of each pharmacologically well-characterized site on LI. Identification of the agonist and of the antagonist site will, respectively, aid in understanding the molecular basis for FASD and accelerate the development of drugs that block the toxic effects of ethanol on the developing nervous system.
| Arevalo, Enrique; Shanmugasundararaj, Sivananthaperumal; Wilkemeyer, Michael F et al. (2008) An alcohol binding site on the neural cell adhesion molecule L1. Proc Natl Acad Sci U S A 105:371-5 |
