Neurexins are a large family of proteins that act as neuronal cell-surface receptors. The function and localization of the various neurexins, however, have not yet been clarified. Beta-neurexins are candidate receptors for neuroligin-1, a postsynaptic membrane protein that can trigger synapse formation at axon contacts. Here we report that neurexins are concentrated at synapses and that purified neuroligin is sufficient to cluster neurexin and to induce presynaptic differentiation. Oligomerization of neuroligin is required for its function, and we find that beta-neurexin clustering is sufficient to trigger the recruitment of synaptic vesicles through interactions that require the cytoplasmic domain of neurexin. We propose a two-step model in which postsynaptic neuroligin multimers initially cluster axonal neurexins. In response to this clustering, neurexins nucleate the assembly of a cytoplasmic scaffold to which the exocytotic apparatus is recruited.

We report the full cDNA sequence encoding the human homologue of murine PA2.26 (T1␣-2, podoplanin), a small mucin-type transmembrane glycoprotein originally identified as a cell-surface antigen induced in keratinocytes during mouse skin carcinogenesis. The human PA2.26 gene is expressed as 2 transcripts of 0.9 and 2.7 kb in several normal tissues, such as the placenta, skeletal muscle, heart and lung. Using a specific polyclonal antibody raised against a synthetic peptide of the protein ectodomain, PA2.26 was immunohistochemically detected in about 25% (15/61) of human early oral squamous cell carcinomas. PA2.26 distribution in the tumours was heterogeneous and often restricted to the invasive front. Double immunofluorescence and confocal microscopy analysis showed that PA2.26 colocalized with the membrane cytoskeleton linker ezrin at the surface of tumour cells and that its presence in vivo was associated with downregulation of membrane E-cadherin protein expression. Ectopic expression of human PA2.26 in HeLa carcinoma cells and immortalized HaCaT keratinocytes promoted a redistribution of ezrin to the cell edges, the formation of cell-surface protrusions and reduced Ca 2؉ -dependent cell-cell adhesiveness. These results point to PA2.26 as a novel biomarker for oral squamous cell carcinomas that might be involved in migration/invasion.Key words: mucin; PA2.26; ezrin; E-cadherin; microvilli; OSCC Squamous cell carcinomas (SCCs) of the oral cavity, pharynx and larynx remain a significant public health problem. They represent 2-3% of all malignancies, and their incidence, particularly that of oral SCCs (OSCCs), is increasing in Western countries. 1 In spite of improved therapeutic procedures, the prognosis of OSCC patients remains poor and considerably lower than that of other neoplasias. 2 This fact can be attributed to several factors: failure to respond to available chemotherapy, late presentation of the lesions and lack of suitable markers for early detection and prognosis. 3,4 Hence, the finding of novel tumour markers, particularly those associated with tumour cell invasion and spreading, can help provide a more accurate evaluation of prognosis and a more efficient management of the disease.PA2.26 antigen was identified in our laboratory as a cell-surface protein induced in murine epidermal keratinocytes and dermal fibroblast-like cells during wound healing and chemical carcinogenesis. 5 Sequence analysis of the isolated cDNA and biochemical characterization of the protein revealed that murine PA2.26 is a small mucin-like transmembrane glycoprotein of about 45 kDa, 6 highly homologous to the rat alveolar type I cell marker T1␣ and the podocyte-associated glycoprotein podoplanin. 7,8 Murine PA2.26 nucleotide sequence is almost identical to that of OTS-8 and gp38, markers of the osteoblastic cell lineage and stromal cells in peripheral lymphoid tissues, respectively, 9,10 and completely matches the nucleotide sequence of RANDAM-2, a recently discovered membrane glycoprotein expressed in neuronal cells during m...

The formation of neuronal circuits during development involves a combination of synapse stabilization and elimination events. Synaptic adhesion molecules are thought to play an important role in synaptogenesis, and several trans-synaptic adhesion systems that promote the formation and maturation of synapses have been identified. The neuroligin-neurexin complex is a heterophilic adhesion system that promotes assembly and maturation of synapses through bidirectional signaling. In this protein complex, postsynaptic neuroligins are thought to interact trans-synaptically with presynaptic neurexins. However, the subcellular localization of neurexins has not been determined. Using immunoelectron microscopy, we found that endogenous neurexins and epitope-tagged neurexin-1␤ are localized to axons and presynaptic terminals in vivo. Unexpectedly, neurexins are also abundant in the postsynaptic density. cis-expression of neurexin-1␤ with neuroligin-1 inhibits trans-binding to recombinant neurexins, blocks the synaptogenic activity of neuroligin-1, and reduces the density of presynaptic terminals in cultured hippocampal neurons. Our results demonstrate that the function of neurexin proteins is more diverse than previously anticipated and suggest that postsynaptic cis-interactions might provide a novel mechanism for silencing the activity of a synaptic adhesion complex.

The monoclonal antibody PA2.26, produced against mouse epidermal keratinocytes transformed with 7,12-dimethylbenz[a]anthracene (DMBA), recognizes a 43- to 47-kDa cell-surface protein that was absent from non-tumorigenic epidermal keratinocytes but present in transformed epidermal cell lines as well as cultured normal fibroblasts. In vivo, the antigen was absent from normal epidermis but induced in basal-like epidermal keratinocytes and dermal fibroblasts during tissue regeneration after wounding and treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The PA2.26 protein was also expressed in basal-like cells of differentiated papillomas and carcinomas generated in mice treated with DMBA and TPA. In addition, the antigen was abundantly synthesized by stromal cells of the tumors. These results suggest that PA2.26 antigen is involved in reactive processes during skin remodeling and carcinogenesis.

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Autism spectrum disorders (ASDs) comprise a group of clinical phenotypes characterized by repetitive behavior and social and communication deficits. Autism is generally viewed as a neurodevelopmental disorder where insults during embryonic or early postnatal periods result in aberrant wiring and function of neuronal circuits. Neurexins are synaptic proteins associated with autism. Here, we generated transgenic βNrx1ΔC mice in which neurexin function is selectively impaired during late postnatal stages. Whole-cell recordings in cortical neurons show an impairment of glutamatergic synaptic transmission in the βNrx1ΔC mice. Importantly, mutant mice exhibit autism-related symptoms, such as increased self-grooming, deficits in social interactions, and altered interaction for nonsocial olfactory cues. The autistic-like phenotype of βNrx1ΔC mice can be reversed after removing the mutant protein in aged animals. The defects resulting from disruption of neurexin function after the completion of embryonic and early postnatal development suggest that functional impairment of mature circuits can trigger autism-related phenotypes.

SUMMARY: PA2.26 antigen is a small mucin-type transmembrane glycoprotein induced in mouse epidermal keratinocytes during carcinogenesis. It is located at plasma membrane projections, such as microvilli and ruffles, where it interacts with the actin cytoskeleton. Previous studies revealed that ectopic expression of PA2.26 in epidermal MCA3D keratinocytes induces cell surface extensions and increased motility. Here, we show that PA2.26-expressing MCA3D (3D2.26) cell transfectants undergo a phenotypic conversion linked to the acquisition of malignant characteristics. The 3D2.26 cells down-regulate basal keratin K14 and up-regulate vimentin and keratin K8 expression. Immunofluorescence analysis in 3D2.26 cell cultures showed loss of cortical actin filaments and destabilization of adherens junctions mediated by E-and P-cadherin, although both cadherin mRNAs were expressed in the transfectants. When the cadherin protein levels were analyzed in Western blots, no P-cadherin protein or smaller polypeptide E-cadherin forms were detected, suggesting that E-and P-cadherin synthesized in 3D2.26 cells was unstable and proteolytically degraded. Transplantation of 3D2.26 cells into athymic nude mice induced tumors, whereas MCA3D cells and control (3DN) transfectants were not tumorigenic after 72 days postinjection. The phenotype of the tumors was undifferentiated, with mixed regions exhibiting a glandular differentiation pattern in which the presence of numerous surface microvilli was observed at the ultrastructural level. Interestingly, PA2.26 antigen was highly expressed in these microvillous cell surfaces. Tumor cells were vimentin-and K8-positive and showed an aberrant pattern of E-cadherin protein expression in which large cytoplasmic aggregates were found close to the nucleus. Infiltration of tumor cells into lymphatic vessels and the presence of frequent regional lymph node metastases were also observed in the tumors. These results indicate that expression of PA2.26 antigen in premalignant keratinocytes induces a fully transformed and metastatic phenotype, and they suggest an involvement of PA2.26 in malignant progression. (Lab Invest 2000, 80:1749 -1759. P A2.26 antigen was identified as a cell-surface protein of about 45 kDa induced in basal-like keratinocytes and dermal fibroblasts during mouse epidermal carcinogenesis and skin remodeling processes. PA2.26 is absent from cultured nontumorigenic keratinocytes, but it is expressed in carcinoma cell lines and cultured active fibroblasts (Gandarillas et al, 1997). The molecular and biochemical characterization of PA2.26 revealed that it is a small mucin-like transmembrane sialoglycoprotein of 172 amino acids, located at microvilli, filopodia, lamellipodia, and ruffles. In normal tissues, PA2.26 is expressed in different type of cells: in the epithelia of choroid plexuses, ependyma, glomeruli, and alveoli, in all mesothelia, and in endothelia of lymphatic vessels, always concentrated at the apical plasma membranes and microvillous projections (Scholl et al, 1999).OTS-8 an...

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