Calmodulin binds to specific sequences in the cytoplasmic domain of C-CAM and down-regulates C-CAM self-association.
C-CAM is a cell adhesion molecule belonging to the immunoglobulin supergene family and is known to mediate calcium-independent homophilic cell-cell binding. Two major isoforms, C-CAM1 and C-CAM2, which differ in their cytoplasmic domains, have been identified. Previous investigations have demonstrated that both cytoplasmic domains can bind calmodulin in a calcium-dependent reaction. In this investigation, peptides corresponding to the cytoplasmic domains of C-CAM were synthesized on cellulose membranes and used to map the binding sites for 125I-labeled calmodulin. Both C-CAM1 and C-CAM2 had one strong calmodulin-binding site in the membrane-proximal region. Those binding regions were conserved in C-CAM from rat, mouse, and man. In addition, C-CAM1 from rat and mouse contained a weaker binding site in the distal region of the cytoplasmic domain. Biosensor experiments were performed to determine rate and equilibrium constants of the C-CAM/calmodulin interaction. An association rate constants of 3.3 x 10(5) M-1 s-1 and two dissociation rate constants of 2.2 x 10(-2) and 3.1 x 10(-5) s-1 were determined. These correspond to equilibrium dissociation constants of 6.7 x 10(-8) and 9.4 x 10(-11) M, respectively. In dot-blot binding experiments, it was found that binding of calmodulin causes a down-regulation of the homophilic self-association of C-CAM. This suggests that calmodulin can regulate the functional activity of C-CAM.
Edlund
M
M
Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden.
Blikstad
I
I
Obrink
B
B
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002272:Carcinoembryonic Antigen; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D014644:Genetic Variation; D006023:Glycoproteins; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D013997:Time Factors
D015703:Antigens, CD; C069847:CD66 antigens; D002147:Calmodulin; D002272:Carcinoembryonic Antigen; C486183:Ceacam1 protein, mouse; C087701:Ceacam2 protein, mouse; D015815:Cell Adhesion Molecules; D006023:Glycoproteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D000251:Adenosine Triphosphatases