The CEACAM1 N-terminal Ig domain mediates cis- and trans-binding and is essential for allosteric rearrangements of CEACAM1 microclusters

Cell adhesion molecules (CAMs) sense the extracellular microenvironment and transmit signals to the intracellular compartment. In this investigation, we addressed the mechanism of signal generation by ectodomains of single-pass transmembrane homophilic CAMs. We analyzed the structure and homophilic interactions of carcinoembryonic antigen (CEA)–related CAM 1 (CEACAM1), which regulates cell proliferation, apoptosis, motility, morphogenesis, and microbial responses. Soluble and membrane-attached CEACAM1 ectodomains were investigated by surface plasmon resonance–based biosensor analysis, molecular electron tomography, and chemical cross-linking. The CEACAM1 ectodomain, which is composed of four glycosylated immunoglobulin-like (Ig) domains, is highly flexible and participates in both antiparallel (trans) and parallel (cis) homophilic binding. Membrane-attached CEACAM1 ectodomains form microclusters in which all four Ig domains participate. Trans-binding between the N-terminal Ig domains increases formation of CEACAM1 cis-dimers and changes CEACAM1 interactions within the microclusters. These data suggest that CEACAM1 transmembrane signaling is initiated by adhesion-regulated changes of cis-interactions that are transmitted to the inner phase of the plasma membrane.     

Inside-out Signaling Promotes Dynamic Changes in the Carcinoembryonic Antigen-related Cellular Adhesion Molecule 1 (CEACAM1) Oligomeric State to Control Its Cell Adhesion Properties

Cell-cell contacts are fundamental to multicellular organisms and are subject to exquisite levels of control. The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) can engage in both cis-homophilic (parallel) oligomerization and trans-homophilic (anti-parallel) binding. In this study, we establish that the CEACAM1 transmembrane domain has a propensity to form cis-dimers via the transmembrane-embedded ⁴³²GXXXG⁴³⁶ motif and that this basal state is overcome when activated calmodulin binds to the CEACAM1 cytoplasmic domain. Although mutation of the ⁴³²GXXXG⁴³⁶ motif reduced CEACAM1 oligomerization, it did not affect surface localization of the receptor or influence CEACAM1-dependent cellular invasion by the pathogenic Neisseria. The mutation did, however, have a striking effect on CEACAM1-dependent cellular aggregation, increasing both the kinetics of cell-cell association and the size of cellular aggregates formed. CEACAM1 association with tyrosine kinase c-Src and tyrosine phosphatases SHP-1 and SHP-2 was not affected by the ⁴³²GXXXG⁴³⁶ mutation, consistent with their association with the monomeric form of wild type CEACAM1. Collectively, our results establish that a dynamic oligomer-to-monomer shift in surface-expressed CEACAM1 facilitates trans-homophilic binding and downstream effector signaling.     

CEACAM6 cross-linking induces caveolin-1-dependent, Src-mediated focal adhesion kinase phosphorylation in BxPC3 pancreatic adenocarcinoma cells

Despite lacking transmembrane or intracellular domains, glycosylphosphatidylinositol-anchored proteins can modulate intracellular signaling events, in many cases through aggregation within membrane "lipid raft" microdomains. CEACAM6 is a glycosylphosphatidylinositol-linked cell surface protein of importance in the anchorage-independent survival and metastasis of pancreatic adenocarcinoma cells. We examined the effects of antibody-mediated cross-linking of CEACAM6 on intracellular signaling events and anchorage-independent survival of the CEACAM6-overexpressing pancreatic ductal adenocarcinoma cell line, BxPC3. CEACAM6 cross-linking increased c-Src activation and induced tyrosine phosphorylation of p125(FAK) focal adhesion kinase. Focal adhesion kinase phosphorylation was dependent on c-Src kinase activation, for which caveolin-1 was required. CEACAM6 cross-linking induced a significant increase in cellular resistance to anoikis. These observations represent the first characterization of the mechanism through which this important cell surface oncoprotein influences intracellular signaling events and hence malignant cellular behavior.     

Clustering-induced signaling of CEACAM1 in PC12 cells

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), an Ig-like transmembrane protein, functions in cell adhesion, angiogenesis and epithelial cell morphogenesis, and has been identified as a tumor suppressor. For all of these functions, CEACAM1 requires signaling capabilities. However, the mechanisms of CEACAM1-mediated signaling are only poorly understood. Here we characterized for the first time CEACAM1 expression and signaling in the neuroendocrine rat pheochromocytoma PC12 cell line. Stimulation of CEACAM1 by ligation on the cell surface with antibodies induced formation of large CEACAM1 clusters and a rapid and transient CEACAM1 tyrosine dephosphorylation. Functionally, this dephosphorylation correlated with a reduced association between CEACAM1 and the tyrosine phosphatase SHP2. Clustering also stimulated binding of CEACAM1 to the actin cytoskeleton, measured by a partial translocation of CEACAM1 into the insoluble fraction after detergent extraction. Both tyrosine dephosphorylation and interaction with the cytoskeleton were sensitive to neuronal differentiation of PC12 cells. The first detected downstream activation of the mitogen-activated protein kinases ERK1 and ERK2, but not of JNK or p38, describes a novel target of CEACAM1-mediated signaling and contributes to the understanding of how CEACAM1 regulates cellular function.     

CEACAM1 dynamics during neisseria gonorrhoeae suppression of CD4+ T lymphocyte activation

Neisseria gonorrhoeae colony opacity-associated (Opa) proteins bind to human carcinoembryonic antigen cellular adhesion molecules (CEACAM) found on host cells including T lymphocytes. Opa binding to CEACAM1 suppresses the activation of CD4(+) T cells in response to a variety of stimuli. In this study, we use primary human CD4(+) T cells isolated from peripheral blood to define the molecular events occurring subsequent to Opa-CEACAM1 binding. We establish that, in contrast to other cell types, T cells do not engulf N. gonorrhoeae upon CEACAM1 binding. Instead, the bacteria recruit CEACAM1 from intracellular stores and maintain it on the T cell surface. Upon TCR ligation, the co-engaged CEACAM1 becomes phosphorylated on tyrosine residues within the ITIMs apparent in the cytoplasmic domain. This allows the recruitment and subsequent activation of the src homology domain 2-containing tyrosine phosphatases SHP-1 and SHP-2 at the site of bacterial attachment, which prevents the normal tyrosine phosphorylation of the CD3zeta-chain and ZAP-70 kinase in response to TCR engagement. Combined, this dynamic response allows the bacteria to effectively harness the coinhibitory function of CEACAM1 to suppress the adaptive immune response at its earliest step.     

The role of CEA-related cell adhesion molecule-1 (CEACAM1) in vascular homeostasis

Carcinoembryonic antigen (CEA)-related cell adhesion molecules belong to the immunoglobulin superfamily, are expressed in a broad spectrum of tissues and cell types and exert context-dependent activating as well as inhibitory effects. Among these molecules, the CEA-related cell adhesion molecule-1 (CEACAM1) is a transmembrane molecule with an extracellular, a transmembrane and a cytoplasmic domain. The latter contains immunoreceptor tyrosine-based inhibitory motifs and functions as a signaling molecule. CEACAM1 can form homo- and heterodimers which is relevant for its signaling activities. CEACAM1 acts as co-receptor that modulates the activity of different receptor types including VEGFR-2, and B and T cell receptors. CEACAM1 is expressed in endothelial cells, in pericytes of developing and newly formed immature blood vessels and in angiogenically activated adult vessels, e.g., tumor blood vessels. However, it is either undetectable or only weakly expressed in quiescent blood vessels. Recent studies indicated that CEACAM1 is involved in the regulation of the endothelial barrier function. In CEACAM1 ^?/? mice, increased vascular permeability and development of small atherosclerotic lesions was observed in the aortae. CEACAM1 is also detectable in activated lymphatic endothelial cells and plays a role in tumor lymphangiogenesis. This review summarizes the vascular effects of CEACAM1 and focuses on its role in vascular morphogenesis and endothelial barrier regulation.     

Soluble CEACAM8 Interacts with CEACAM1 Inhibiting TLR2-Triggered Immune Responses

Lower respiratory tract bacterial infections are characterized by neutrophilic inflammation in the airways. The carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 8 is expressed in and released by human granulocytes. Our study demonstrates that human granulocytes release CEACAM8 in response to bacterial DNA in a TLR9-dependent manner. Individuals with a high percentage of bronchial lavage fluid (BALF) granulocytes were more likely to have detectable levels of released CEACAM8 in the BALF than those with a normal granulocyte count. Soluble, recombinant CEACAM8-Fc binds to CEACAM1 expressed on human airway epithelium. Application of CEACAM8-Fc to CEACAM1-positive human pulmonary epithelial cells resulted in reduced TLR2-dependent inflammatory responses. These inhibitory effects were accompanied by tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM) of CEACAM1 and by recruitment of the phosphatase SHP-1, which could negatively regulate Toll-like receptor 2-dependent activation of the phosphatidylinositol 3-OH kinase-Akt kinase pathway. Our results suggest a new mechanism by which granulocytes reduce pro-inflammatory immune responses in human airways via secretion of CEACAM8 in neutrophil-driven bacterial infections.     

The cell adhesion molecule CEACAM1-L is a substrate of caspase-3-mediated cleavage in apoptotic mouse intestinal cells

The CEACAM1 cell adhesion molecule is a member of the carcinoembryonic antigen family. In the mouse, four distinct isoforms are generated by alternative splicing. These encode either two or four immunoglobulin domains linked through a transmembrane domain to a cytoplasmic domain that encompasses either a short 10-amino acid tail or a longer one of 73 amino acids. Inclusion of exon 7, well conserved in evolution, generates the long cytoplasmic domain. A potential caspase recognition site in mouse, rat, and human CEACAM1-L also becomes available within the peptide encoded by exon 7. We used CEACAM1-L-transfected mouse colon carcinoma CT51 cells treated with three different apoptotic agents to study its fate during cell death. We found that CEACAM1-L is cleaved resulting in rapid degradation of most of its 8-kDa cytoplasmic domain. Caspase-mediated cleavage was demonstrated using purified recombinant caspases. The long cytoplasmic domain was cleaved specifically by caspase-3 in vitro but not by caspase-7 or -8. Moreover cleavage of CEACAM1-L in apoptotic cells was blocked by addition of a selective caspase-3 inhibitor to the cultures. Using point and deletion mutants, the conserved DQRD motif in the membrane-proximal cytoplasmic domain was identified as a caspase cleavage site. We also show that once CEACAM1-L is caspase-cleaved it becomes a stronger adhesion molecule than both the shorter and the longer expressing isoforms.     

c-Src-dependent cross-talk between CEACAM6 and alphavbeta3 integrin enhances pancreatic adenocarcinoma cell adhesion to extracellular matrix components

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an immunoglobulin superfamily member with a diversity of extracellular ligands that is implicated in the initiation and progression of a variety of malignancies. We sought to characterize the effects of CEACAM6 crosslinking on pancreatic adenocarcinoma cellular interaction with the extracellular matrix (ECM) components fibronectin and vitronectin. Antibody-mediated CEACAM6 crosslinking was performed and the ability of BxPC3 cells, which inherently overexpress CEACAM6, to adhere to fibronectin and vitronectin was quantified. The roles of the archetypal fibronectin (alpha5beta1 integrin) and vitronectin (alphavbeta3 integrin) receptors were determined. The effects of c-Src inhibition were investigated using the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and c-Src specific RNA interference. CEACAM6 crosslinking initiates c-Src-dependent cross-talk between CEACAM6 and alphavbeta3 integrin, leading to increased ECM component adhesion. CEACAM6-mediated signaling events may contribute to the invasive and metastatic potential of pancreatic adenocarcinoma cells by promoting their interaction with ECM components.     

Serum CEACAM1 Level Is Associated with Diagnosis and Prognosis in Patients with Osteosarcoma

Carcinoembryonic antigen related cell adhesion molecule 1 (CEACAM1) is a trans-membrane multifunctional cell adhesion molecule associated with tumor cell proliferation, apoptosis, angiogenesis, invasion, and migration during tumor development. In the present study, we evaluated serum CEACAM1 level in osteosarcoma patients to explore its diagnostic and prognostic value for this particular malignancy. Sera from 113 patients with primary osteosarcoma, 98 patients with benign bone tumors and 126 healthy controls were obtained. Serum CEACAM1 level was measured with ELISA and correlation with clinicopathological characteristics was further analyzed. Receiver operating curves (ROC), Kaplan-Meier curves, and log-rank analyses as well as Cox proportional hazard models were used to evaluate diagnostic and prognostic significance. The results revealed that serum CEACAM1 level was significantly higher in osteosarcoma patients compared to benign bone tumors and healthy controls (455.2 ± 179.9 vs 287.4 ± 103.2, 260.8 ± 109.7 pg/ml, respectively). Osteosarcoma patients with larger tumors, later-tumor stages, low tumor grades, and distant metastases had much higher CEACAM1 compared to those with smaller tumors, earlier tumor stages, high tumor grades and non-distant metastases (P < 0.05 for all). Multivariate logistic regression analysis confirmed that high serum CEACAM1 level was an independent risk factor for distant metastases (OR = 3.02, 95%CI 1.65–4.17). To distinguish osteosarcoma patients from those with benign bone tumor and healthy controls, ROC/AUC analysis indicated an AUC of 0.81 (sensitivity 0.61; specificity 0.89) and an AUC of 0.77 (sensitivity 0.57; specificity 0.92), respectively. Osteosarcoma patients with higher CEACAM1 had relatively lower survival compared to those with low CEACAM1 (P < 0.01), and multivariate analyses for overall survival revealed that high serum CEACAM1 level was an independent prognostic factor for osteosarcoma (HR = 1.56, 95%CI 1.23–3.28). The present study suggested that elevated serum CEACAM1 level might be a novel diagnostic and prognostic biomarker for osteosarcoma patients.     

The CEACAM1-L glycoprotein associates with the actin cytoskeleton and localizes to cell-cell contact through activation of Rho-like GTPases

Associations between plasma membrane-linked proteins and the actin cytoskeleton play a crucial role in defining cell shape and determination, ensuring cell motility and facilitating cell-cell or cell-substratum adhesion. Here, we present evidence that CEACAM1-L, a cell adhesion molecule of the carcinoembryonic antigen family, is associated with the actin cytoskeleton. We have delineated the regions involved in actin cytoskeleton association to the distal end of the CEACAM1-L long cytoplasmic domain. We have demonstrated that CEACAM1-S, an isoform of CEACAM1 with a truncated cytoplasmic domain, does not interact with the actin cytoskeleton. In addition, a major difference in subcellular localization of the two CEACAM1 isoforms was observed. Furthermore, we have established that the localization of CEACAM1-L at cell-cell boundaries is regulated by the Rho family of GTPases. The retention of the protein at the sites of intercellular contacts critically depends on homophilic CEACAM1-CEACAM1 interactions and association with the actin cytoskeleton. Our results provide new evidence on how the Rho family of GTPases can control cell adhesion: by directing an adhesion molecule to its proper cellular destination. In addition, these results provide an insight into the mechanisms of why CEACAM1-L, but not CEACAM1-S, functions as a tumor cell growth inhibitor.     

Distinct Rho GTPase activities regulate epithelial cell localization of the adhesion molecule CEACAM1: involvement of the CEACAM1 transmembrane domain

CEACAM1 is an intercellular adhesion glycoprotein. As CEACAM1 plays an important role in epithelial cell signaling and functions, we have examined its localization in epithelial cells. We have observed that distribution at cell contacts is not always seen in these cells, suggesting that CEACAM1 localization might be regulated. In Swiss 3T3 cells, the targeting of CEACAM1 at cell-cell boundaries is regulated by the Rho GTPases. In the present study, we have used the MDCK epithelial cells to characterize the effects of the Rho GTPases and their effectors on CEACAM1 intercellular targeting. Activated Cdc42 and Rac1 or their downstream effector PAK1 targeted CEACAM1 to sites of cell-cell contacts. On the other hand, neither activated RhoA nor activated Rho kinase directed CEACAM1 to cell boundaries, resulting in a condensed distribution of CEACAM1 at the cell surface. Interestingly, inhibition of this pathway resulted in CEACAM1 intercellular localization suggesting that a tightly regulated balance of Rho GTPase activities is necessary to target CEACAM1 at cell-cell boundaries. In addition, using CEACAM1 mutants and chimeric fusion constructs containing domains of the colony-stimulating factor receptor, we have shown that the transmembrane domain of CEACAM1 is responsible for the Cdc42-induced targeting at cell-cell contacts.     

Carcinoembryonic antigen-related cell adhesion molecule 1 inhibits proximal TCR signaling by targeting ZAP-70

The long cytoplasmic tail (CT) isoforms of carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1) are expressed on activated human T cells and possess two ITIM motifs in the CT. These isoforms of CEACAM1 are inhibitory for T cell responses initiated by the TCR/CD3 complex with the inhibition dependent upon the ITIMs of CEACAM1 and Src homology 2 domain-containing phosphatase 1 (SHP-1). However, the mechanism by which this inhibition occurs in T cells is unknown. We demonstrate here that the Src family kinase, Lck, and the ability of CEACAM1 to bind homophilically are required for the ITIM phosphorylation of CEACAM1 that is a prerequisite for CEACAM1 association with SHP-1. We further show that CEACAM1 associates with and recruits SHP-1 to the TCR/CD3 complex leading to decreased phosphorylation of CD3-zeta and ZAP-70 and consequently decreased activation of the elements downstream of ZAP-70. This is physiologically relevant because extinction of SHP-1 expression or blockade of homophilic binding by CEACAM1 using a Fab that specifically recognizes the homophilic binding region of human CEACAM1 increases the cytolytic function initiated by the TCR/CD3 complex. These studies show that long CT isoforms of CEACAM1 orchestrate an inhibitory program that abrogates extremely proximal events downstream of the TCR/CD3 complex by focusing on the activation of ZAP-70.     

Carcinoembryonic antigen cell adhesion molecule 1 directly associates with cytoskeleton proteins actin and tropomyosin

CEA cell adhesion molecule 1 (CEACAM1), a type 1 transmembrane and homotypic cell adhesion protein belonging to the carcinoembryonic antigen (CEA) gene family and expressed on epithelial cells, is alternatively spliced to produce four major isoforms with three or four Ig-like ectodomains and either long (CEACAM1-L) or short (CEACAM1-S) cytoplasmic domains. When murine MC38 (methylcholanthrene-induced adenocarcinoma 38) cells were transfected with human CEACAM1-L and stimulated with sodium pervanadate, actin was found to co-localize with CEACAM1-L at cell-cell boundaries but not in untreated cells. When CEACAM1-L was immunoprecipitated from pervanadate-treated MC38/CEACAM1-L cells and the associated proteins were analyzed by two-dimensional gel analysis and mass spectrometry, actin and tropomyosin, among other proteins, were identified. Whereas a glutathione S-transferase (GST) fusion protein containing the l-isoform (GST-Cyto-L) bound poorly to F-actin in a co-sedimentation assay, the S-isoform fusion protein (GST-Cyto-S) co-sedimented with F-actin, especially when incubated with G-actin during polymerization (K(D) = 7.0 microm). Both GST-Cyto-S and GST-Cyto-L fusion proteins bind G-actin and tropomyosin by surface plasmon resonance studies with binding constants of 0.7 x 10(-8) and 1.0 x 10(-7) m for GST-Cyto-L to G-actin and tropomyosin, respectively, and 3.1 x 10(-8) and 1.3 x 10(-7) m for GST-Cyto-S to G-actin and tropomyosin, respectively. Calmodulin or EDTA inhibited binding of the GST-Cyto-L fusion protein to G-actin, whereas calmodulin and G-actin, but not EDTA, stimulated binding to tropomyosin. A biotinylated 14-amino acid peptide derived from the juxtamembrane portion of the cytoplasmic domain of CEACAM1-L associated with both G-actin and tropomyosin with K(D) values of 1.3 x 10(-5) and 1.8 x 10(-5) m, respectively. These studies demonstrate the direct interaction of CEACAM1 isoforms with G-actin and tropomyosin and the direct interaction of CEACAM1-S with F-actin.     

Widespread Divergence of the CEACAM/PSG Genes in Vertebrates and Humans Suggests Sensitivity to Selection

In mammals, carcinoembryonic antigen cell adhesion molecules (CEACAMs) and pregnancy-specific glycoproteins (PSGs) play important roles in the regulation of pathogen transmission, tumorigenesis, insulin signaling turnover, and fetal–maternal interactions. However, how these genes evolved and to what extent they diverged in humans remain to be investigated specifically. Based on syntenic mapping of chordate genomes, we reveal that diverging homologs with a prototypic CEACAM architecture–including an extracellular domain with immunoglobulin variable and constant domain-like regions, and an intracellular domain containing ITAM motif–are present from cartilaginous fish to humans, but are absent in sea lamprey, cephalochordate or urochordate. Interestingly, the CEACAM/PSG gene inventory underwent radical divergence in various vertebrate lineages: from zero in avian species to dozens in therian mammals. In addition, analyses of genetic variations in human populations showed the presence of various types of copy number variations (CNVs) at the CEACAM/PSG locus. These copy number polymorphisms have 3–80% frequency in select populations, and encompass single to more than six PSG genes. Furthermore, we found that CEACAM/PSG genes contain a significantly higher density of nonsynonymous single nucleotide polymorphism (SNP) compared to the chromosome average, and many CEACAM/PSG SNPs exhibit high population differentiation. Taken together, our study suggested that CEACAM/PSG genes have had a more dynamic evolutionary history in vertebrates than previously thought. Given that CEACAM/PSGs play important roles in maternal–fetal interaction and pathogen recognition, these data have laid the groundwork for future analysis of adaptive CEACAM/PSG genotype-phenotypic relationships in normal and complicated pregnancies as well as other etiologies.     

Phosphorylation of human CEACAM1-LF by PKA and GSK3β promotes its interaction with β-catenin

CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a binding site for β-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the ¹⁵N labeled cytoplasmic domain peptide. Incubation with a variety of Ser/Thr kinases revealed phosphorylation of Ser508 by GSK3bβ but not by PKC. The lack of phosphorylation by PKC is likely due to evolutionary sequence changes between the rodent and human genes. Phosphorylation site assignment by mass spectrometry and NMR revealed phosphorylation of Ser472, Ser461 and Ser512 by PKA, of which Ser512 is part of a conserved consensus site for GSK3β binding. We showed here that only after phosphorylation of Ser512 by PKA was GSK3β able to phosphorylate Ser508. Phosphorylation of Ser512 by PKA promoted a tight association with the armadillo repeat domain of β-catenin at an extended region spanning the ITIMs of CEACAM1. The kinetics of phosphorylation of the ITIMs by Src, as well dephosphorylation by SHP2, were affected by the presence of Ser508/512 phosphorylation, suggesting that PKA and GSK3β may regulate the signal transduction activity of human CEACAM1-LF. The interaction of CEACAM1-LF with β-catenin promoted by PKA is suggestive of a tight association between the two ITIMs of CEACAM1-LF.     

Mapping the binding domains on meningococcal Opa proteins for CEACAM1 and CEA receptors

The opacity (Opa) proteins of pathogenic Neisseria spp. are adhesins, which play an important role in adhesion and invasion of host cells. Most members of this highly variable family of outer membrane proteins can bind to the human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). Several studies have identified the Opa-binding region on the CEACAM receptors; however, not much is known about the binding sites on the Opa proteins for the corresponding CEACAM-receptors. The high degree of sequence variation in the surface-exposed loops of Opa proteins raises the question how the binding sites for the CEACAM receptors are conserved. Neisseria meningitidis strain H44/76 possesses four different Opa proteins, of which OpaA and OpaJ bind to CEACAM1, while OpaB and OpaD bind to CEACAM1 and CEA. A sequence motif involved in binding to CEACAM1 was identified by alanine scanning mutagenesis of those amino acid residues conserved within the hypervariable (HV) regions of all four Opa proteins. Hybrid Opa variants with different combinations of HV-1 and HV-2 derived from OpaB and OpaJ showed a reduced binding to CEACAM1 and CEA, indicating that particular combinations of HV-1 and HV-2 are required for the Opa binding capacity. Homologue scanning mutagenesis was used to generate more refined hybrids containing novel combinations of OpaB and OpaJ sequences within HV-1 and HV-2. They could be used to identify residues determining the specificity for CEA binding. The combined results obtained with mutants and hybrids strongly suggest the existence of a conserved binding site for CEACAM receptors by the interaction of HV-1 and HV-2 regions.     

CEACAM1 (CD66a) promotes human monocyte survival via a phosphatidylinositol 3-kinase- and AKT-dependent pathway

CEACAM1 (also known as CD66a) is a transmembrane glycoprotein that mediates homophilic intercellular interactions that influence cellular growth, immune cell activation, and tissue morphogenesis. Various studies have suggested a link between CEACAM1 and cellular apoptosis, including a recent demonstration that ERK1/2 signaling is triggered downstream of CEACAM1. In this study, we reveal that CEACAM1-long binding confers survival signals to human peripheral blood mononuclear cells. CEACAM-specific antibodies effectively protected peripheral blood mononuclear cells from apoptosis, with this effect being particularly dramatic for primary monocytes that undergo spontaneous apoptosis during in vitro culture. This protective effect was reiterated when using soluble CEACAM1, which binds to cell-surface CEACAM1 via homophilic interactions. Monocyte survival correlated with a CEACAM1-dependent up-regulation of the cellular inhibitor of apoptosis Bcl-2 and the abrogation of caspase-3 activation. CEACAM1 binding triggered a phosphatidylinositol 3-kinase-dependent activation of the protein kinase Akt without influencing the activity of extracellular signal-related kinase ERK, whereas the phosphatidylinositol 3-kinase-specific inhibitor LY294002 effectively blocked the protective effect of CEACAM1. Together, this work indicates that CEACAM1 confers a phosphatidylinositol 3-kinase- and Akt-dependent survival signal that inhibits mitochondrion-dependent apoptosis of monocytes. By controlling both ERK/MEK and PI3K/Akt pathways, CEACAM1 functions as a key regulator of contact-dependent control of cell survival, differentiation, and growth.     

Regulation of CEACAM1 transcription in human breast epithelial cells

Background  

Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is a transmembrane protein with multiple functions in different cell types. CEACAM1 expression is frequently mis-regulated in cancer, with down-regulation reported in several tumors of epithelial origin and de novo expression of CEACAM1 in lung cancer and malignant melanoma. In this report we analyzed the regulation of CEACAM1 expression in three breast cancer cell lines that varied in CEACAM1 expression from none (MCF7) to moderate (MDA-MB-468) to high (MCF10A, comparable to normal breast).     

The transmembrane domain of CEACAM1-4S is a determinant of anchorage independent growth and tumorigenicity

CEACAM1 is a multifunctional Ig-like cell adhesion molecule expressed by epithelial cells in many organs. CEACAM1-4L and CEACAM1-4S, two isoforms produced by differential splicing, are predominant in rat liver. Previous work has shown that downregulation of both isoforms occurs in rat hepatocellular carcinomas. Here, we have isolated an anchorage dependent clone, designated 253T-NT that does not express detectable levels of CEACAM1. Stable transfection of 253-NT cells with a wild type CEACAM1-4S expression vector induced an anchorage independent growth in vitro and a tumorigenic phenotype in vivo. These phenotypes were used as quantifiable end points to examine the functionality of the CEACAM1-4S transmembrane domain. Examination of the CEACAM1 transmembrane domain showed N-terminal GXXXG dimerization sequences and C-terminal tyrosine residues shown in related studies to stabilize transmembrane domain helix-helix interactions. To examine the effects of transmembrane domain mutations, 253-NT cells were transfected with transmembrane domain mutants carrying glycine to leucine or tyrosine to valine substitutions. Results showed that mutation of transmembrane tyrosine residues greatly enhanced growth in vitro and in vivo. Mutation of transmembrane dimerization motifs, in contrast, significantly reduced anchorage independent growth and tumorigenicity. 253-NT cells expressing CEACAM1-4S with both glycine to leucine and tyrosine to valine mutations displayed the growth-enhanced phenotype of tyrosine mutants. The dramatic effect of transmembrane domain mutations constitutes strong evidence that the transmembrane domain is an important determinant of CEACAM1-4S functionality and most likely by other proteins with transmembrane domains containing dimerization sequences and/or C-terminal tyrosine residues.