Structure-function analysis of Nel, a thrombospondin-1-like glycoprotein involved in neural development and functions.

Nel (neural epidermal growth factor (EGF)-like molecule) is a multimeric, multimodular extracellular glycoprotein with heparin-binding activity and structural similarities to thrombospondin-1. Nel is predominantly expressed in the nervous system and has been implicated in neuronal proliferation and differentiation, retinal axon guidance, synaptic functions, and spatial learning. The Nel protein contains an N-terminal thrombospondin-1 (TSP-N) domain, five cysteine-rich domains, and six EGF-like domains. However, little is known about the functions of specific domains of the Nel protein. In this study, we have performed structure-function analysis of Nel, by using a series of expression constructs for different regions of the Nel protein. Our studies demonstrate that the TSP-N domain is responsible for homo-multimer formation of Nel and its heparin-binding activity. In vivo, Nel and related Nell1 are expressed in several regions of the mouse central nervous system with partly overlapping patterns. When they are expressed in the same cells in vitro, Nel and Nell1 can form hetero-multimers through the TSP-N domain, but they do not hetero-oligomerize with thrombospondin-1. Whereas both the TSP-N domain and cysteine-rich domains can bind to retinal axons in vivo, only the latter causes growth cone collapse in cultured retinal axons, suggesting that cysteine-rich domains interact with and activate an inhibitory axon guidance receptor. These results suggest that Nel interacts with a range of molecules through its different domains and exerts distinct functions.

In vitro guidance of retinal axons by a tectal lamina-specific glycoprotein Nel.

Nel is a glycoprotein containing five chordin-like and six epidermal growth factor-like domains and is strongly expressed in the nervous system. In this study, we have examined expression patterns and in vitro functions of Nel in the chicken retinotectal system. We have found that in the developing tectum, expression of Nel is localized in specific laminae that retinal axons normally do not enter, including the border between the retinorecipient and non-retinorecipient laminae. Nel-binding activity is detected on retinal axons both in vivo and in vitro, suggesting that retinal axons express a receptor for Nel. In vitro, Nel inhibits retinal axon outgrowth and induces growth cone collapse and axon retraction. These results indicate that Nel acts as an inhibitory guidance cue for retinal axons, and suggest its roles in the establishment of the lamina-specificity in the retinotectal projection.

Biphasic functions of the kinase-defective Ephb6 receptor in cell adhesion and migration.

EphB6 is a unique member in the Eph family of receptor tyrosine kinases in that its kinase domain contains several alterations in conserved amino acids and is catalytically inactive. Although EphB6 is expressed both in a variety of embryonic and adult tissues, biological functions of this receptor are largely unknown. In the present study, we examined the function of EphB6 in cell adhesion and migration. We demonstrated that EphB6 exerted biphasic effects in response to different concentrations of the ephrin-B2 ligand; EphB6 promoted cell adhesion and migration when stimulated with low concentrations of ephrin-B2, whereas it induced repulsion and inhibited migration upon stimulation with high concentrations of ephrin-B2. A truncated EphB6 receptor lacking the cytoplasmic domain showed monophasic-positive effects on cell adhesion and migration, indicating that the cytoplasmic domain is essential for the negative effects. EphB6 is constitutively associated with the Src family kinase Fyn. High concentrations of ephrin-B2 induced tyrosine phosphorylation of EphB6 through an Src family kinase activity. These results indicate that EphB6 can both positively and negatively regulate cell adhesion and migration, and suggest that tyrosine phosphorylation of the receptor by an Src family kinase acts as the molecular switch for the functional transition.