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.
Hunter
D D
DD
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Cashman
N
N
Morris-Valero
R
R
Bulock
J W
JW
Adams
S P
SP
Sanes
J R
JR
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