Structure and function of C-CAM1: effects of the cytoplasmic domain on cell aggregation

Abstract

C-CAMs are epithelial cell-adhesion molecules of the immunoglobulin supergene family with sequences highly homologous to carcinoembryonic antigen (CEA). C-CAMs and their human homologues, biliary glycoproteins, are unique among the CEA-family proteins in that they have cytoplasmic domains. Furthermore, alternative splicing generates C-CAM isoforms with different cytoplasmic domains, suggesting that the cytoplasmic domains of C-CAM may play important roles in regulating the function or functions of C-CAM. By using both sense and antisense approaches, we have shown that C-CAM1 is a tumour suppressor in prostate carcinogenesis. This observation raises the possibility that the cytoplasmic domain of C-CAM1 may be involved in signal transduction or interaction with cytoskeletal elements to elicit the tumour suppressor function. The cytoplasmic domain of C-CAM1 contains several potential phosphorylation sites, including putative consensus sequences for cyclic AMP-dependent kinase and tyrosine kinase. One of the potential tyrosine phosphorylation sites is located within the antigen-receptor homology (ARH) domain. The ARH domain of the membrane-bound IgM molecule is necessary for signal transduction in B-cells. These structural features suggest that the cytoplasmic domain of C-CAM1 may be important for signal transduction. To test this possibility, we generated several site-directed C-CAM1 mutants and tested their ability to support adhesion and their abilities to be phosphorylated in vivo. Results from these studies revealed that Tyr-488 is phosphorylated in vivo. However, replacing this tyrosine with phenylalanine did not significantly compromise its adhesion function. Similarly, Ser and Thr residues are phosphorylated in vivo, but deletion of the potential cyclic AMP-dependent kinase site did not significantly reduce the adhesion function. These results suggest that the kinase phosphorylation sites in the cytoplasmic domain of C-CAM1 are not required for the adhesion function. However, these phosphorylation sites are probably involved in the regulation of C-CAM-mediated signal transduction. Thus, there are probably distinct structural requirements for the adhesion and the signal transduction functions of C-CAM. Incidentally, a C-CAM1 deletion mutant containing a 10-amino-acid cytoplasmic domain was able to support adhesion activity. This is in contrast to our previous finding that a C-CAM isoform, C-CAM3, with a 6-amino-acid cytoplasmic domain could not support cell adhesion. This result indicates that the extra four amino acids, which are absent in C-CAM3 and contain a potential Ser/Thr phosphorylation site, are important for the adhesion function.

Carcinoembryonic Antigen Cell Adhesion Molecule 1 Directly Associates with Cytoskeleton Proteins Actin and Tropomyosin

“…In the case of CEACAM1-L, its cytoplasmic domain has been shown to be phosphorylated on Ser/ Thr residues by PKC (49) and on Tyr residues in its ITIM motif by Src kinases (19,23). Attempts to disrupt cell-cell association by mutation of the Tyr residues to Phe in the ITIM motif failed to change the cell-cell adhesion phenotype (50), and the large number of Ser/Thr residues in the CEACAM1-L domain have discouraged investigators from performing further mutational analysis. Although some light was thrown on the problem when Obrink and co-workers (27,28,46) showed that the cytoplasmic domain of rat CEACAM1-S and the juxtamembrane residues of the CEACAM1-L cytoplasmic domain bound calmodulin, little was known about further downstream targets or the consequences of the PKC phosphorylation events.…”

Association of an 80 kDa protein with C-CAM1 cytoplasmic domain correlates with C-CAM1-mediated growth inhibition

“…As predicted from its cDNA sequence, C-CAM1 contains four extracellular Ig-like domains, a transmembrane domain, and a cytoplasmic domain. By site-directed mutagenesis, we have shown that the ®rst Ig-domain is essential for adhesion, whereas the second, third, and fourth Ig-domains and the cytoplasmic domain are not (Cheung et al, 1993b;Lin et al, 1995;Olsson et al, 1995). The fact that C-CAM1 has both cell-adhesion and growth-suppressive activities suggests that these two functions may be related.…”

Homophilic adhesion of human CEACAM1 involves N-terminal domain interactions: structural analysis of the binding site

“…Previous studies indicate that rat CEACAM1 and human CEACAM1-4L, CEACAM1-3L, CEACAM1-4S, and CEACAM1-1L transfectants adhere homophilically. 5,6,22,23,30,[43][44][45] We have examined the ability of human CEACAM1-4L, CEACAM1-4S, and CEACAM1-1S transfectants to adhere directly to immobilized recombinant proteins carrying the entire extracellular domain CEACAM1-4L/S. Our results show that, despite the higher level of CEACAM1-1S expression on CHO-CEACAM1-1S transfectants, only the CEACAM1-4L and CEACAM1-4S transfectants were able to bind significantly (30%-62% of the input cells added) to immobilized CEACAM1-4-Fc molecules ( Figure 2).…”