Interactive reportGas6, a ligand for the receptor protein-tyrosine kinase Tyro-3, is widely expressed in the central nervous system1
Introduction
Receptor protein tyrosine kinases (PTKs) and their ligands play multiple roles in the developing and mature nervous system. First described for their role in cell growth and proliferation, receptor PTKs are known to also function in cell survival, neural cell-type determination, cell migration and axonal pathfinding. In this study, we explore the distribution of Gas6, a ligand for the receptor PTK Tyro-3, in order to identify relevant ligand-expressing and receptor-expressing sets of cells.
Perhaps the best studied example of receptor PTK mediated signal transduction in the nervous system is the trk subfamily of receptors, which includes trk A, B and C and their ligands, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin 4/5 (NT-4/5) [10]. The analysis of these molecules has improved our understanding of the signaling events underlying neuronal survival and plasticity. The largest subfamily of receptor PTKs, the Eph subfamily, has been implicated in a number of roles including the establishment of topographic maps in the developing nervous system and it appears that the Eph ligands, the ephrins, serve as positional labels in the retino–tectal system [76]. Another receptor PTK which plays a critical role in the nervous system is MuSK; mice lacking MuSK, which serves as a receptor for agrin, fail to form neuromuscular junctions [20]. Our efforts are directed toward understanding the role of the receptor PTK Tyro-3 and its ligands in the rat brain.
Tyro-3 is a member of the Axl subfamily of receptor PTKs, which includes Axl, Mer and Tyro-3 [15]. The kinase rek, which has been identified in chick retina, is more closely related to Tyro-3 than to Axl and it may represent the avian homologue of Tyro-3 [9]. Axl was originally isolated from a patient with myeloproliferative disorders [58]and Mer was obtained from a neoplastic B cell line [28]suggesting a possible role in cellular transformation. Tyro-3 appears to possess the ability to transform cells 41, 74, 75. As with many receptor families, the Axl subfamily nomenclature is complicated by the independent identification of these genes by several groups. Axl is also known as Ark, UFO, and Tyro-7 36, 42, 58, 66. Mer is also called Nyk, eyk and Tyro-12 37, 38, 42, 45and Tyro-3 has been designated Rse, tif, Sky, etk2 and Dtk 8, 16, 19, 42, 50, 60. These receptors exhibit a similar extracellular domain structure that is composed of two immunoglobulin (Ig)-like domains [80]followed by two fibronectin (FN) type-III repeats [63]. This combination of domains is also present in several neural cell adhesion molecules [17]and suggests a role in cell–cell interactions for members of this family. Axl has been implicated in homophilic binding events [6]. Axl is most abundantly expressed in the ovarian follicles, hematopoietic system, skeletal muscle and the heart 21, 56. Mer is predominantly expressed in the ovary, testes, prostate, lung, and kidney 27, 28, 37and at lower levels in the spleen, placenta, thymus, small intestine, colon and liver. Outside of the nervous system, Tyro-3 is detected in the kidney, ovary and testes 42, 50, 60.
In the adult rat and mouse brain, in situ hybridization and Northern blot analyses have shown that Tyro-3 mRNA is present at high levels in the brain where it is expressed maximally in the olfactory bulb, the piriform cortex, tenia tecta, the subiculum and the granule neurons of the cerebellum. It is present in the cerebral cortex, primarily in layers II–VI, and in the hippocampus, where strong expression sharply defines area CA1, with lower expression levels in area CA3 41, 42, 59, 69, 77. The receptors Axl and Mer are only expressed at low levels in the CNS 6, 27, 28, and in studies to be described elsewhere (Prieto et al., in preparation), we show that both of these receptors appear to be co-localized in the cerebellum.
Gas6 serves as a ligand for all known members of the Axl family of receptors including Axl, Tyro-3 (Rse) and c-mer 11, 49, 52, 61, 78. Gas6 has a demonstrable affinity for these receptors and induces receptor phosphorylation. Another putative ligand for Tyro-3 is protein S, a Gas6-related protein (see below). The assignment of protein S as a ligand has been the subject of controversy with the primary concern being the failure to demonstrate Tyro-3 receptor activation with protein S derived from the same species as the receptor. Human and bovine protein S have been reported to activate murine Tyro-3 57, 71, but high concentrations of human protein S have failed to activate human Tyro-3 24, 61. Human Gas6 can activate both human Tyro-3 and Axl. Protein S is well documented as an anticoagulant in the blood coagulation cascade 30, 33, 40where it is known to bind to the complement C4b-binding protein (C4BP), and to act as a cofactor for the activated protein C complex, a serine protease that inactivates coagulation factors Va and VIIIa 18, 39, 46, 67, 79. We have described the expression of both these putative Tyro-3 ligands.
Gas6 and protein S exhibit 44% amino acid identity and share a complex domain structure (Fig. 1D). The amino-terminal Gla domain, rich in the modified amino acid, γ carboxyl–glutamic acid (gla), is followed by a loop region (B-Box), 4 epidermal growth factor (EGF)-like repeats, and a carboxyl-terminal domain bearing significant amino acid sequence similarity to the steroid hormone binding protein (SHBP) [47]. The Gla domain and EGF-like repeats are the most highly conserved regions between protein S and Gas6. The SHBP-like domain of Gas6 is necessary for receptor activation as chimeric proteins which contain this domain are able to induce tyrosine phosphorylation while proteins which lack this region fail to do so. The Gla domain potentiates this effect, possibly through its ability to promote cell surface binding mediated by its γ-carboxylated glutamic acid residues 54, 73.
Recently, an alternatively spliced Gas6 variant has been characterized 26, 48that contains a 43 amino acid inserted sequence which allows the proteolytic cleavage of this Gas6 protein into two polypeptides, an amino-terminal 36 kDa fragment containing the Gla domain, B-Box and EGF-like repeats and a carboxyl-terminal 50 kDa fragment encompassing the SHBP-like domain. Gas6 may therefore exist in both intact and cleaved forms, which may possess distinct properties.
Gas6 was originally isolated as a gene induced under growth-arrest conditions [68]. The addition of Gas6 can rescue NIH-3T3 7, 25and vascular smooth muscle cells (VSMCs) from apoptosis induced by serum withdrawal [55]. Gas6 has also been shown to induce the proliferation of human Schwann cells, which express both Tyro-3 and Axl [44]. Gas6 is therefore capable of promoting the proliferation of at least one neural cell type but its possible role in cell survival in the nervous system remains to be explored.
We have analyzed the distribution of the putative ligands Gas6 and protein S in the CNS in order to gain insight into the function of Tyro-3 in the nervous system. We have generated specific anti-Gas6 antibodies and used these to identify the sites of Gas6 protein expression. We have also determined the sites of mRNA expression in an in situ hybridization survey examining both developmental and mature neural tissue. Gas6 is widely expressed in the CNS starting at early postnatal stages with maximal levels persisting in the adult. In contrast, the sites of protein S expression are extremely limited. Gas6 and Tyro-3 are both expressed in many brain regions and their patterns, while distinguishable, do overlap. These studies provide the neuroanatomical framework on which models for the interaction of Gas6 and Tyro-3 in the nervous system can be developed.
Section snippets
Fusion proteins
A Gas6–GST fusion protein was produced for use as antigen in the generation of Gas6-specific antibodies. To obtain precisely defined Gas6 fusion proteins with minimal amino acid sequence relatedness with protein S, we used PCR to amplify the loop region, also designated B-Box (see Fig. 1D), extending from amino acids 87 to 114 [47], from cDNA clone Gas6-5 (C. Lai, unpublished). This clone was obtained by screening a λ ZAP cDNA library [41]with a small Gas6 cDNA fragment (gift of Dr. J.
Characterization of anti-GAS6 antibodies
We have generated specific antibodies directed against the B-Box or loop region of Gas6 (amino acids 87–114) (Fig. 1D). A GST–Gas6 fusion protein containing this region, selected for its limited similarity with protein S (16% amino acid sequence identity), was produced [47]and used as immunogen. In a Western blotting analysis, these antibodies detected an 85 kDa protein in both cell extracts (Fig. 1A, lane 2) and culture supernatants (Fig. 1A, lane 4) prepared from 293 cells transfected with a
Discussion
Gas6 has been previously identified as a ligand for members of the Axl subfamily of receptor PTKs and is capable of activating all of the three subfamily members, Axl, Tyro-3 and Mer 12, 24, 49, 52, 61, 71, 78. Because of its ability to stimulate each of these receptors, Gas6 could activate more than one signaling pathway in cells that express multiple receptors. This may involve receptor heterodimerization, although this has not yet been demonstrated for receptors of this subfamily. Because of
Acknowledgements
We wish to thank Drs. P. Sanna and J. Griffin for helpful suggestions throughout this work. We also wish to thank Dr. M. Buchmeier for use of his Zeiss microscope and Dr. S. Heinemann and Dr. D. Vetter for use of their Wild microscope. We thank Dr. H. Fox for a GAPDH cDNA clone. We thank E. Battenberg, D. Bigley, and T. Fischer for assistance in the preparation of the figures. This work has been supported in part by grant NIH R01 NS32367 to C.L. and NIH RO1 HL21544 (M.J.H.)
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Published on the World Wide Web on 2 December 1998.