ASSOCIATION OF AN 80 KDA PROTEIN WITH C-CAM1 CYTOPLASMIC DOMAIN CORRELATES WITH C-CAM1-MEDIATED GROWTH-INHIBITION

Decreased expression of C-CAM, a member of the CEA family of immunoglo bulin like cell adhesion molecules, occurs in carcinomas of the colon, liver and prostate. Down regulation of C-CAM during the early stages of carcinogenesis in rat liver and human prostate has also been report ed, We have recently shown that restoration of the expression of the i soform with long cytoplasmic domain, C-CAM1, leads to suppression of t he tumorigenicity of prostatic carcinoma cells in vivo and growth supp ression in vitro. These observations suggest that C-CAM1 may play an i mportant role in regulating cell growth in normal tissues, Previous st udies have demonstrated that the function of many members of the Ig-su pergene family is dependent on interactions with cytoplasmic proteins, Tn the present study, we have used a bifunctional cross-linker to ide ntify cellular proteins that interact directly with C-CAM1, Immunoblot analysis of WGA bound membrane proteins crosslinked with DSS identifi ed a 180 kDa complex composed of C-CAM1 and an 80 kDa protein designat ed CAP-80 (C-CAM Associated Protein), Immunoprecipitation with anti-C- CAM antibodies showed that CAP-80 was co-precipitated with C-CAM from detergent solubilized, WGA-purified proteins, To assess the specificit y of CAP-80 binding, the ability of CAP-80 to form stable complexes wi th C-CAM1 mutants expressed in insect cells was tested, Deletion of th e cytoplasmic domain of C-CAM1 abolished complex formation whereas del etion of the extracellular Ig domains had no effect, These results sug gest that a CAP-80 homologue (ICAP-80) is present in insect cells and ICAP-80 interacts with the cytoplasmic domain of C-CAM1. Replacement o f Tyr488, a residue in the cytoplasmic domain known to be phosphorylat ed in vivo, with Phe did not diminish the association between C-CAM1 a nd ICAP-80, suggesting that Tyr488 phosphorylation is not required for association, The ability of various C-CAM1 mutants to associate with ICAP-80 correlated with their growth inhibitory activities, suggesting that ICAP-80/CAP-80 may play an important role in C-CAM1-mediated gro wth inhibition.