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 supe rgene family and is known to mediate calcium-independent hemophilic ce ll-cell binding, Two major isoforms, C-CAM1 and C-CAM2, which differ i n their cytoplasmic domains, have been identified, Previous investigat ions have demonstrated that both cytoplasmic domains can bind calmodul in in a calcium-dependent reaction, In this investigation, peptides co rresponding to the cytoplasmic domains of C-CAM were synthesized on ce llulose membranes and used to map the binding sites for I-125-labeled calmodulin, Both C-CAM1 and C-CAMS had one strong calmodulin binding s ite in the membrane-proximal region. These binding regions were conser ved in C-CAM from rat, mouse, and man, In addition, C-CAM1 from rat an d mouse contained a weaker binding site in the distal region of the cy toplasmic domain, Biosensor experiments were performed to determine ra te and equilibrium constants of the C-CAM/calmodulin interaction, An a ssociation rate constant of 3.3 x 10(5) M(-1) s(-1) and two dissociati on rate constants of 2.2 x 10(-2) and 3.1 x 10(-5) s(-1) were deter mi ned, These correspond to equilibrium dissociation constants of 6.7 x 1 0(-8) and 9.4 x 10(-11) M, respectively, In dot-blot binding experimen ts, it was found that binding of calmodulin causes a down-regulation o f the hemophilic self-association of C-CAM. This suggests that calmodu lin can regulate the functional activity of C-CAM.