A study of the cytoplasmic domains of C-CAM isoforms : calmodulin binding and phosphorylation

Abstract: A STUDY OF THE CYTOPLASMIC DOMAINS OF C-CAM ISOFORMS: CALMODULIN BINDING AND PHOSPHORYLATION Magnus Edlund Laboratory of Medical Cell Biology, Department of Cell and Molecular Biology Karolinska Institutet, Stockholm, Sweden C-CAM, a member of the Immunoglobulin Superfamily, was originallyidentified as an intercellular adhesion molecule, and can mediate cell-cell adhesion byhomophilic binding. However, it also appears in high concentrations in luminal,microvillar surfaces. Although its functions in these areas are still unclear, it is likelythat C-CAM's intra- and extracellular affinities are modulated via interactions withcytoplasmic factors, and that these factors are themselves part of calcium and kinase-mediated regulatory pathways. Such inside-out affinity regulation has been seen inother adhesion families, where it often involves modulation of cytoskeletal interactionsand molecular clustering. We have identified a new C-CAM isoform in rat liver (C-CAM2). This isoformdiffers from C-CAMl in the most N-terminal, Ig-like domain, and in the C-terminal,cytoplasmic domain. The lack of 53 nuceotides in C-CAM2 results in a frame shift, anew stop codon, and a shortened cytoplasmic tail. We conclude from northern blotanalyses that the extracellular differences come from variance between out-bred ratstocks. 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 thecytoplasmic domains of both C-CAMl and C-CAM2 and is thus a good candidateregulator of C-CAM affinity. To locate the calmodulin-binding site, membrane-boundsynthetic peptides, corresponding to C-CAM's cytoplasmic domains in rat, mouse, andhuman were constructed. Despite large sequence differences between peptides, themembrane-proximal region bound calmodulin in all species. The rate and equilibriumconstants of these interactions were determined using biosensor technology. C-CAM2-transfected Chinese hamster ovary (CHO) cells were used inaggregation assays, before and after treatment with the kinase activator PMA. Thecalcium 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 additionof PMA and A23187 prevented the calcium-mediated reduction in cell aggregation. Inin vitro studies, calmodulin-bound C-CAM showed reduced self-association, andphosphorylated C-CAM showed reduced calmodulin binding. Phosphorylation could,therefore, function in a multi-protein regulatory mechanism to render C-CAMinsensitive to calmodulin down-regulation.Key words: adhesion molecules, adhesion regulation, BGP, Bgp, biliary glycoprotein,Calcium, Calmodulin, CaM, CAMs, carcinoembryonic antigen gene family, C-CAM,CEA, cell adhesion, Immunoglobulin super-family, PKC, protein kinase C.ISBN 96-628-1950-X Stockholm 1996