BIOLOGICAL OVERVIEW

ROR proteins are conserved receptor tyrosine kinases (RTKs) characterized by an extracellular Fz domain [also called cysteine-rich-domain (CRD)], an immunoglobulin (Ig) domain, and a kringle domain. Mutations in ROR genes cause developmental defects including skeletal abnormalities in mice and humans. Studies of vertebrate RORs showed that the ROR CRD, like the Fz CRD, can bind to Wnts (Billiard, 2005; Hikasa, 2002; Kani, 2004; Mikels, 2006a; Oishi, 2003). In cell culture, ROR2 abrogates expression of a canonical Wnt reporter (Billiard, 2005; Mikels, 2006a; Green, 2007 and references therein).

Drosophila Drorencodes a putative receptor tyrosine kinase (RTK) andmaps to cytological location 31B/C on the second chromosome.In embryos, this gene is expressed specifically in the developingnervous system. The Dror protein appears to be a homolog oftwo human RTKs, Rorl and Ror2. Dror and Rorl proteinsshare 36% amino acid identity in their extracellular domainsand 61% identity in their catalytic tyrosine kinase (TK) domains.Rorl and Ror2 were originally identified on the basis ofthe similarity of their TK domains to the TK domains ofmembers of the Trk family of neurotrophin receptors. TheDror protein shows even greater similarity to the Trk proteinswithin this region than do the human Ror proteins. In light ofits similarity to trk and its neural-specific expression pattern,it is suggestd that Dror may encode a neurotrophic receptor thatfunctions during early stages of neural development in Drosophila (Wilson, 1993).

Dror shows extensive sequencesimilarity to vertebrate Ror proteins throughout its length;however, Rorl and Ror2 include additional motifs at theextreme amino-terminal and carboxyl-terminal ends.Like the vertebrate Rorproteins, the extracellular domain of Dror contains akringle domain and a Ror-like cysteine-containing domain.Other RTKs, such as the members of the EGF receptorfamily and insulin receptor family, are also characterized byextracellular cysteine-containing domains, butthese are not obviously related to the domains in the Rorfamily of RTKs. A unique feature of the cysteine-containingdomain in Dror is that it is interrupted by a 55-aa lysine-richinsertion of unknown function. The role of the single kringledomain adjacent to the transmembrane domain is also unclear (Wilson, 1993).

Generally genes encode clusters of kringle domainsand, unlike the Ror RTKs, these are associated with a serineprotease-like domain. The presence ofa kringle domain in Dror is of particular interest, since proteins with kringle domains have not previouslybeen identified in invertebrates (Wilson, 1993).

The Dror TK domain is most similar to the TK domains ofvertebrate Ror and Trk proteins, showing 7%-16% greateridentity with these proteins than with TK domains in theclosely related insulin receptor family. In fact, theRor and Trk TK domains show more similarity to the TKdomain of Dror than they do to each other. However,surprisingly, the small number of amino acid substitutionsthat are specific either to the members of the Ror family orto the members of the Trk family and have therefore beensuggested to be characteristic of a particular receptor groupwithin the Trk-like superfamily are generally not present inDror. One possible interpretation of these data is that the Rorand Trk families of RTKs developed their limited number ofunique features after the chordate line diverged from arthropodsin evolution. Searches for additional trk-related genes inDrosophila with Dror probes, employing conditions that havepreviously been used successfully to identify vertebrate trkgenes and Dtrk, a second Drosophila Trk-family member (off-track), have thus far failed to identify asecond ror gene or any additional trk-related genes in flies (Wilson, 1993).

The previously characterized Drosophila gene Dtrk appears to encode a distant relative of the Trk family, with 9 of the 40 most conserved amino acids in TK domains altered and <40% identity with vertebrate Trk TK domains.However, unlike Dror, Dtrk does show limited similarity(<25% identity) to the Trk proteins in its extracellulardomain, even though its activation may be mediated by celladhesion rather than by a neurotrophic factor (Wilson, 1993 and references therein).

The expression patterns of the vertebrateror genes have not been reported, although ror cDNAclones were originally isolated from a human neuroblastomacell line. The relatedness of the TK domains of both thevertebrate and Drosophila Ror proteins to Trk TK domainsmakes it attractive to speculate that the ror genes play a rolein neural development. The observation that Dror expressionin embryos is restricted to the nervous system is consistentwith this idea. It is suggested that at least some of the aminoacids that are found in both Trk and Ror TK domains, butrarely in other TK domains, have therefore probably evolvedto mediate neural-specific functions. However, in the electricray Torpedo californica, a RTK with a Trk-like TK domainhas recently been identified that is expressed specifically inmuscle and not in neurons. One untested explanation forthis discrepancy is that the Torpedo receptor may be localizedto the neuromuscular junction, a subcellular specializationthat shares several functional properties with neurons (Wilson, 1993).

Since the vertebrate and Drosophila Ror proteins are sosimilar in their extracellular domains, it is likely that theirligands are also structurally related. It is not known whetherthese ligands are diffusible, like the neurotrophins, or are cellsurface molecules with a more limited signaling range. Further,although the similarity of Trk and Ror TK domainssuggests that some of their intracellular neural-specific functionsare related, it is not clear which functions the Rorproteins may share with neurotrophin receptors. The peak ofembryonic Dror expression (8-12 hr) starts before and overlapswith the period when early processes of neural differentiation,such as axonogenesis, occur in flies (9-10 hronwards). Interestingly, vertebrate ror genes are alsoexpressed relatively early in development, some time beforethe trk genes. Therefore, the Ror proteins probably playan important role in early neural differentiation and may beless involved in the later processes of neuronal cell survivalthat are commonly associated with the neurotrophin receptors (Wilson, 1993).

It is anticipated that a genetic analysis of Dror in Drosophilashould address these issues and contribute to an understanding of the function of the Trk-like receptor superfamily in all higher eukaryotes (Wilson, 1993).

A third receptor tyrosine kinase has been characterized and termed 'Dnrk,' for Drosophila Neurospecific receptor kinase. Dnrk is expressed specifically in the developing nervous system following germ band elongation. The expression is restricted to the layer of neural progenitor cells between the epidermal and mesodermal cell layers. Expression is found in the brain, the ventral cord and late in embryogenesis in the peripheral nervous system. The distribution of transcripts after germ band shortening matches the profile of developing commisures and connectives. The extracellular domain of Dnrk protein exhibits a high degree of homology with those of Dror and human Rors. Dnrk possesses two conserved extracellular cysteine-containing domains and an extracellular kringle domain, resembling those observed in the Ror family of RTKs. All 16 cysteins in Dnrk are found in equivalent positions in Dror, Ror1 and Ror2. Dnrk protein contains the catalytic tyrosine kinase domain with two putative ATP-binding motifs, resembling those observed in Dtrk. The TK domain of Dnrk exhibits autophosphorylation activities in vitro. The TK domain shows about 40-45% identity to the corresponding domains of TrkB, Ror1, Ror2 and Dror. An altered TK domain, lacking the distal ATP-binding motif, also exhibits autophosphorylation activities, but to a lesser extent than wild type Dnrk. In addition to its TK activity, there are several putative tyrosine-containing motifs that upon phosphorylation may interact with Src homology 2 regions of other signaling molecules (Oishi, 1997).

GENE STRUCTURE

Bases in 5' UTR -129

Bases in 3' UTR - 88

PROTEIN STRUCTURE

Amino Acids - 685

Structural Domains

The protein consists of an extracellular cysteine-containing region, a kringle domain (a type of protein interactin domain), five potential N-glycosylation sites, a hydrophobic transmembrane domain and an intracellular putative tyrosine kinase domain. The amino terminal portion has a hydrophobic signal peptide involved in secretion across microsomal membranes during protein synthesis (Wilson, 1993).

The tyrosine kinase domain is similar to the TK domain of the vertebrate Trk family and the two human Ror proteins. Dror intracellular segment does not include a tyrosine for autophosphorylation. It shares a kringle domain with vertebrate Ror, but Dror has no immunoglobulin domain homologous to that of vertebrate Ror, nor does it contain the carboxyl-terminal serine (threonine-rich and proline rich domains present in Ror1 and Ror2) (Wilson, 1993).

date revised: 10 April 2008

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