Abstract. Wild-type and mutant chicken integrin/31 subunit (/31o) cDNAs were expressed in NIH 3T3 cells and assayed for localization in focal adhesions of cells plated on fibronectin substrates. Focal adhesion localization in stable transfected cells was assayed by indirect immunofluorescent staining with chicken-specific anti-/3L: antibodies. Mutant/31c integrins containing internal deletions of 13 amino acids adjacent to the membrane, A759-771, and 20 centrally located amino acids, A771-790, localized in focal adhesions demonstrating that sequences required for direction to focal adhesion structures were not limited to one region of the cytoplasmic domain. Point mutations revealed three clusters of amino acids which contribute to localization in focal adhesions. These three clusters or signals are: cyto-1 (764-774), cyto-2 (785-788), and cyto-3 (79%800). The l 1-residue cyto-1 signal is only found on integrin/3 subunit sequences, except/34. Four residues within this region, 13764, F768, F771, and E774, could not be altered without reducing focal adhesion staining intensities, and likely form a signal that occupies one side of an tx helix. Mutations involving two cyto-1 residues, K770 and F771, also appeared to affect heterodimer affinity and specificity. Cyto-2 (785-788,), NPIY, is an NPXY signal that forms a tight turn motif. Cyto-2 provides a structural conforrnation, which when perturbed by proline removal or addition, inhibits integrin localization in focal adhesions. Cyto-3 (79%800), NPKY, resembles cyto-2, however, the nonconserved proline residue can be replaced without alteration of the localization phenotype. Cyto-3, therefore, constitutes a unique integrin signal, NXXY. Both serine and tyrosine residues at positions 790 and 788, respectively, which have been implicated in integrin phosphorylation/regulation, were conservatively replaced without detectable effect on focal adhesion localization. However, acidic replacements for these amino acids reduced focal adhesion staining intensities, suggesting that phosphorylation at these sites may negatively regulate integrin function.T H~ integrin superfamily of heterodimeric cell surface receptors associates with extracellular matrix (ECM) I proteins and with cytoskeletal-associated proteins such as talin and c~-actinin (Burridge et al., 1988;Albeda and Buck, 1990). There are at least four integrin families, each defined by a different common/5 subunit, and at least four other/5 subunits that associate with only one or. At least 12 ot subunits have been identified which associate with one or more of the eight/5 subunits. The ot subunits generally confer ECM substrate specificity (Hemler, 1990; A1-beda and Buck, 1990;Springer, 1990), although otv, which associates with several different 13 subunlts, does not follow this rule (Smith et al., 1990;Bodary and McLean, 1990;Vogel et al., 1990).Both ot and/5 integrin subunits have large extracellular, single membrane-spanning, and small cytoplasmic domains. The avian/51 subunit (/31o) is 803 amino acids long Yokichi...

Abstract. Chicken integrin B1 eDNA and its sitedirected mutants were cloned into a mammalian expression vector and introduced into mouse NIH 3T3 cells. Stable transfectants expressing the chicken/31 subunit or its site-directed mutants were identified by immunostaining with antibodies specific for the chicken integrin/31 subunit. The chicken/~1 proteins were expressed predominately in the endoplasmic reticulum of transfectants and to a lesser degree in the plasma membrane. Immunoblots and immunoprecipitations, using anti-chicken integrin antibodies, revealed three different sizes of the chicken subunit (90, 95, and 120 kD) and a mouse 140-kD cz subunit. Immunoprecipitations of the cell surface receptors showed only two peptides, an 120-kD/31 and an 140-kD ot subunit. Antibodies perturbing mouse and chicken integrin-specific cell adhesions were used to demonstrate that the chimeric receptors functioned in adhesion to both laminin and fibronectin. Immunofluorescent staining with antibodies specific for either the chicken or mouse receptors showed that both the wild type and the chimeric receptors localized in focal contacts. Several mutations in the cytoplasmic domain were synthesized and used in the transfection experiments. In one mutant the tyrosine (Tyr 788) in the consensus sequence for phosphorylation was replaced by a phenylalanine. In another the lysine (Lys 757) at the end of the membrane spanning region was replaced by a leucine. Both of these mutants formed dimers with mouse ~ subunits, participated in adhesion, localized in focal contacts, and displayed biological properties indistinguishable from the wild-type transfection. In contrast, mutants containing deletions greater than 5-15 amino acids nearest the carboxyl end in the cytoplasmic domain neither promoted adhesion nor localized in focal contacts. They did, however, form heterodimers that were expressed on the cell surface.

Neural activity results in long term changes that underlie synaptic plasticity. To examine the molecular basis of activity-dependent plasticity, we have used differential cloning techniques to identify genes that are rapidly induced in brain neurons by synaptic activity. Here, we identify a novel cadherin molecule Arcadlin (activity-regulated cadherin-like protein). arcadlin mRNA is rapidly and transiently induced in hippocampal granule cells by seizures and by N-methyl-D-aspartatedependent synaptic activity in long term potentiation. The extracellular domain of Arcadlin is most homologous to protocadherin-8; however, the cytoplasmic region is distinct from that of any cadherin family member. Arcadlin protein is expressed at the synapses and shows a homophilic binding activity in a Ca 2؉ -dependent manner. Furthermore, application of Arcadlin antibody reduces excitatory postsynaptic potential amplitude and blocks long term potentiation in hippocampal slices. Its close homology with cadherins, its rapid inducibility by neural activity, and its involvement in synaptic transmission suggest that Arcadlin may play an important role in activity-induced synaptic reorganization underlying long term memory.Glutamate receptor stimulation leads to a rapid Ca 2ϩ influx into neurons with associated protein phosphorylation events that underlie short term memory. In contrast, long term memory can be distinguished from short term memory in that it requires new mRNA and protein synthesis (1). To analyze components of the gene expression program underlying long term memory in the vertebrate brain, we and others have employed differential cloning techniques to identify mRNAs that are rapidly induced by excitatory activity. In addition to transcription factors, this approach has identified a number of immediate early genes that encode enzymes that may directly modify cellular function, including tissue-plasminogen activator, cyclooxygenase-2, a novel small molecular weight G-protein, and a cytoskeleton-associated protein (2-6). These proteins presumably interact with neuronal proteins and indirectly affect long term changes in connections and the efficacy thereof.LTP 1 provides a widely adopted mammalian model for activitydependent changes in synaptic efficacy. The mechanisms contributing to long term changes in synaptic transmission are still contentious. Among many possibilities, one of the hypotheses that has been proposed is that neural activity could lead to modifications in synaptic structure and eventually changes in synaptic connectivity. In support of this idea, numerous morphological studies have provided evidence that neural activity such as kindling or electrical stimulation induces modifications in dendritic arborization, spine densities, or synaptic morphology (7-10).Adhesion molecules are known to be involved in many aspects of cell-cell interactions, including cell migration, axonal growth, pathfinding, sprouting, and regeneration (11, 12). Recent reports have demonstrated that some adhesion molecules are expressed ...

A pathological hallmark of Alzheimer's disease is the deposition of amyloid fibrils in the brain. The principal component of amyloid fibrils is beta/A4 amyloid protein, which can be generated by the aberrant processing of a large membrane-bound glycoprotein, the beta/A4 amyloid protein precursor (APP)3. To test whether overexpression of APP generates abnormally processed derivatives that affect the viability of neurons, we stably transfected full-length human APP complementary DNA into murine embryonal carcinoma P19 cells. These cells differentiate into post-mitotic neurons and astrocytes after exposure to retinoic acid. When differentiation of the APP cDNA-transfected P19 cells was induced, all neurons showed severe degenerative changes and disappeared within a few days. The degenerating neurons contained large amounts of APP derivatives that were truncated at the amino terminus and encompassed the entire beta/A4 domain. These results suggest that post-mitotic neurons are vulnerable to overexpressed APP, which undergoes aberrant processing to generate potentially amyloidogenic fragments.