containing promotes growth.

After denervation of muscle, motor axons reinnervate original synaptic sites. A recombinant fragment of the synapse specific laminin 132 chain (s-laminin) was reported to inhibit motor axon growth. Consequently, a specific sequence (leucine-arginineglutamate, LR E) of the laminin 132 chain was proposed to act as a stop signal and to mediate specific reinnervation at the neuromuscular junction (Porter, B.E., J. Weis, and J.R. Sanes. 1995. Neuron. 14:549-559). We demonstrate here that native chick laminin-4, which contains the 132 chain and is present in the synaptic basement membrane, does not inhibit but rather promotes motor axon growth. In native heterotrimeric laminin, the L R E sequence of the [32 chain is found in a triple coiled-coil region that is formed by all three subunits. We show here that the effect of L R E depends on the structural context. Whereas a recombinant randomly coiled L R E peptide indeed inhibited outgrowth by chick motoneurons, a small recombinant triple coiledcoil protein containing this sequence did not. EGENERATING motor axons preferentially reinnervate original synaptic sites that cover only ~0.1% of the muscle fiber surface (Letinsky et al., 1976). Factors responsible for this selectivity are likely to be components of the synaptic basement membrane, because, if the muscle fibers have been killed as well, motor axons still grow back to former synaptic sites on basement membrane "ghosts" (Sanes et al., 1978). The laminin 132 chain (s-laminin) which is enriched at the synaptic basement membrane of the neuromuscular junction was claimed to be involved in this targeting (Hunter et al., 1989b; for nomenclature see Burgeson et al., 1994). Laminins are glycoproteins of ~600-900 kD molecular mass which are abundant in basement membranes (Timpl and Brown, 1994). Each laminin molecule is composed of one a, one 13, and one ~/chain which have been classified according to their sequence homology and domain organization (Engel et al., 1994). Several members encoded by different genes have been cloned for any of these three types of subunits (see Ryan et al., 1994; Timpl and Brown, 1994; Iivanainen et al., 1995; Miner et al., 1995), and at least seven heterotrimeric laminin isoforms have been isolated so far (Timpl and Brown, 1994). The different subunits, and consequently, the resulting laminin isoforms, have distinct tissue Address all correspondence to Matthias Chiquet, M.E. Mfiller-lnstitut far Biomechanik, Universit~t Bern, Murtenstrasse 35, Postfach 30, CH-3010 Bern, Switzerland. Tel. : 41 31 6324953. Fax: 41 31 6324999. The present address of R. Brandenberger is Howard Hughes Medical Institute, University of California School of Medicine, Parnassus and Third Avenues, San Francisco, CA 94143-0724. distributions and their expression is developmentally regulated (e.g., Ekblom et al., 1990; Engvall et al., 1990; Sanes et al., 1990; Noakes et al., 1995b). For example, laminin with a2 and ~/1 chains are found all over the muscle basement membrane surface whereas those with 131 and 132 chains are distributed in a mutually exclusive manner. The 131 subunit is found only extrasynaptically whereas the 132 chain is specific for the basement membrane of the synaptic cleft and of the myotendinous junction (Engvall et al., 1990; Sanes et al., 1990). The 132 chain has originally been discovered by the use of antibodies that specifically recognize synaptic basement membranes (Hunter et al., 1989b). Subsequently, it was shown that a laminin 132 chain-derived peptide, Leu-Arg-Glu (LRE) 1, is selectively adhesive for motoneurons while inhibiting their neurite growth (Hunter et al., 1989a; Porter et al., 1995). Moreover, recombinant laminin 132 chain was not adhesive for sensory neurons or PC12 cells, nor did it inhibit their neurite outgrowth (Hunter et al., 1989a; Porter et al., 1995). These studies led to the hypothesis that the 132 chain could act as stop signal specific for growing motor axons, and that this might at least in part explain the preferential reinnervation of original muscle endplates. The subsequent differentiation into a nerve terminal might be a consequence of growth cone arrest, or it might also be mediated by LRE (Porter et al., 1995). Laminin 132 chain deficient mice show aberrant differentiation of motor nerve terminals 1. Abbreviat ions used in this paper: BDNF, brain derived neurotrophic factor; CD, circular dichroism; CG, ciliary ganglion; CMP, cartilage matrix protein; LRE, Leu-Arg-Glu. © The Rockefeller University Press, 0021-9525/96/12/1583/10 $2.00 The Journal of Cell Biology, Volume 135, Number 6, Part 1, December 1996 1583-1592 1583 (Noakes et al., 1995a), implying that this subunit is indeed involved in their differentiation. However, the in vitro data concerning the adhesivity and inhibitory action of the laminin 132 chain have been generated by the use of recombinant fragments and synthetic peptides of a single laminin chain. The LRE sequence lies within the triple-stranded coiled-coil region of the assembled, trimeric laminin molecule. It is therefore important to test the activity of native laminin containing the 132 chain. The laminin isoform present at the neuromuscular junction is likely to be laminin-4, composed of the ~2, the -y1, and the 132 chain (Sanes et al., 1990). We report here that native chick laminin-4 does not inhibit but rather promotes neurite outgrowth by motoneurons. Moreover, this study investigates the influence of the structural context of the LRE sequence on its inhibitory activity. Materials and Methods