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Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a dendrite-expressed membrane glycoprotein of telencephalic neurons in the mammalian brain. By deletion of the cytoplasmic and membrane-spanning domains of ICAM-5, we observed that the membrane distribution of ICAM-5 was determined by the cytoplasmic portion. Therefore we have characterized the intracellular associations of ICAM-5 by using a bacterially expressed glutathione S-transferase (GST) fusion protein encompassing the cytoplasmic part of ICAM-5. One of the main proteins in the neuronal cell line Paju that bound to the ICAM-5 cytodomain was alpha-actinin. ICAM-5 expressed in transfected Paju cells was found in alpha-actinin immunoprecipitates, and ICAM-5 colocalized with alpha-actinin both in Paju cells and in dendritic filopodia and spines of primary hippocampal neurons. We were also able to coprecipitate alpha-actinin from rat brain homogenate. Binding to alpha-actinin appeared to be mediated mainly through the N-terminal region of the ICAM-5 cytodomain, as the ICAM-5(857-861) cytoplasmic peptide (KKGEY) mediated efficient binding to alpha-actinin. Surface plasmon resonance analysis showed that the turnover of the interaction was rapid. In a mutant cell line, Paju-ICAM-5-KK/AA, the distribution was altered, which implies the importance of the lysines in the interaction. Furthermore, we found that the ICAM-5/alpha-actinin interaction is involved in neuritic outgrowth and the ICAM-5(857-861) cytoplasmic peptide induced morphological changes in Paju-ICAM-5 cells. In summary, these results show that the interaction between ICAM-5 and alpha-actinin is mediated through binding of positively charged amino acids near the transmembrane domain of ICAM-5, and this interaction may play an important role in neuronal differentiation.

Amphoterin, a major form of HMG (high mobility group) 1 proteins, is highly expressed in immature and malignant cells. A role in cell motility is suggested by the ability of amphoterin to promote neurite extension through RAGE (receptor of advanced glycation end products), an immunoglobulin superfamily member that communicates with the GTPases Cdc42 and Rac. We show here that cell contact with the laminin matrix induces accumulation of both amphoterin mRNA and protein close to the plasma membrane, which is accompanied by extracellular export of amphoterin. A role for amphoterin in extracellular matrix-dependent cell regulation is further suggested by the finding that specific decrease of amphoterin mRNA and protein, using antisense oligonucleotides transfected into cells, inhibits cell migration to laminin in a transfilter assay whereas the oligonucleotides in the culture medium have no effect. Moreover, affinity-purified anti-amphoterin antibodies inhibit cell migration to laminin, supporting an extracellular role for the endogenous amphoterin in cell motility. The finding that amphoterin expression is more pronounced in cells with a motile phenotype as compared to cells of dense cultures, is consistent with the results of the cell migration assays. Our results strongly suggest that amphoterin is a key player in the migration of immature and transformed cells.