Extracellular N-domain alone can mediate specific heterophilic adhesion between members of the carcinoembryonic antigen family, CEACAM6 and CEACAM8

The domain(s) responsible for the specific heterophilic adhesion between two members of the carcinoembryonic antigen (CEA) family, CEACAM6 and CEACAM8, both of which with three extracellular domains, were investigated using Chinese hamster ovary (CHO) transfectants expressing chimeric antigens. Using a chimeric antigen in which the N-domain, a sole extracellular domain, of CEACAM3 was substituted with that of CEACAM6, it was shown that the N-domain of CEACAM6 alone was able to mediate specific adhesion to CEACAM8. Furthermore, the chimeric antigen was shown to bind significantly to chimeric CEA whose N-domain was substituted with that of CEACAM8, but not to unsubstituted CEA. These results demonstrate that the N-domain alone is sufficient and other domains of CEACAM6 or CEACAM8 are not required for this specific binding. We therefore propose a model of heterophilic interaction between the N-domains, which is distinct from that of CEA-CEA homophilic binding.     

The critical role of residues 43R and 44Q of carcinoembryonic antigen cell adhesion molecules-1 in the protection from killing by human NK cells

The multifunctional carcinoembryonic Ag cell adhesion molecule (CEACAM)1 protein has recently become the focus of intense immunological research. We have previously shown that the CEACAM1 homophilic interactions inhibit the killing activity of NK cells. This novel inhibitory mechanism plays a key role in melanoma immune evasion, inhibition of decidual immune response, and controlling NK autoreactivity in TAP2-deficient patients. These roles are mediated mainly by homophilic interactions, which are mediated through the N-domain of the CEACAM1. The N-domain of the various members of the CEACAM family shares a high degree of similarity. However, it is still unclear which of the CEACAM family members is able to interact with CEACAM1 and what are the amino acid residues that control this interaction. In this study we demonstrate that CEACAM1 interacts with CEACAM5, but not with CEACAM6. Importantly, we provide the molecular basis for CEACAM1 recognition of various CEACAM family members. Sequence alignment reveals a dichotomy among the CEACAM family members: both CEACAM1 and CEACAM5 contain the R and Q residues in positions 43 and 44, respectively, whereas CEACAM3 and CEACAM6 contain the S and L residues, respectively. Mutational analysis revealed that both 43R and 44Q residues are necessary for CEACAM1 interactions. Implications for differential expression of CEACAM family members in tumors are discussed.     

Synthetic peptides from the N-domains of CEACAMs activate neutrophils.

Four members of the carcinoembryonic antigen family, CEACAM1, CEACAM8, CEACAM6 and CEACAM3, recognized by CD66a, CD66b, CD66c and CD66d monoclonal antibodies (mAb), respectively, are expressed on human neutrophils. CD66a, CD66b, CD66c and CD66d mAb binding to neutrophils triggers an activation signal that regulates the adhesive activity of CD11/CD18, resulting in an increase in neutrophil adhesion to human umbilical vein endothelial cells. Molecular modeling of CEACAM1 using IgG and CD4 as models has been performed, and three peptides from the N-terminal domain were found to increase neutrophil adhesion to human umbilical vein endothelial cell monolayers. The peptides were 14 amino acids in length and were predicted to be present at loops and turns between beta-sheets. To better understand the amino acid sequences critical for this biological activity, in the present study we examined the other neutrophil CEACAMs and the highly homologous CEACAM, CEA. Molecular modeling of the N-terminal domains of human CEACAM8, -6, -3 and CEA was performed. Twenty peptides, each 14 amino acids in length, that were homologous to the previously reported peptides from the N-domains of CEACAM1, were synthesized and tested for their ability to alter neutrophil adhesion. Only one new peptide, from the N-domain of CEA, was found to increase neutrophil adhesion, and this peptide differed from the corresponding CEACAM1 peptide by only a single conservative amino acid substitution. Importantly, minor amino acid differences between active and inactive homologous peptides suggest regions of these peptides that are critical for biological activity. The data suggest that the regions SMPF of peptide CD66a-1, QLFG of peptide CD66a-2 and NRQIV of peptide CD66a-3 are critical for the activities of these peptides, and for the native CEACAMs.     

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.     

Several acidic amino acids in the N-domain of insulin-like growth factor-binding protein-5 are important for its transactivation activity

Insulin-like growth factor-binding protein (IGFBP)-5 is a secreted protein that binds to IGFs and modulates IGF actions. IGFBP-5 is also found in the nuclei of cultured cells and has transactivation activity. Here we report the nuclear localization of endogenous IGFBP-5 in mouse embryonic skeletal cells. Chromatin immunoprecipitation experiments indicated that IGFBP-5 interacts with the nuclear histone-DNA complex. Using a series of deletion mutants, the transactivation domain of IGFBP-5 was mapped to its N-terminal region. Intriguingly, the transactivation activity of IGFBP-5 is masked by negative regulatory elements located in the L- and C-domains. Among the other IGFBPs, the N-domains of IGFBP-2 and -3 also had strong transactivation activity, whereas those of IGFBP-1 and -6 had no activity. The IGFBP-4 N-domain had modest activity. Sequence analysis revealed several amino acids in the IGFBP-5 N-domain that are not present in IGFBP-1. The activities of mutants in which these residues were changed to the corresponding IGFBP-1 sequence were determined. Mutations that changed acidic residues to neutral residues (e.g. E8A, D11S, E12A, E30S/P31A, E43L, and E52A) or a polar to a basic residue (e.g. Q56R) significantly reduced transactivation activity. The E8A/D11S/E12A triple mutant and E52A/Q56R double mutants showed further reduced activity. The combinatory mutants had essentially no transactivation activity. Taken together, our results indicate that there are several conserved residues in the IGFBP-5 N-terminal region that are critical for transactivation and that IGFBP-2 and -3 also have strong transactivation activity in their N-domains.     

Carcinoembryonic antigen-related cell adhesion molecule 1 expression and signaling in human, mouse, and rat leukocytes: evidence for replacement of the short cytoplasmic domain isoform by glycosylphosphatidylinositol-linked proteins in human leukocytes

Carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1), the primordial carcinoembryonic Ag gene family member, is a transmembrane cell adhesion molecule expressed in leukocytes, epithelia, and blood vessel endothelia in humans and rodents. As a result of differential splicing, CEACAM1 occurs as several isoforms, the two major ones being CEACAM1-L and CEACAM1-S, that have long (L) or short (S) cytoplasmic domains, respectively. The L:S expression ratios vary in different cells and tissues. In addition to CEACAM1, human but not rodent cells express GPI-linked CEACAM members (CEACAM5-CEACAM8). We compared the expression patterns of CEACAM1-L, CEACAM1-S, CEACAM6, and CEACAM8 in purified populations of neutrophilic granulocytes, B lymphocytes, and T lymphocytes from rats, mice, and humans. Human granulocytes expressed CEACAM1, CEACAM6, and CEACAM8, whereas human B lymphocytes and T lymphocytes expressed only CEACAM1 and CEACAM6. Whereas granulocytes, B cells, and T cells from mice and rats expressed both CEACAM1-L and CEACAM1-S in ratios of 2.2-2.9:1, CEACAM1-S expression was totally lacking in human granulocytes, B cells, and T cells. Human leukocytes only expressed the L isoforms of CEACAM1. This suggests that the GPI-linked CEACAM members have functionally replaced CEACAM1-S in human leukocytes. Support for the replacement hypothesis was obtained from experiments in which the extracellular signal-regulated kinases (Erk)1/2 were activated by anti-CEACAM Abs. Thus, Abs against CEACAM1 activated Erk1/2 in rat granulocytes, but not in human granulocytes. Erk1/2 in human granulocytes could, however, be activated by Abs against CEACAM8. We demonstrated that CEACAM1 and CEACAM8 are physically associated in human granulocytes. The CEACAM1/CEACAM8 complex in human cells might accordingly play a similar role as CEACAM1-L/CEACAM1-S dimers known to occur in rat cells.     

The carcinoembryonic antigen (CEA) gene family in non-human primates

Carcinoembryonic antigen (CEA) is a tumor marker of wide clinical use though its function remains unknown. The CEA counterpart and some related macromolecules cannot be demonstrated in mice, thus prohibiting studies of CEA function by gene disruption strategies. In an attempt to find a relevant animal model for functional studies of CEA we have investigated the occurrence of CEA subgroup members in baboon and African green monkey at the genomic and mRNA levels. The investigation was focused on the characteristic immunoglobulin-variable region-like (IgV-like) N-terminal domain of the family members. Based on N-domain sequences 3 and 4 different CEA subgroup genes, respectively, were identified. One sequence in each monkey species corresponded to human CEACAM8, while it was not possible to assign an obvious human counterpart for the other N-domain sequences. However, studies of cDNAs from African green monkey COS-1 cells identified one of the sequences as CEACAM1. Expression of CEACAM1 mRNA and protein was upregulated by IFNgamma as has previously been demonstrated for human CEACAM1. Presence of GPI-linked CEA subgroup members in African green monkey was suggested by sequencing. Both monkey species would thus seem suitable for functional studies of selected CEA subgroup members.     

Functional interaction between the pro-apoptotic DAP3 and the glucocorticoid receptor

The widely expressed adhesion receptor CEACAM1 is a member of the carcinoembryonic antigen (CEA) family within the immunoglobulin (Ig) superfamily of glycoproteins. While the expression of transmembrane isoforms has been described in detail, only little is known about soluble isoforms. By RT-PCR characterization of rat pheochromocytoma PC12 and mammary adenocarcinoma MTC cell lines, two novel splice variants, designated CEACAM1-4C1 and CEACAM1-4C2, lacking the transmembrane region, were identified. In addition, we demonstrate the expression of transmembrane CEACAM1-4L and CEACAM1-4S with a truncated cytoplasmic domain. The C-termini of CEACAM1-4C2 and CEACAM1-L are identical, which allowed the specific in vitro and in vivo detection of the soluble CEACAM1-4C2 protein by an antiserum generated against the CEACAM1-L cytoplasmic part. Functionally, soluble CEACAM1 could inhibit CEACAM1-mediated aggregation of CHO cells. In conclusion, our data define a new mechanism for the appearance of functionally active rat CEACAM1 protein in body fluids.     

CEACAM1, a cell-cell adhesion molecule, directly associates with annexin II in a three-dimensional model of mammary morphogenesis

The epithelial cell adhesion molecule CEACAM1 (carcinoembryonic antigen cell adhesion molecule-1) is down-regulated in colon, prostate, breast, and liver cancer. Here we show that CEACAM1-4S, a splice form with four Ig-like ectodomains and a short cytoplasmic domain (14 amino acids), directly associates with annexin II, a lipid raft-associated molecule, which is also down-regulated in many cancers. Annexin II was identified using a glutathione S-transferase pull-down assay in which the cytoplasmic domain of CEACAM-4S was fused to glutathione S-transferase, the fusion protein was incubated with cell lysates, and isolated proteins were sequenced by mass spectrometry. The interaction was confirmed first by reciprocal immunoprecipitations using anti-CEACAM1 and anti-annexin II antibodies and second by confocal laser microscopy showing co-localization of CEACAM1 with annexin II in mammary epithelial cells grown in Matrigel. In addition, CEACAM1 co-localized with p11, a component of the tetrameric AIIt complex at the plasma membrane, and with annexin II in secretory vesicles. Immobilized, oriented peptides from the cytoplasmic domain of CEACAM1-4S were shown to directly associate with bovine AIIt, which is 98% homologous to human AIIt, with average KD values of about 30 nM using surface plasmon resonance, demonstrating direct binding of functionally relevant AIIt to the cytoplasmic domain of CEACAM1-4S.     

Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

CEACAM1, CEA/CEACAM5, and CEACAM6 are cell adhesion molecules (CAMs) of the carcinoembryonic antigen (CEA) family that have been shown to be deregulated in lung cancer and in up to 50% of all human cancers. However, little is known about the functional impact of these molecules on undifferentiated cell growth and tumor progression. Here we demonstrate that cell surface expression of CEACAM1 on confluent A549 human lung adenocarcinoma cells plays a critical role in differentiated, contact-inhibited cell growth. Interestingly, CEACAM1-L, but not CEACAM1-S, negatively regulates proliferation via its ITIM domain, while in proliferating cells no CEACAM expression is detectable. Furthermore, we show for the first time that CEACAM6 acts as an inducer of cellular proliferation in A549 cells, likely by interfering with the contact-inhibiting signal triggered by CEACAM1-4L, leading to undifferentiated anchorage-independent cell growth. We also found that A549 cells expressed significant amounts of non-membrane anchored variants of CEACAM5 and CEACAM6, representing a putative source for the increased CEACAM5/6 serum levels frequently found in lung cancer patients. Taken together, our data suggest that post-confluent contact inhibition is established and maintained by CEACAM1-4L, but disturbances of CEACAM1 signalling by CEACAM1-4S and other CEACAMs lead to undifferentiated cell growth and malignant transformation.     

Mutational analysis of the cytoplasmic domain of CEACAM1-4L in humanized mammary glands reveals key residues involved in lumen formation: Stimulation by Thr-457 and inhibition by Ser-461

CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a type I transmembrane glycoprotein involved in cell-cell adhesion, undergoes extensive alternative splicing, resulting in isoforms with 1-4 Ig-like extracellular domains (ECDs) with either long or short cytoplasmic tails. We have previously shown that CEACAM1-4L (4 ECDs with a long cytoplasmic domain) formed glands with lumena in humanized mammary mouse fat pads in NOD/SCID mice. In order to identify the key residues of CEACAM1-4L that play essential roles in lumen formation, we introduced phosphorylation mimic (e.g., Thr-457 or Ser-461 to Asp) or null mutations (Thr-457 or Ser-461 to Ala) into the cytoplasmic domain of CEACAM1-4L and tested them in both the in vivo mouse model and in vitro Matrigel model of mammary morphogenesis. MCF7 cells stably expressing CEACAM1-4L with the single mutation T457D or the double mutant T457D+S461D, but not the null mutants induced central lumen formation in 3D Matrigel and in humanized mammary fat pads. However, the single phosphorylation mimic mutation S461D, but not the null mutation blocked lumen formation in both models, suggesting that S461 has inhibitory function in glandular lumen formation. Compared to our results for the -4S isoform (Chen et al., J. Biol. Chem, 282: 5749-5760, 2008), the T457A null mutation blocks lumen formation for the -4L but not for the -4S isoform. This difference is likely due to the fact that phosphorylation of S459 (absent in the -4L isoform) positively compensates for loss of T457 in the -4S isoform, while S461 (absent in the -4S isoform) negatively regulates lumen formation in the -4L isoform. Thus, phosphorylation of these key residues may exert a fine control over the role of the -4L isoform (compared to the -4S isoform) in lumen formation.     

Identification of Lewis x structures of the cell adhesion molecule CEACAM1 from human granulocytes

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed on epithelia, blood vessel endothelia, and leukocytes. A variety of physiological functions have been assigned to CEACAM1. It is involved in the formation of glands and blood vessels, in immune reactions, and in the regulation of tumor growth. As a homophilic and heterophilic adhesion receptor, it signals through different cellular pathways. The existence of special oligosaccharide structures such as Lewis x or sialyl-Lewis x glycans within this highly glycosylated protein has been postulated, but chemical proof is missing so far. Because such structures are known to be essential for different cell-cell recognition and adhesion processes, characterizing the CEACAM1 glycan structure is of pivotal importance in revealing the biological function of CEACAM1. We examine the terminal glycosylation pattern of CEACAM1 from human granulocytes, focusing on Lewis x epitopes. Lewis x-specific antibodies react with immunoaffinity-purified native CEACAM1. Antibody binding was completely abolished by treatment with fucosidase III, confirming a terminal alpha(1-3,4) fucose linkage to the N-acetylglucosamine of lactosamine residues, a key feature of Lewis epitopes. To verify these data, MALDI-TOF MS analysis after stepwise exoglycosidase digestion of the CEACAM1 N-glycan mixture was performed. A complex mixture of CEACAM1-bound oligosaccharides could be characterized with an unusually high amount of fucose. The sequential digestions clearly identified several different Lewis x glycan epitopes, which may modulate the cell adhesive functions of CEACAM1.     

Cloning and Localization of Exon 5-Containing Isoforms of the NMDAR1 Subunit in Human and Rat Brains

Abstract: Nine isoforms of the rat NMDAR1 receptor subunit have been previously identified, of which several have an alternatively spliced N-terminal insert believed to be important in proton sensitivity of the receptor. The cloning of the human homologues of NMDAR1-3b (hNMDA1-1) and NMDAR1-4b (hNMDA1-2), both bearing the insert, is reported here. A monoclonal antibody generated against the N-terminal region of these isoforms showed reactivity with at least two distinct human brain proteins of ∼115 kDa. This antibody was further characterized by using a series of truncated fusion proteins and splice variants of NMDAR1 demonstrating its specific recognition of an epitope within the 21-amino acid N-terminal insert, encoded by exon 5. Western blot and immunocytochemical studies were performed to examine the expression of the exon 5-containing isoforms of the NMDAR1 subunit in both rat and human brain.     

Human antibodies react with an epitope of the human papillomavirus type 6b L1 open reading frame which is distinct from the type-common epitope.

Recombinant proteins encoded by the human papillomavirus type 6b (HPV6b) L1 open reading frame react with sera from patients with condylomata acuminata and also react with rabbit antiserum raised against sodium dodecyl sulfate-disrupted bovine papillomavirus type 1 (BPV1) virions. To map the immunoreactive epitopes, a series of procaryotic expression plasmids was made which contained a nested set of 3' to 5' deletions in the HPV6b L1 open reading frame. The deleted plasmids expressed a set of carboxy to amino terminus truncated fusion proteins. Regions containing the immunoreactive epitopes were mapped by determining which of the deleted fusion proteins retained reactivity with sera in Western immunoblot assays. The coding sequence for a human antibody-reactive linear epitope mapped between HPV6b nucleotide coordinates 7045 and 7087, and the rabbit anti-BPV1-reactive epitope coding sequence mapped between coordinates 6377 and 6454. Synthetic peptides derived from the epitope mapping were reacted with sera in enzyme-linked immunosorbent assay. Human sera reacted with synthetic peptide QSQAITCQKPTPEKEKPDPYK (HPV6b L1 amino acids 417 through 437). Rabbit anti-BPV1 and rabbit antisera raised against HPV16 L1 recombinant proteins reacted with the synthetic peptide DGDMVDTGFGAMNFADLQTNKSDVPIDI (HPV6b L1 amino acids 193 through 220). Human sera which reacted with HPV6b L1 fusion proteins cross-reacted with an HPV11 L1 fusion protein but did not react with fusion proteins encoded by HPV1a, HPV16, or HPV18. Rabbit anti-BPV1 reacted with L1 fusion proteins encoded by all of these HPV types. In contrast to the type-common (rabbit anti-BPV1-reactive) epitope, the human antibody-reactive epitope appears to be relatively HPV type specific.     

The CEACAM1 transmembrane domain, but not the cytoplasmic domain, directs internalization of human pathogens via membrane microdomains

Several bacterial pathogens exploit carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to promote attachment and uptake into eukaryotic host cells. The widely expressed isoform CEACAM1 is involved in cell–cell adhesion, regulation of cell proliferation, insulin homeostasis, and neo-angiogenesis, processes that depend on the cytoplasmic domain of CEACAM1. By analysing the molecular requirements for CEACAM1-mediated internalization of bacteria, we surprisingly find that the CEACAM1 cytoplasmic domain is completely obsolete for bacterial uptake. Accordingly, CEACAM1-4L as well as a CEACAM1 mutant with a complete deletion of the cytoplasmic domain (CEACAM1 ΔCT) promote equivalent internalization of several human pathogens. CEACAM1-4L- and CEACAM1 ΔCT-mediated uptake proceeds in the presence of inhibitors of actin microfilament dynamics, which is in contrast to CEACAM3-mediated internalization. Bacteria-engaged CEACAM1-4L and CEACAM1 ΔCT, but not CEACAM3, localize to a gangliosid GM1- and GPI-anchored protein-containing portion of the plasma membrane. In addition, interference with cholesterol-rich membrane microdomains severely blocks bacterial uptake via CEACAM1-4L and CEACAM1 ΔCT, but not CEACAM3. Similar to GPI-anchored CEACAM6, both CEACAM1-4L as well as CEACAM1 ΔCT partition into a low-density, Triton-insoluble membrane fraction upon receptor clustering, whereas CEACAM3 is not detected in this fraction. Bacterial uptake by truncated CEACAM1 or chimeric CEACAM1/CEACAM3 molecules reveals that the transmembrane domain of CEACAM1 is responsible for its association with membrane microdomains. Together, these data argue for a functional role of lipid rafts in CEACAM1-mediated endocytosis that is promoted by the transmembrane domain of the receptor and that might be relevant for CEACAM1 function in physiologic settings.     

Apical expression of human full-length hCEACAM1-4L protein renders the Madin Darby Canine Kidney cells responsive to lipopolysaccharide leading to TLR4-dependent Erk1/2 and p38 MAPK signalling

CEACAM1 expressed by granulocytes and epithelial cells is recognized as a membrane-associated receptor by some Gram-negative pathogens. Here we report a previously unsuspected role of human CEACAM1-4L (hCEACAM1-4L) in polarized epithelial cells. We find that in contrast with non-transfected cells, Madin Darby Canine Kidney strain II (MDCK) engineered for the apical expression of the long cytoplasmic chain protein hCEACAM1-4L showed a serum-independent increase in the phosphorylation of the extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 mitogen-activated protein kinases (MAPKs) after treatment with lipopolysaccharide (LPS) of wild-type, diffusely adhering Afa/Dr Escherichia coli (Afa/Dr DAEC) strain IH11128. Aggregates of FITC-LPS bind the apical domain of MDCK-hCEACAM1-4L cells colocalizing with the apically expressed hCEACAM1-4L protein and do not bind MDCK-pCEP cells, and surface plasmon resonance analysis shows that LPS binds to the extracellular domain of the CEACAM1-4L protein. We showed that cell polarization and lipid rafts positively control the LPS-IH11128-induced phosphorylation of Erk1/2 in MDCK-hCEACAM1-4L cells. Structure-function analysis using mutated hCEACAM1-4L protein shows that the cytoplasmic domain of the protein is needed for LPS-induced MAPK signalling, and that phosphorylation of Tyr-residues is not increased in association with MAPK signalling. The hCEACAM1-4L-dependent Erk1/2 phosphorylation develops in the presence of lipid A and does not develop in the presence of penta-acylated LPS. Finally, small interfering RNA (siRNA) silencing of canine TLR4 abolishes the hCEACAM1-4L-dependent, LPS-induced phosphorylation of Erk1/2. Collectively, our results support the notion that the apically expressed, full-length hCEACAM1-4L protein functions as a novel LPS-conveying molecule at the mucosal surface of polarized epithelial cells for subsequent MD-2/TLR4 receptor-dependent MAPK Erk1/2 and p38 signalling.     

Intron retention generates a novel isoform of CEACAM6 that may act as an adhesion molecule in the ectoplasmic specialization structures between spermatids and sertoli cells in rat testis

By differential display technique followed by RT-PCR and DNA sequence analyses, we isolated carcinoembryonic antigen-related cell adhesion molecule 6 (Ceacam6) and its novel spliced variant Ceacam6-Long (Ceacam6-L) from rat testis. Ceacam6-L mRNA was generated by retention of 67 nucleotide-length third intron in Ceacam6 gene. Ceacam6-L is a member of an immunoglobulin superfamily and encodes a protein of 50 kDa with a signal sequence at the N-terminus, one immunoglobulin (Ig)-like domain, three IgCAM domains, a transmembrane region, and a short intracellular region. Expression analyses by RT-PCR and Northern blot showed that Ceacam6-L was exclusively expressed in rat testis and first detectable at 5 wk during postnatal development of testis. We performed immunoblot analyses and immunohistochemistry using the anti-CEACAM6-L antibody. Confocal laser scanning microscopy revealed that CEACAM6-L was not present at blood-testis barrier junctions between Sertoli cells but localized at the interface between Sertoli cells and germ cells, possibly to work as an adhesion molecule in the apical compartment of the seminiferous epithelium. At stages VII-VIII, at which all of the elongated spermatids migrated to the luminal surface of the seminiferous tubules, CEACAM6-L was found to locate at the concave side of elongated spermatid heads, following the curvature of their sickle-shaped nuclei, suggesting that CEACAM6-L might be involved in the anchoring of spermatids to Sertoli cells and spermiation. We concluded that CEACAM6-L might be a novel adhesion molecule constructing the apical ectoplasmic specialization in testis.