Abstract
C-CAM, a member of the Immunoglobulin Superfamily, was originally identified as an intercellular adhesion molecule, and can mediate cell-cell adhesion by homophilic binding. However, it also appears in high concentrations in luminal, microvillar surfaces. Although its functions in these areas are still unclear, it is likely that C-CAM's intra- and extracellular affinities are modulated via interactions with cytoplasmic factors, and that these factors are themselves part of calcium and kinase-mediated regulatory pathways. Such inside-out affinity regulation has been seen in other adhesion families, where it often involves modulation of cytoskeletal interactions and molecular clustering.
We have identified a new C-CAM isoform in rat liver (C-CAM2). This isoform differs from C-CAMl in the most N-terminal, Ig-like domain, and in the C-terminal, cytoplasmic domain. The lack of 53 nucleotides in C-CAM2 results in a frame shift, anew stop codon, and a shortened cytoplasmic tail. We conclude from northern blot analyses that the extracellular differences come from variance between out-bred rat stocks. Each rat strain has both C-CAM 1 and C-CAM2, so there are at least four rat C-CAM isoforms (C-CAMla, C-CAMlb, C-CAM2a, C-CAM2b).
The calcium-activated protein, calmodulin, was found to interact with the cytoplasmic domains of both C-CAMl and C-CAM2 and is thus a good candidate regulator of C-CAM affinity. To locate the calmodulin-binding site, membrane-bound synthetic peptides, corresponding to C-CAM's cytoplasmic domains in rat, mouse, and human were constructed. Despite large sequence differences between peptides, the membrane-proximal region bound calmodulin in all species. The rate and equilibrium constants of these interactions were determined using biosensor technology.
C-CAM2-transfected Chinese hamster ovary (CHO) cells were used in aggregation assays, before and after treatment with the kinase activator PMA. The calcium ionophore A23187 was also added, alone or in combination with PMA. C-CAM-mediated cell aggregation was unaffected by PMA-stimulated phosphorylation, while aggregation was reduced by calcium influx. However, the simultaneous addition of PMA and A23187 prevented the calcium-mediated reduction in cell aggregation. In in vitro studies, calmodulin-bound C-CAM showed reduced self-association, and phosphorylated C-CAM showed reduced calmodulin binding. Phosphorylation could, therefore, function in a multi-protein regulatory mechanism to render C-CAM insensitive to calmodulin down-regulation.
