ABSTRACT
The amyloid precursor protein (APP) plays a central role in Alzheimer's disease, but its actions in normal development are not well understood. Here, a tagged APP ectodomain was used to identify extracellular binding partners in developing chick brain. Prominent binding sites were seen in the olfactory bulb and on retinal axons growing into the optic tectum. Co-precipitation from these tissues and tandem mass spectrometry led to the identification of two associated proteins: contactin 4 and NgCAM. In vitro binding studies revealed direct interactions among multiple members of the APP and contactin protein families. Levels of the APP processing fragment, CTFalpha, were modulated by both contactin 4 and NgCAM. In the developing retinotectal system, APP, contactin 4 and NgCAM are expressed in the retina and tectum in suitable locations to interact. Functional assays revealed regulatory effects of both APP and contactin 4 on NgCAM-dependent growth of cultured retinal axons, demonstrating specific functional interactions among these proteins. These studies identify novel binding and functional interactions among proteins of the APP, contactin and L1CAM families, with general implications for mechanisms of APP action in neural development and disease.
Subject(s)
Amyloid beta-Protein Precursor/metabolism , Cell Adhesion Molecules, Neuron-Glia/metabolism , Cell Adhesion Molecules, Neuronal/metabolism , Retina/embryology , Retina/metabolism , Superior Colliculi/embryology , Superior Colliculi/metabolism , Amyloid beta-Protein Precursor/chemistry , Amyloid beta-Protein Precursor/genetics , Animals , Axons/metabolism , Base Sequence , Binding Sites , Cell Adhesion Molecules, Neuron-Glia/genetics , Cell Adhesion Molecules, Neuronal/antagonists & inhibitors , Cell Adhesion Molecules, Neuronal/genetics , Chick Embryo , Contactins , DNA/genetics , Gene Expression Regulation, Developmental , Humans , Mice , Neural Cell Adhesion Molecule L1/genetics , Neural Cell Adhesion Molecule L1/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Binding , Protein Structure, Tertiary , RNA Interference , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Retinal Ganglion Cells/metabolismABSTRACT
Some endocytosis receptors related to the low-density lipoprotein receptor, including low-density lipoprotein receptor-related protein-1A, very-low-density lipoprotein receptor, and sorting protein-related receptor, bind protease-inhibitor complexes, including urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and the uPA-PAI-1 complex. The unique capacity of these receptors for high-affinity binding of many structurally unrelated ligands renders mapping of receptor-binding surfaces of serpin and serine protease ligands a special challenge. We have mapped the receptor-binding area of the uPA-PAI-1 complex by site-directed mutagenesis. Substitution of a cluster of basic residues near the 37-loop and 60-loop of uPA reduced the receptor-binding affinity of the uPA-PAI-1 complex approximately twofold. Deletion of the N-terminal growth factor domain of uPA reduced the affinity 2-4-fold, depending on the receptor, and deletion of both the growth factor domain and the kringle reduced the affinity sevenfold. The binding affinity of the uPA-PAI-1 complex to the receptors was greatly reduced by substitution of basic and hydrophobic residues in alpha-helix D and alpha-helix E of PAI-1. The localization of the implicated residues in the 3D structures of uPA and PAI-1 shows that they form a continuous receptor-binding area spanning the serpin as well as the A-chain and the serine protease domain of uPA. Our results suggest that the 10-100-fold higher affinity of the uPA-PAI-1 complex compared with the free components depends on the bonus effect of bringing the binding areas on uPA and PAI-1 together on the same binding entity.
