Sylvia G. Kachalsky

The ligand binding domain of the nicotinic acetylcholine receptor: Immunological analysis

The interaction of the acetylcholine receptor (AChR) binding site domain with specific antibodies and with α‐bungarotoxin (α‐BTX) has been compared. The cloned and expressed ligand binding domain of the mouse AChR α‐subunit binds α‐BTX, whereas the mongoose‐expressed domain is not recognized by α‐BTX. On the other hand, both the mouse and mongoose domains bind to the site‐specific monoclonal antibody 5.5. These results demonstrate that the structural requirements for binding of α‐BTX and mcAb 5.5, both of which interact with the AChR binding site, are distinct from each other.

Pervanadate-induced adhesion of CD4+ T cell to fibronectin is associated with tyrosine phosphorylation of paxillin.

The initial stages of T cell activation involve tyrosine protein kinase‐mediated intracellular signaling events. Integrin‐mediated adhesion pf CD4+ T lymphocytes to extracellular matrix glycoproteins, such as fibronectin, is an activation‐dependent process. The involvement of tyrosine protein kinases in the adhesion of CD4+ T cells to fibronectin was examined using pervanadate, a protein‐tyrosine phosphatase inhibitor. Pervanadate induced the adhesion of human CD4+ T cells to immobilized fibronectin in a β1integrin‐mediated fashion, and adhesion was associated with an increase of protein tyrosine phosphorylation. Tyrosine protein kinase inhibitors abrogated both T cell adhesion and intracellular protein tyrosine phosphorylation. Participation of cytoskeletal proteins in the pervanadate‐induced T cell adhesion was indicated because cytoskeleton disruption by cytochalasin B inhibited cell adhesion to fibronectin. We demonstrate that the cytoskeletal protein paxillin underwent time‐dependent tyrosine phosphorylation simultaneously with pervanadate‐induced T cell adhesion to fibronectin. Tyrosine phosphorylation of paxillin was related to cell adhesion, since pretreatment of T cells with cytochalasin B abrogated both adhesion and phosphorylation. This study demonstrates a correlation between activation of protein tyrosine kinases, tyrosine phosphorylation of paxillin, and integrin‐mediated T cell adhesion to extracellular matrix glycoproteins. J. Leukoc. Biol. 62: 405–413; 1997.

Molecular Evolution of the Binding Site of the Nicotinic Acetylcholine Receptor

The nicotinic acetylcholine receptor (AChR) is an oliganeric protein of four homologous subunits present in a stoichianetry of α 2 β S δ The α-subunit has been demonstrated to contain the cholinergic binding site. We have previously shown that a synthetic dodecapeptide from the cc-subunit which includes the two tandem cysteines at positions 192 and 193, contains the essential elements of the ligand binding site. In an attempt to elucidate the molecular basis for the ligand binding site of AChR we chose to study non-classical muscle AChRs from animals that are resistant to oc-neurotoxins and whose receptors do not bind oc-bungarotoxin (α-BTX). cDNA fragments of the AChR α-subunit from three species of snakes and from a mongoose which encode a domain corresponding to amino acid residues 122–205, were cloned, sequenced and expressed in E.coli. Most substitutions in the snake and mongoose fragments, which are overall highly homologous to other muscle AChR cc-subunits, are present in the presumed ligand-binding site in the vicinity of cysteines 192 and 193. Interestingly, both snakes and mongoose have a putative glycosylation site in the binding site domain. Sequence comparison suggests that substitutions at positions 187,189 and 194, which occur in both snake and mongoose AChR, are important in determining their resistance to α-BTX. On the other hand, amino acid residues which were reported to participate in acetylcholine binding are conserved in these animals as well.