We report the identification of ligands for Tyro 3 (alternatively called Sky, rse, brt, or tif) and Axl (alternatively, Ark or UFO), members of a previously orphan family of receptor-like tyrosine kinases. These ligands correspond to protein S, a protease regulator that is a potent anticoagulant, and Gas6, a protein related to protein S but lacking any known function. Our results are reminiscent of recent findings that the procoagulant thrombin, a protease that drives clot formation by cleaving fibrinogen to form fibrin, also binds and activates intracellular signaling via a G protein-coupled cell surface receptor. Proteases and protease regulators that also activate specific cell surface receptors may serve to integrate coagulation with associated cellular responses required for tissue repair and growth, as well as to coordinate protease cascades and associated cellular responses in other systems, such as those involved in growth and remodeling of the nervous system.

Formation of th neuromuscular junction depends upon reciprocal inductive interactions between the developing nerve and muscle, resulting in the precise juxtaposition of a differentiated nerve terminal with a highly specialized patch on the muscle membrane, termed the motor endplate. Agrin is a nerve-derived factor that can induced molecular reorganizations at the motor endplate, but the mechanism of action of agrin remains poorly understood. MuSK is a receptor tyrosine kinase localized to the motor endplate, seemingly well positioned to receive a key nerve-derived signal. Mice lacking either agrin or MuSK have recently been generated and exhibit similarly profound defects in their neuromuscular junctions. Here we demonstrate that agrin acts via a receptor complex that includes MuSK as well as a myotube-specific accessory component.

While a number of growth factors have been described that are highly specific for particular cell lineages, neither a factor nor a receptor uniquely specific to the skeletal muscle lineage has previously been described. Here we identify a receptor tyrosine kinase (RTK) specific to skeletal muscle, which we term "MuSK" for muscle-specific kinase. MuSK is expressed at low levels in proliferating myoblasts and is induced upon differentiation and fusion. In the embryo, it is specifically expressed in early myotomes and developing muscle. MuSK is then dramatically down-regulated in mature muscle, where it remains prominent only at the neuromuscular junction; MuSK is thus the only known RTK that localizes to the neuromuscular junction. Strikingly, MuSK expression is dramatically induced throughout the adult myofiber after denervation, block of electrical activity, or physical immobilization. In humans, MuSK maps to chromosome 9q31.3-32, which overlaps with the region reported to contain the Fukuyama muscular dystrophy mutation. Identification of MuSK introduces a novel receptor-factor system that seems sure to play an important and selective role in many aspects of skeletal muscle development and function.

Nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 (NT-3) are the three members of the neurotrophin family known to exist in mammals. Recently, a fourth neurotrophin (designated neurotrophin-4 or NT-4), which shares all of the features found in the mammalian neurotrophins, has been identified in Xenopus and viper. We used sequences specific to the Xenopus/viiper NT-4 to isolate a neurotrophin from both human and rat genomic DNA that appears to represent the mammalian counterpart of Xenopus/viper NT-4. Human NT-4 as well as a human NT-4 pseudogene colocalize to chromosome 19 band q13.3. Mammalian NT-4 has many unusual features compared to the previously identified neurotrophins and is less conserved evolutionarily than the other neurotrophins. However, mammalian NT-4 displays bioactivity and trk receptor specificity similar to that of Xenopus NT-4.Nerve growth factor (NGF) is the prototypical member of a family of mammalian neuronal survival and differentiation factors (1), known as the neurotrophins, that also includes brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) (2-10). These three neurotrophins are all initially synthesized as larger precursors that are proteolytically cleaved to release the mature neurotrophins. The mature regions of the three neurotrophins display 50-55% amino acid identity to each other, with the major regions of structural similarity bordering six invariant cysteine residues that, in active NGF, have been shown to form three intrachain disulfide bonds.The neurotrophins can be distinguished based on their distinct patterns of spatial and temporal expression (11-15) as well as their differing effects on neuronal targets (3-7). The ability of a cell to respond to a particular neurotrophin appears to be dependent on the presence of the appropriate trk receptor. The three known trk receptors (designated here as trkA, trkB, and trkC) are transmembrane tyrosine protein kinases that specifically bind to the neurotrophins; trkA binds and can be activated by , trkB binds and can mediate functional responses to , and trkC seems relatively specific for .Recently, a fourth neurotrophin (designated neurotrophin-4 or NT-4) has been molecularly cloned from Xenopus (26); a gene segment encoding part of the mature NT-4 from viper was also isolated. Xenopus NT-4 shares all the important features that characterize the mammalian neurotrophins. Here we describe the molecular cloning and characterization of a neurotrophin that apparently corresponds to the mammalian counterpart of Xenopus/viper NT-4. § (The D-number assignment for humNT-4 chromosomal location is D19S202E.)

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