The amyloid precursor protein (APP) plays a central role in Alzheimer's disease, but its physiological function and that of its mammalian paralogs, the amyloid precursor-like proteins 1 and 2 (APLPs), is still poorly understood. APP has been proposed to form dimers, a process that could promote cell adhesion via trans-dimerization. We investigated the dimerization and cell adhesion properties of APP/APLPs and provide evidence that all three paralogs are capable of forming homo-and heterocomplexes. Moreover, we show that trans-interaction of APP family proteins promotes cell-cell adhesion in a homo-and heterotypic fashion and that endogenous APLP2 is required for cell-cell adhesion in mouse embryonic fibroblasts. We further demonstrate interaction of all the three APP family members in mouse brain, genetic interdependence, and molecular interaction of APP and APLPs in synaptically enriched membrane compartments. Together, our results provide evidence that homo-and heterocomplexes of APP/APLPs promote trans-cellular adhesion in vivo.

The formation of postsynaptic GABAA and glycine receptor clusters requires the receptor-associated peripheral membrane protein gephyrin. Here we describe two splice variants of a novel gephyrin-binding protein, termed collybistin I and II, which belong to the family of dbl-like GDP/GTP exchange factors (GEFs). Co-expression of collybistin II with gephyrin induced the formation of submembrane gephyrin aggregates that accumulate hetero-oligomeric glycine receptors. Our data suggest that collybistin II regulates the membrane deposition of gephyrin by activating a GTPase of the Rho/Rac family. Therefore, this protein may be an important determinant of inhibitory postsynaptic membrane formation and plasticity.

The sequential enzymatic actions of ␤-APP cleaving enzyme 1 (BACE1), presenilins (PS), and other proteins of the ␥-secretase complex liberate ␤-amyloid (A␤) peptides from larger integral membrane proteins, termed ␤-amyloid precursor proteins (APPs). Relatively little is known about the normal function(s) of APP or the neuronal compartment(s) in which APP undergoes proteolytic processing. Recent studies have been interpreted as consistent with the idea that APP serves as a kinesin-1 cargo receptor and that PS and BACE1 are associated with the APP-resident membranous cargos that undergo rapid axonal transport. In this report, derived from a collaboration among several independent laboratories, we examined the potential associations of APP and kinesin-1 using glutathione S-transferase pull-down and coimmunoprecipitation assays. In addition, we assessed the trafficking of membrane proteins in the sciatic nerves of transgenic mice with heterozygous or homozygous deletions of APP. In contrast to previous reports, we were unable to find evidence for direct interactions between APP and kinesin-1. Furthermore, the transport of kinesin-1 and tyrosine kinase receptors, previously reported to require APP, was unchanged in axons of APP-deficient mice. Finally, we show that two components of the APP proteolytic machinery, i.e., PS1 and BACE1, are not cotransported with APP in the sciatic nerves of mice. These findings suggest that the hypothesis that APP serves as a kinesin-1 receptor and that the proteolytic processing machinery responsible for generating A␤ is transported in the same vesicular compartment in axons of peripheral nerves requires revision.

The clustering of glycine receptors and major subtypes of GABA(A) receptors at inhibitory synapses is mediated by the tubulin-binding protein gephyrin. In an attempt to identify additional components of inhibitory postsynaptic specializations, we performed a yeast two-hybrid screen using gephyrin as bait. Multiple positive clones encoded either the dynein light chain-1 (Dlc-1), also known as dynein LC8 and protein inhibitor of neuronal nitric oxide synthase, or its homolog Dlc-2. Dlc-1 protein bound efficiently to gephyrin in in vitro binding assays and colocalized with gephyrin during coexpression in HEK293 cells. The binding site for Dlc was mapped to a fragment of 63 amino acids within the central linker domain of gephyrin. In hippocampal neurons, endogenous Dlc protein was enriched at synaptic sites identified by synaptophysin and gephyrin immunostaining. Immunoelectron microscopy in spinal cord sections revealed Dlc immunoreactivity at the edges of postsynaptic differentiations, in close contact with cytoskeletal structures and at the periphery of the Golgi apparatus. Because Dlc-1 and Dlc-2 have been described as stoichiometric components of cytoplasmic dynein and myosin-Va complexes, our results suggest that motor proteins are involved in the subcellular localization of gephyrin.

Accumulating evidence suggests that the copper-binding amyloid precursor protein (APP) has an essential synaptic function. APP synaptogenic function depends on trans-directed dimerization of the extracellular E1 domain encompassing a growth factor-like domain (GFLD) and a copper-binding domain (CuBD). Here we report the 1.75 Å crystal structure of the GFLD in complex with a copper ion bound with high affinity to an extended hairpin loop at the dimerization interface. In coimmunoprecipitation assays copper binding promotes APP interaction, whereas mutations in the copper-binding sites of either the GFLD or CuBD result in a drastic reduction in APP cisorientated dimerization. We show that copper is essential and sufficient to induce trans-directed dimerization of purified APP. Furthermore, a mixed culture assay of primary neurons with HEK293 cells expressing different APP mutants revealed that APP potently promotes synaptogenesis depending on copper binding to the GFLD. Together, these findings demonstrate that copper binding to the GFLD of APP is required for APP cis-/trans-directed dimerization and APP synaptogenic function. Thus, neuronal activity or diseaseassociated changes in copper homeostasis likely go along with altered APP synaptic function.