Trends in Cell Biology
ReviewRor receptor tyrosine kinases: orphans no more
Introduction
Receptor tyrosine kinases (RTKs) have crucial roles in many cellular processes including differentiation, proliferation, migration, angiogenesis and survival. Therefore, it is not surprising that dysfunctional RTKs cause severe developmental defects and diseases such as cancer. Receptor tyrosine kinase-like orphan receptor (Ror) proteins are no exception and disruptions of human Ror proteins are associated with skeletal deformities and with leukemia. RTKs normally enable communication between a cell and its environment by binding to an extracellular ligand and initiating an intracellular signaling cascade. For a long time, the Ror family of RTKs was one of the few types of RTK for which the ligand and signaling pathway remained elusive, giving rise to their ‘orphan’ nomenclature; however, recent work has greatly advanced the understanding of Ror function. In particular, Ror proteins have emerged as central regulators of Wnt signaling, an important developmental signaling pathway.
Ror proteins are type-I transmembrane receptor tyrosine kinases (Figure 1). Like other RTKs, they are predominantly located in the plasma membrane [1]. The extracellular region of vertebrate Ror proteins contains an immunoglobulin (Ig) domain, a Cys-rich domain (CRD), also called a Frizzled domain and a Kringle (Kr) domain. Intracellularly, Ror proteins possess a tyrosine kinase (TK) domain and a proline-rich domain (PRD) straddled by two Ser/Thr-rich domains, Ser/Thr1 and Ser/Thr2 [2].
Vertebrates have two ROR family members encoded by ROR1 and ROR2 [formerly known as neurotrophic tyrosine kinase receptor (NTRKR)1 and NTRKR2, respectively], first identified in a human neuroblastoma cell line by a polymerase chain reaction (PCR)-based search for tyrosine kinases similar to Trk neurotrophic receptors [2]. Splice variants of ROR1 that encode truncated proteins lacking either the extracellular domains [3] or the transmembrane and intracellular domains (GenBank locus NM_001083592; http://www.ncbi.nlm.nih.gov/sites/entrez) have been described. Given that the former, called truncated ROR1 (t-ROR1), might be artefactual [4] and that the latter has not been analyzed in detail, in this review we consider only full-length ROR proteins. Despite their lack of several amino acids that are highly conserved in protein tyrosine kinases, ROR1 and ROR2 each have kinase activity in vitro2, 5. ROR orthologs have been identified in fruit flies (Drosophila melanogaster; dROR) [6], roundworms (Caenorhabditis elegans; cam-1) 7, 8, sea slugs (Aplysia californica; Apror) [9], zebrafish (Danio rerio; Ror2 and Ror2) [10], chickens (Gallus gallus; cRor1 and cRor2) 11, 12, frogs (Xenopus laevis; XRor1 and XRor2) [13] and mice (Mus musculus; mRor1 and mRor2) [5]. Drosophila neurospecific receptor kinase (Dnrk), an RTK with similarity to Trk and Ror RTKs [14], has been excluded here because it is now thought of as a muscle-specific kinase (MuSK) ortholog [15] (Box 1). Whereas the CRD, Kr and TK domains are characteristic of all ROR proteins, the architecture of the other domains varies between species (Figure 1).
The extracellular CRD of Ror is similar to the Wnt-binding domain that is found in Frizzled receptors 16, 17, 18, 19, 20, indicating that Ror proteins also bind to Wnt ligands (see later). Wnt proteins are a family of secreted glycoproteins that have crucial roles in development and disease (for a review, see Ref. [21]). In the classic model of Wnt signaling, a Wnt ligand binds to a Frizzled (Fzd) receptor and to the Lrp5/6 co-receptor. This interaction results in the stabilization of cytoplasmic β-catenin, enabling it to accumulate, translocate to the nucleus and function as a transcriptional co-activator with T-cell factor (TCF), a DNA-binding protein. Other mechanisms of Wnt signaling include Wnt–calcium signaling, Wnt–c-Jun N-terminal kinase (JNK) signaling and the planar cell polarity (PCP) pathway (for a review, see Ref. [22]). The degree to which these pathways overlap is presently unclear.
Several studies report diverse, and sometimes conflicting, interactions of Ror with Wnt signaling. It is likely that the discrepancies reflect the diversity of systems tested, with Ror proteins in fact having multiple functions depending on the cellular context (the coexistence of other Wnt pathway components operating in a given cell). In this review, we have clustered compatible observations into a handful of signaling mechanisms, which are discussed in detail, after a brief account of Ror function during development. We conclude with a description of other Ror interactions that are not yet associated with Wnt signaling.
In humans, Ror protein functions are known primarily in skeletal development. hROR2 mutations cause well-characterized skeletal defects such as dominant brachydactyly type B (BDB), a condition of shortened or missing digits 23, 24 and recessive Robinow syndrome (RRS), a form of short-limbed dwarfism 25, 26. hROR2 polymorphisms are also associated with variations in human bone length and mineral density [27]. In mouse and chick, Ror genes have a partially redundant role in skeletal development and are also required for development of the cardiac and respiratory systems 1, 4, 5, 28, 29, 30, 31, 32. Notably, the skeletal defects of mRor2 mutant mice – dwarfism, shortened limbs and facial abnormalities – resemble the deformities of the human disease RRS. Although mutations in hROR1 have not been linked to any human disease, hROR1 is overexpressed in chronic lymphocytic leukemia (CLL) and confers a survival advantage to these cells in vitro33, 34. Consistent with there being a role for hROR1 in cancer, ROR1 was identified as a potent survival kinase in HeLa cervical carcinoma cells [35]. The signaling events downstream of Ror receptors, which are still largely unknown, need to be deciphered to treat Ror-based diseases and malignancies. Table 1 presents a summary of the biological functions and expression patterns of Ror proteins.
Although Ror proteins are strongly expressed in the developing nervous systems of many species (Table 1), the role of Ror proteins in neuronal development remains unclear. The mutant phenotype of dROR, which is expressed exclusively in the developing nervous system of Drosophila[6] has not been described. In mice, although the largely non-overlapping expression patterns of mROR1 and mROR2 in the developing nervous system makes redundancy unlikely, mROR2 knockout mice do not display obvious neurological defects 4, 5; however, it is possible that a subtle phenotype is masked by the early lethality of these mice.
Despite the apparent lack of a vertebrate neuronal phenotype, strong neuronal expression and structural similarity to MuSK protein, which is an RTK required for synapse formation [36], indicate that Ror proteins are involved in neuronal development. Evidence supporting this comes from C. elegans, in which CAM-1 regulates neuronal migration, axon outgrowth and axon guidance 8, 37, 38, 39. One neuron regulated by CAM-1 is the canal-associated neuron, hence the name canal-associated neuron migration defective 1 (CAM-1). CAM-1 also regulates the localization of acetylcholine receptors at the neuromuscular synapse [40], a function performed by the MuSK receptor in mammals [36]. Interestingly, although the closest homolog of CAM-1 is Ror, CAM-1 turns up as the closest homolog for both Ror and MuSK in C. elegans (Box 1), thereby raising the possibility that CAM-1 fulfills the roles of both Ror and MuSK. Although it is unknown whether Ror proteins perform neuronal functions in species that have a distinct MuSK protein, Ror proteins display a localization pattern in cultured mammalian neurons consistent with functions in neurite extension and the organization of neuronal subdomains 9, 41, 42, 43.
Section snippets
ROR proteins sequester Wnt ligands
A series of studies led to the discovery that CAM-1, the C. elegans Ror protein, inhibits the function of a C. elegans Wnt ligand, EGL-20 8, 39, 44. It was first shown that cam-1 mutations cause defects in the migration of several neurons along the anterior–posterior axis [8]. One of these neurons is CAM-1. This migration phenotype was later determined to be reciprocal to the phenotype caused by loss of the Wnt ligand egl-20[39]. Further investigation revealed that cam-1 overexpression mimics
Potentially Wnt-independent Ror functions
A yeast-two-hybrid (Y2H) protein–protein interaction screen using the mRor1 and mRor2 C termini as bait identified Dlxin-1 as a protein that interacts with Ror2, but not with Ror1 [64] (Table 3a). Dlxin-1 is a melanoma-associated-antigen (MAGE) family member and was confirmed to bind to Ror2, but not to Ror1, by co-immunoprecipitation. Ror2 kinase activity is not required for this association. Ror2, by means of its C-terminal proline-rich or Ser/Thr2 domains, recruits Dlxin-1 from the cytoplasm
Concluding remarks and future perspectives
Ror proteins are involved in a multitude of cellular processes and signaling events. A striking theme, however, is that Ror proteins function as Wnt receptors. In particular, there is a general consensus that Wnt5a binds to and activates Ror2; therefore, Wnt5a can be considered a bona fide Ror2 ligand. Although much progress has been made in understanding Ror2 function as a Wnt receptor, several key questions remain unanswered. What is the connection between Ror and the PCP pathway? What is the
Acknowledgements
P.W.S. is an investigator with the HHMI. J.L.G. was supported by the Thomas Hunt Morgan Fellowship for graduate study toward the Doctor of Philosophy degree in Biology at the California Institute of Technology. S.G.K. was supported by NIH training grant GM07616.
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