Implications for the domain arrangement of axonin-1 derived from the mapping of its NgCAM binding site.
The neuronal cell adhesion molecule axonin-1 is composed of six immunoglobulin and four fibronectin type III domains. Axonin-1 promotes neurite outgrowth, when presented as a substratum for neurons in vitro, via a neuronal receptor that has been identified as the neuron-glia cell adhesion molecule, NgCAM, based on the blocking effect of polyclonal antibodies directed to NgCAM. Here we report the identification of axonin-1 domains involved in NgCAM binding. NgCAM-conjugated microspheres were tested for binding to COS cells expressing domain deletion mutants of axonin-1. In addition, monoclonal antibodies directed to axonin-1 were assessed for their ability to block the axonin-1-NgCAM interaction, and their epitopes were mapped using the domain deletion mutants. The results suggest that the four amino-terminal immunoglobulin domains of axonin-1 form a domain conglomerate which is necessary and sufficient for NgCAM binding. Surprisingly, NgCAM binding to membrane-bound axonin-1 was increased strongly by deletion of the fifth or sixth immunoglobulin domains of axonin-1. Based on these results and on negative staining electron microscopy, we propose a horseshoe-shaped domain arrangement of axonin-1 that obscures the NgCAM binding site. Neurite outgrowth studies with truncated forms of axonin-1 show that axonin-1 is a neurite outgrowth-promoting substratum in the absence of the NgCAM binding site.
Rader
C
C
Institute of Biochemistry, University of Zurich, Switzerland.
Kunz
B
B
Lierheimer
R
R
Giger
R J
RJ
Berger
P
P
Tittmann
P
P
Gross
H
H
Sonderegger
P
P
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019062:Cell Adhesion Molecules, Neuron-Glia / Q000378:metabolism*; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002642:Chick Embryo; D058969:Contactin 2; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016501:Neurites; D009838:Oligodeoxyribonucleotides; D011485:Protein Binding; D017510:Protein Folding
D019062:Cell Adhesion Molecules, Neuron-Glia; D015816:Cell Adhesion Molecules, Neuronal; D058969:Contactin 2; D009838:Oligodeoxyribonucleotides