An LRE (leucine-arginine-glutamate)-dependent mechanism for adhesion of neurons to S-laminin.
S-laminin is a homolog of laminin that is concentrated in the synaptic cleft of the neuromuscular junction. We previously showed that the tripeptide LRE is a crucial determinant for binding of ciliary motoneurons to recombinant s-laminin. Here, we describe a neuroblastoma-spinal neuron hybrid cell line, NSC-34, that binds to an LRE-containing s-laminin fragment and to a synthetic LRE-protein conjugate. NSC-34 cells exhibit several properties of motoneurons; other cell lines tested were not motoneuron-like and did not display LRE-dependent adhesion. We therefore used NSC-34 cells to characterize the LRE-dependent adhesion mechanism. Inhibition studies with a series of 20 tripeptide LRE analogs showed that the cells exhibit a high degree of selectivity for LRE, and suggested that ligand binding requires a combination of electrostatic and hydrophobic interactions. The effects of cations on LRE-dependent adhesion are unlike those of previously described adhesion molecules including the integrins, a family of receptors for extracellular matrix proteins, including laminin. Specifically, adhesion to LRE does not require divalent cations and is inhibited by Ca2+ (but not by Mg2+) in the physiological range. In contrast, adhesion of NSC-34 cells to laminin is LRE- and Ca2+ independent but Mg2+ dependent, and appears to be mediated by integrins. Additionally, experiments using mixed substrates demonstrated that LRE-protein conjugates inhibit neurite outgrowth promoted by laminin. Finally, we show that, under ionic conditions that minimize integrin-dependent adhesion, NSC-34 cells bind to s-laminin-rich basal laminae in tissue sections in an LRE-dependent manner. Together, these results suggest that LRE comprises a motoneuron-selective adhesion site that is accessible in native basal laminae and that acts to inhibit neurite outgrowth.
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110.
D D
DD
Hunter
N
N
Cashman
R
R
Morris-Valero
J W
JW
Bulock
S P
SP
Adams
J R
JR
Sanes
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000502:physiology*; D001485:Basement Membrane / Q000502:physiology; D002448:Cell Adhesion; D002460:Cell Line; D005971:Glutamates / Q000502:physiology*; D018698:Glutamic Acid; D007477:Ions; D007797:Laminin / Q000502:physiology*; D007930:Leucine / Q000502:physiology*; D008969:Molecular Sequence Data; D009046:Motor Neurons / Q000502:physiology; D016501:Neurites / Q000502:physiology; D009474:Neurons / Q000502:physiology*
D005971:Glutamates; D007477:Ions; D007797:Laminin; C058783:laminin beta2; D018698:Glutamic Acid; D001120:Arginine; D007930:Leucine