Piotr Masiakowski

trkB encodes a functional receptor for brain-derived neurotrophic factor and neurotrophin-3 but not nerve growth factor

A variety of findings seem to functionally link brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), while distinguishing both of these factors from the third member of the neurotrophin family, nerve growth factor (NGF). Here we demonstrate that all three of these neuronal survival molecules bind similarly to the low affinity NGF receptor, but that BDNF and NT-3, unlike NGF, do not act via the high affinity NGF receptor. However, both BDNF and NT-3, but not NGF, bind to full-length and truncated forms of a receptor-like tyrosine kinase, trkB, for which no ligand had previously been identified. In addition to binding BDNF and NT-3, trkB can mediate functional responses to both of these neurotrophins when it is expressed in PC12 cells, although BDNF appears to be the more effective ligand. Thus trkB encodes an essential component of a functional receptor for BDNF and NT-3, but not for NGF. Further evidence predicts the existence of additional functional receptors for the neurotrophins.

The anticoagulation factor protein S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases

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.

Ror2, encoding a receptor-like tyrosine kinase, is required for cartilage and growth plate development.

Receptor tyrosine kinases often have critical roles in particular cell lineages by initiating signalling cascades in those lineages. Examples include the neural-specific TRK receptors, the VEGF and angiopoietin endothelial-specific receptors, and the muscle-specific MUSK receptor. Many lineage-restricted receptor tyrosine kinases were initially identified as ‘orphans’ homologous to known receptors, and only subsequently used to identify their unknown growth factors. Some receptor-tyrosine-kinase–like orphans still lack identified ligands as well as biological roles. Here we characterize one such orphan, encoded by Ror2 (ref. 12). We report that disruption of mouse Ror2 leads to profound skeletal abnormalities, with essentially all endochondrally derived bones foreshortened or misshapen, albeit to differing degrees. Further, we find that Ror2 is selectively expressed in the chondrocytes of all developing cartilage anlagen, where it essential during initial growth and patterning, as well as subsequently in the proliferating chondrocytes of mature growth plates, where it is required for normal expansion. Thus, Ror2 encodes a receptor-like tyrosine kinase that is selectively expressed in, and particularly important for, the chondrocyte lineage.

Recombinant Human and Rat Ciliary Neurotrophic Factors

The human ciliary neurotrophic factor (CNTF) gene was identified and cloned, based on homology with the recently cloned rat cDNA. The gene encodes a protein of 200 amino acids, which shares about 80% sequence identity with rat and rabbit CNTF and, like these homologues, lacks an apparent secretion signal sequence. The human CNTF gene, like the rat gene, appears to contain a single intron separating two protein coding exons. An intronless human CNTF gene was constructed by the use of polymerase chain reactions and introduced into vectors designed for expression of foreign proteins in E. coli. The rat CNTF gene was also introduced into similar vectors. Both the human and rat proteins were expressed at exceptionally high levels, at 20–40% and 60–70% of total protein, respectively. Extraction of the recombinant proteins from inclusion bodies by guanidinium chloride, followed by two column chromatography steps, produced high yields of pure CNTF that supported survival and neurite outgrowth from embryonic chick ciliary neurons in culture. The biological activity of both recombinant proteins was comparable to that of native rat CNTF.

The Wnt receptor CRD domain is also found in MuSK and related orphan receptor tyrosine kinases

The receptor tyrosine kinase (RTK) MuSK is part of the receptor complex that initiates neuromuscular junction formation in response to agrin [[1]xReceptor tyrosine kinase specific for the skeletal muscle lineage: expression in embryonic muscle, at the neuromuscular junction, and after injury. Valenzuela, DM, Stitt, TN, DiStefano, PS, Rojas, E, Mattsson, K, Compton, DL et al. Neuron. 1995; 15: 573–584Abstract | Full Text PDF | PubMed | Scopus (296)See all References, [2]xThe receptor tyrosine kinase MuSK is required for neuromuscular junction formation in vivo. DeChiara, TM, Bowen, DC, Valenzuela, DM, Simmons, MV, Poueymirou, WT, Thomas, S et al. Cell. 1996; 85: 501–512Abstract | Full Text | Full Text PDF | PubMed | Scopus (590)See all References, [3]xAgrin acts via a MuSK receptor complex. Glass, DJ, Bowen, DC, Stitt, TN, Radziejewski, C, Bruno, J, Ryan, TE et al. Cell. 1996; 85: 513–523Abstract | Full Text | Full Text PDF | PubMed | Scopus (493)See all References]. MuSK, as well as the related orphan RTK-like proteins Ror1 and Ror2 [4xA novel family of cell surface receptors with tyrosine kinase-like domain. Masiakowski, P and Carroll, RD. J Biol Chem. 1992; 267: 26181–26190PubMedSee all References][4], and their homologs in lower organisms, have an extracellular region composed of varying combinations of immunoglobulin and Kringle domains, but they all also have a domain defined by a pattern of 10 cysteine residues.We have noticed that the spacing of cysteines, and a number of other amino acids, is shared between this domain and a cysteine-rich domain (CRD) that serves as the ligand-binding portion of the Frizzled (Fz) family of seven-pass transmembrane receptors for the Wnt signaling molecules [5xA new member of the frizzled family from Drosophila functions as a Wingless receptor. Bhanot, P, Brink, M, Samos, CH, Hsieh, JC, Wang, Y, Macke, JP et al. Nature. 1996; 382: 225–230Crossref | PubMed | Scopus (990)See all References][5]. As seen in other receptor families, the Wnt-binding domain can also be a part of secreted Frizzled-related proteins (sFRPs) [6xA family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors. Rattner, A, Hsieh, JC, Smallwood, PM, Gilbert, DJ, Copeland, NG, Jenkins, NA, and Nathans, J. Proc Natl Acad Sci USA. 1997; 94: 2859–2863Crossref | PubMed | Scopus (404)See all References][6].The Fz CRD-like sequences have also been identified in Smoothened (Smo), as well as in the α1 chain of type XVIII collagen (Col18), and carboxypeptidase Z (CPZ). Smo is the signaling partner in the Sonic hedgehog (Shh) receptor system. Smo is inactivated by unoccupied Patched (Ptc) protein. Binding of Shh to Ptc relieves this inhibition. The similarity between Smo and Fz, including the CRD, prompted a suggestion [7xPatching up Hedgehog. Nusse, R. Nature. 1996; 384: 119–120Crossref | PubMed | Scopus (15)See all References][7] that the activity of Smo may in addition be modulated by a Wnt-related ligand, though no support for this possibility has been presented.Alternatively, the 10-cysteine domain found in these diverse proteins may be thought of as a general module for protein–protein interactions, evolving in different cases to bind very different counterparts (just like immunoglobulin domains). A computer alignment of the CRDs from representative proteins (Figure 1Figure 1) shows that the Wnt-binding Fz receptors and sFRPs form a distinct group, well separated from the Smo and MuSK/Ror receptors. Thus, determination of the binding specificity of CRDs in these receptors, as well as in Col18 and CPZ, seems likely to reveal new binding capabilities of the 10-cysteine module.Figure 1(a) Multiple sequence alignment of 10-cysteine domains and (b) a phylogenetic tree showing the clustering relationships used to create the alignment. The sequences were downloaded through NCBI Entrez and processed using the programs in the GCG Wisconsin Package [8xSee all References][8].View Large Image | View Hi-Res Image | Download PowerPoint Slide

Genomic organization and chromosomal localization of the human and mouse genes encoding the α receptor component for ciliary neurotrophic factor

Ciliary neurotrophic factor (CNTF) has recently been found to share receptor components with, and to be structurally related to, a family of broadly acting cytokines, including interleukin-6, leukemia inhibitory factor, and oncostatin M. However, the CNTF receptor complex also includes a CNTF-specific component known as CNTF receptor alpha (CNTFR alpha). Here we describe the molecular cloning of the human and mouse genes encoding CNTFR. We report that the human and mouse genes have an identical intron-exon structure that correlates well with the domain structure of CNTFR alpha. That is, the signal peptide and the immunoglobulin-like domain are each encoded by single exons, the cytokine receptor-like domain is distributed among 4 exons, and the C-terminal glycosyl phosphatidylinositol recognition domain is encoded by the final coding exon. The position of the introns within the cytokine receptor-like domain corresponds to those found in other members of the cytokine receptor superfamily. Confirming a recent study using radiation hybrids, we have also mapped the human CNTFR gene to chromosome band 9p13 and the mouse gene to a syntenic region of chromosome 4.