Mutational analysis of the CD6 binding site in activated leukocyte cell adhesion molecule.
The interaction between CD6 and its ligand activated leukocyte cell adhesion molecule (ALCAM) mediates adhesion of thymocytes to thymic epithelial cells. The extracellular region of ALCAM includes five Ig-like domains, and its N-terminal V-like domain specifically binds to the membraneproximal scavenger receptor cysteine-rich domain of CD6. Previously, six ALCAM residues were identified by alanine scanning mutagenesis to contribute to the interaction with CD6. All of these residues mapped to the predicted A'GFCC'C" face of ALCAM's N-terminal domain. Here we describe the results of experiments designed to further study the CD6 binding site. Other mutagenesis experiments at four previously studied sites were carried out to better understand their importance for the interaction with CD6, and different receptor binding assays were employed to compare the contribution of these and other ALCAM residues to the CD6-ligand interaction. A total of ten new ALCAM mutants were prepared, and three additional residues were identified as critical for CD6 binding. These studies have enabled us to classify ALCAM residues according to their importance for binding and to describe the CD6 binding site in some detail.
Skonier
J E
JE
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, USA.
Bowen
M A
MA
Emswiler
J
J
Aruffo
A
A
Bajorath
J
J
D020410:Activated-Leukocyte Cell Adhesion Molecule; D015703:Antigens, CD / Q000378:metabolism*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D016296:Mutagenesis; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D020410:Activated-Leukocyte Cell Adhesion Molecule; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; C062279:CD6 antigen; D015815:Cell Adhesion Molecules; D006023:Glycoproteins; D008024:Ligands; D011993:Recombinant Fusion Proteins