Cloning of Receptor Tyrosine Kinases from Anvsia

Cloning of Receptor Tyrosine Kinases From ANvsia

Jonathan Hislop Neurology and Neurosurgery, McGill University, Montreal. August, 1998

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements of the degree of Master of Science

@Jonathan Hislop, 1998 National Library Biblioth$ue nationale du Cana a Acquisitions and Acquisitions et Bibbgraphic Semices m~icesbibliographiques 395 Wdlngton Street 395, rue Weilinglori OItawaON K1AW OnawaON K1AON4 CariPds Canada

The author has granted a non- L'auteur a accordé une licence non exclusive licence ailowing the exclusive permettant a la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or seU reproduire, prêter, distribuer ou copies of this thesis in rnicrofonn, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substaatial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or othenirise de celle-ci ne doivent être imprimes reproduced without the author's ou autrement reproduits sans son permission. autorisation. Abstract I have cloned two putative receptor tyrosine kinases (RTKs) fiom Aplysia californica, designated Aror and Anif. Aror is homologous to RTKs fiom the Ror subfarnily. Furthemore, Aror contains an extracellular immunoglobulin-like domain, cysteine-rich region, and kringie domain. These domains are d characteristic of Ror recepton. Amf is homologous to members of the Ret and FGF receptor subfamilies of RTKs. However, Aruf does not have the extracellular domains typical of those receptors. Furthemore the degree of identity between Anif and either the Ret or FG F recepton is closer to that between members of different RTK subfamilies, han between membea of the sarne subfamily. Therefore, Amf encodes an RTK belonging to a novel subfamily related to the Ret/FGFR subfamily. J'ai cloné deux molécules qui sont de presum6s récepteurs tyrosine kinase (RTK) provenant de Aplysia cal@rrnica, et désignées Aror et Ad. Aror est homologue aw membres de la sous-famille Ror des RTK. De plus, Aror comprend un domaine extra- cellulaire apparenté aux immunoglobulines, une région riche en cystéine et un domaine kringle. Ces domaines sont tous caractéristiques des récepteurs Ror. Anif est homologue aux membres des sous-familles Ret et FGFR des RTK. Cependant, Anif n'a pas le domaine extra-cellulaire qui est typique de ces récepteurs. De plus, le degré d'identité entre Anif et l'un ou l'autre de Ret ou FGFR est plus prb du degré d'identité partagé entre les membres des diffdrentes sous-familles RTK que de celui partagé entre les membres d'une même sous-famille. Donc, Adencode un RTK appartenant à une nouvelle sous-famille reliée à celles de Ret et de FGFR, Acknowledgements 1 would like to thank my supervisor Wayne Sossin for his time and patience as a teacher. I'd also like to thank the other members of my advisory cornmittee, Phi1 Barker and David Kaplan, for their helpful comments and direction throughout this project. Thanks also go to David Ragsdale, and Peter MacPhearson for their encouragement and support. My biggest surprise about doing research has ken how much i've enjoyed workine- with everyone in our lab. John Dyer showed me how to perfom many of the procedures for the project, and was a great teacher. 1 enjoyed working with him tremendously, and listening to Monty Python with him. A number of other people €rom the lab also helped me learn various procedures, including Tony Pepio, Xiao-Tang Fan, and Stefanie Yanow. 1 had a great tirne working with al1 of them as well as others from the lab, such as Hedy Chung, Tina Vouloumanos, Darcy Scott, and Arash Nakhost. Poking fun at Arash was particularly rewarding. My family and fnends were also always supportive of me during the project. But my cat Checkers objected to this entire flair and wants lots of attention to make up for it. He also wants to try one of the Aplysia.

III Statement of Originalitv The cloning of each of the receptoa discussed in this study (designated Aror, and Anif) constitute original scholmhip. Neither of these receptors have been previously cloned. 1 pexformed aii the procedures discussed in the study with the exception that most of the DNA sequencing was perfomed by our laboratory technician Xiao-Tang Fan, and by Bio S&T Inc., and The W.M. Keck Biotechnology Resource Laboratory. The rationale for the project was devised by Dr. Wayne Sossin. Table of Contents

Abstract ...... 1

Résumé ...... II

Acknow ledgements...... 111

Staternent of Originality ...... iV

Table of Contents ...... V

List of Tables and Figures ...... VI1

List of Abbreviations ...... VI11 introduction ...... 1

Receptor Tyrosine Kinases: Structure and Function ...... 1 Oved Structure...... 1 The Extracellular Domain ...... -3 The Kinase Domain ...... 3 RTK Activation and Signal Transduction ...... 4 RTKs Have Diverse Roles in Many Organisms ...... 7 RTKs in Invertebrates ...... 1 O RTKs and Memory Formation in Aplysia californica ...... 1 O Serotonin Induces Translation and Intemediate Memory in Aplysia ...... I 1 RTKs and Intermediate Memory in Aplysia ...... 12 Rationale and Objectives ...... 1 2 Cloning of RTKs fiom Aplysia ...... 1 3

Materials and Methods ...... 1 8

Construction of cDNA Library ...... 18 PCR Based Screen and Design of Oligodegenerate bers...... 18 Screening for Full Length Clows ...... 20 Cloning of Aplysia RTKs ...... 25 Amplification of PCR Eragments ...... 25 Aror Hy bridization Screen ...... 26 Aror encodes a putative RTK related to the Ror subfamily ...... 27 Anif Hybridization Screen ...... ,...... *..*,...*,.*.*....30 Anif encodes a putative RTK related to the RetIFGF receptor subfamilies ...... 31

Discussion ...... 45

Summary ...... 45 Additional RTK Fragments Amplified ...... A5 Featwes of Aror ...... 46 Extracellular Domain...... 46 Intracellular Domain ...... 47 Features of Amf ...... -49 Extraceilular Domain ...... 49 intracellular Domain ...... -50 Future Directions ...... 51

References ...... 53 List of Tables and Figures Table 1 - Summary of Clones Obtained Figure 1 - Overall Structure of Various RTKs Figw 2 - Phylogenetic Relationships of Various RTKs Figure 3 - Alignment of RTK Kinase Domains Figure 4 - Crystal Structure of the Insulin Receptor Figure 5 - Cloning of .4pIyssia RTKs Figure 6 - Aror Hybndization Screen Figure 7 - Nucleotide and Amino Acid Sequence of Aror Figure 8 - Similarity of Aror to other Ror Receptors Figure 9 - Sequence Alignment of Aror to Other RTKs Figure 10 - Phylogenetic Relationship of Aror to Other RTKs Figure 1 1 - Aruf Hybridization Screen Figure 12 - Nucleotide and Amino Acid Sequence of Aruf Figure 13 - Similarity of Aruf to Ret/FGF Receptors Figure 14 - Sequence Alignrnent of Aruf to Ret/FGF Receptors Figure 15 - Phylogenetic Relationship of Amf to RetIFGF Receptors List of Abbreviations

RTK = Receptor Tyrosine base Ig = Immunoglobulin EGF = Epidemal Growth Factor FGF = Fibroblast Growth Factor N- = Amino C-= Carboxy M~"= Magnesium ATP = Adenosine Triphosphate PDGF = Platelet Derived Growth Factor 10. GDNF = Glial Cell Line Denved Neurotrophic Factor 1 1. GPI = Glycosyl Phosphatidy 1 Inositol 12. SH2 = Src Homology 2 13. PTB = Phosphotyrosine Binding Domain 14. PI3 Kinase = Phosphoinositol-3 Kinase I 5. IRS- 1 = Insulin Receptor Substrate-1 16. GTP = Guanosine Trinucleotide Phosphate 17. PIP2 = Phosphoinositol-4,5-Bisphosphate 18. IP3 = Inositol-3,4,5-Triphosphate 19. DAG = Diacylglycerol 20, PKC = Protein Kinase C 2 1. CNS = Central Nervous System 22. BDNF = Brain Derived Neurotrophic Factor 23. LTP = Long Term Potentiation 24. NGF = Nerve Growth Factor 25. NT3 = Neurotrophin-3 26. MuSK = Muscle Specific Kinase 27. NMJ = Neuromuscular Junction 28. AchR = Acety lcholine Receptor 29. MASC = Myotube Associated Specificity Component 30. EPSP = Excitatory Postsynaptic Potentials 3 1. 5HT = Serotonin 32. cDNA = Complementary Deoxyribose Nucleic Acid 33. PCR = Polymerase Chain Reaction 34. dCTP = Deoxycitidine 5' -Triphosphate 35. dATP = Deoxyadenosine S'-Triphosphate 36. dTTP = Deoxythyxnidine S'-Triphosphate 3 7. dGTP = Deoxyguanosine 5 '-Triphosphate 38. LB = Luria Bertani 39. ON= Ovemight 40. Bp = Base Pair 41. A.A. = Amino Acids 42. kb = Kilobases 43. PL Cy = Phospholipase Cy

44. Dror = Drosophila Ror 45. Dtrk = Drosophila Trk-like Receptor 46. VEGF = Vascular Endothelid Growth Factor 47. NCAM = Neural Cell Adhesion Molecule 48. CRD = Cysteine Rich Domain 49. Dnrk = Drosophila Neurospecific Receptor Kinase 50. sFRP = Secreted Frizrled-Related Proteins 5 1. HGF = Hepatocyte Growth Factor 52. PDK 1 = Phosphoinositide-Dependent Protein Kinase- l 53. YAP = Yes-Associated Protein 54. nNOS = Neuronal Nitnc Oxide Synthase 55. NMDA = N-methyl-D-aspartate

56. CRNF = Cysteine-Rich Neurotrophic Factor Introduction Receptor Tyrosine Kinases: Structure and Function Cells in multicellular orgaaisms often modulate their activities in response to extrinsic signais from their environment. These signals can be transduced across the target cetls membrane via a receptor on the ceiis surface. One type of receptor is a receptor tyrosine kinase (RTK). Binding of ligand to RTKs induces autophosphorylation of specific inrracellular tyrosine residues. Tyrosine autophosphorylation subsequently stimulates the intrinsic tyrosine kinase activity of the receptor, which phosphorylates substrate molecules and generates recruitment sites for downstream signaling molecules. Activation of these pathways leads to alterations in cellular activity. Together with non-receptor tyrosine kinases and serinelthreonine kinases, they form part of a large farnily of protein kinase molecules. Ligands for RTKs may be diffusable factors, membrane bound proteins, or extracellular matrix proteins. RTKs are important in many cellular processes, such as growth, differentiation, and synaptic development in al1 multicellular organisms.

The structure of RTKs has been extensively revie~ed'~~?',and the findings of these papers are summarized here. It has been found that al1 RTKs share a sirnilar basic structure. Beginning at the N-terminal end, they al1 possess a signal peptide that targeis the protein to the secretory pathway. Following this there is a large extracellular ligand binding domain, which may contain a number of specific structural motifs (discussed below) depending on the individual receptor Figure 11. The extracellular domains of most RTKs often also have a number of N-linked glycosylation sites. The ligand binding domain is comected via a single transmembrane hydrophobic segment to the intracellular portion of the molecule. imrnediately following the transmembrane domain there are several basic residues that hction as a stop-transfer signal, as well as a jwtamembnuie region which precedes the intriacellular catalytic domain. In most RTKs the catalytic domain is approximately 250 amino acids although some receptoa have a hydrophilic insert which intempts the domain, and therefore lengthens it. Finally, foliowing the catalytic domain there is a C-terminal region that varies fiom a few residues up to 200 amho acids depending on the individual receptor. In some cases, such as for the insulin receptor, RTKs are proteolytically processed in the extracellular domain, and are present as disulfide-linked dirners. individual RTKs are often compared to each other on a structural basis, and those receptors that are most similar structutally tend to have functional similarities as well. Al1 RTKs corifonn to the overall structure outlined above, however based on their ligand binding specificity and/or similarity to other RTKs, they cm also be organized into subfamiiies-'4 * 56* '. There are currently at least 14 subfarnilies of RTKs, as well as several additional receptors that have not yet been assigned to a specific subfamily. Examples fiom a number of RTK subfamilies are indicated in figure 1, and the phylogenetic relationships of a variety of RTKs is shown in figure 2.

The Extracellular Domain Despite the overall similarities between RTKs, their extracellular regions are generally the domains that have the least amiw acid identity between individual receptors, and this is thought to refiect the different ligand binding specificities of each receptor. One factor that may lead to similarity in the extracellular domain, and therefore indicate possible assignment of a receptor to a particular subfamily, is the presence of one or more of a set of specific sequence motifs that occur in this region in most of the receptors. These motifs are recognizable segments of sequence ranging in size fiom 10 to over 100 amino acids, and include immunoglobulin (Tg)-like domains, discoidin 1-like domains, fibronectin III repeats, leucine-rich motifs, epidermal growth factor (EGF)-like domains, cadhe~domains, kringle domains, ac id boxes, and a variety of distinct cysteine rich domains Figure 11. There are a number of roles these motifs are thought to have. In sorne cases they have been show to contribute to ligand binding. niis is the case for the third Ig-like domain of the FGF receptor8. Alternatively, some of these motifs, such as kringîe domains, are thought to be involved in protein-protein interactions other than ligand bindingg*lO*''*12". This includes binding to other membrane bound proteins, and extraceîlular rnatrix proteins. Another possibility is that some of these regions are involved in receptor dimerkation, a critical step in RTK signaüng. Finally, some motifs, such as cysteine nch domains, are thought to be important in rnaintaining the structural integrity of the receptor in the exûacellular environment. RTKs may contain various combinations of these motifs. Human Rorl for instance contains an Ig-like domain, a cysteine nch domain, and a kringle dornain near the trammembrane segment". In contrast, the mouse fibroblast growth factor (FGF) receptor contains three immunoglobulin domains and an acid box1', while the only extracellular motif human c-Ret bears is a cadherin domah! Most of these domains are aiso found in a wide variety of other proteins. For instance, kringle domains are also found in hepatocyte growth factor, and in a number of protein involved in blood Ig-like domains, and cadhe~domains are both present in ce11 adhesion molecules, and cysteine rich domains are found in wide variety of molecules?

The Kinase Domain The catalytic (kinase) domain is the region with the greatest identity between al the RTKs Figure 31, and is similar to the catalytic domain of other Ihases, such as serinehhreonine kinases, as Identity between RTK subfamily members is higher still in this region than between members of different subfamilies, and sequence identity within this region can range fiom 32% to as high as 9~%'*~*'.In contrast the degree of identity between the kinase domains of RTKs and se~e/threoninekinases is closer to 20%'"*'. The kinase domain of RTKs is defmed on the basis of sequence similarity, where the ends of the domain correspond to the extreme amino- and carboxy- terminai residues that are conserved through most members of the fa~nil~~~*~~*'.The usefdness of this defuiition has been confïrrned through a series of experiments where deletions of residues near the edges of what was thought to be the domain were made, and the product then tested for kinase activity. In this way the domain was defined as beginnuig at what is usually a hydrophobic residue 7 amino acids on the amino (N)- terminai side of the homologous amho acid sequence motif GxGxxG (standard amino acid code; x = any amino acid) and continuing for appmximately 250 amino acids until a region bearing a conserved aginine (R) followed by a weîî conserved leucine (L) nine residues mertowards the carboxy (C)-tefmiaus, the leucine king the last residue in the d~rnain~~'"*~[Figure 31. Residues falling outside this region have been show to be unnecessary for kinase activi#. In those receptors that contain hydrophilic inserts, the inserts have ais0 been shown to be unnecessary for kinase activity2. nie kinase domain bean a number of specific motifs that are involved in binding to intracellular substrates ador catalytic CO-factorsFigure 31. Most of these motifs are aiso present in serinehhreonine kinases and have similar function~~*'~*'.For instance, the GxGxxG motif together with a well conserved lysine (K)approximately 21 a&o acids towards the C-terminus is involved in binding magnesium (~g~3,and adenosine triphosphate (ATP), both of which are CO-factorsfor substrate phosphorylationL4. Another motif is the sequence HRDLAARN, which is the catalytic site, where the aspartate residue (D) is thought to be the catalytic base2. This residue is conserved amongst al1 the RTKS'*~*~?The crystal structures of the insulin receptor, as well as the FGF receptor have now been determined, and they provide additional evidence as to how the RTKs f~nctionl~*~~*~~.In particular they suggest that the kinase domain of these RTKs is organized into an N-terminai lobe, which includes the M~~'/ATPbinding motif, and a C-terminal lobe which includes the catalytic motif. Magnesium, ATP, and the kinase substrate are thought to be brought together in the clefi between these lobes, leading to substrate phosphorylation [Figure 41'"~'*~~.Based on the strong degree of identity betwecn RTKs in this region, it is likely that some of the structural characteristics that have been elucidated frorn the insuiin receptor and FGFR crystal structure will be shared by other Rï'Ks as well.

RTK Activation and Sienal Transduction The key function of RTKs is to transduce signals across the plasma membrane that will activate intracellular signaiing pethways, and this can be broken down into a number of stepslJoJ1. In the fust step ligand binding causes a conformational change in the extraceliular domain that allows nearby receptors to associate, fomillig a receptor dimerluOJ'. Dimerization may be induced through a number of mechanisms. One mechankm is where two receptor molecules are bound by two ligand molecules, leading to conformational changes in both receptoa that allow di~nerization~~~~~'.In other cases the ligand may have more than one receptor binding site and a single ligand is therefore sufficient to cause dirnerizati~n'~~?Yet another possibility applies to receptors such as the insulin receptor which are already dimers. In these cases ligand binding is thought to induce conformational changes that lead to kinase activati~n'*~~~*~'. In contrast, some ligands such as platelet derived growth factor (PDGF)are thernselves dimers, and each half of the dimer binds to one receptor leacüng to formation of a receptor dimer'tJo21. Another rnechanism that is observed for the FGF receptor, is where an additional factor (in this case hep- sulfate proteoglycan) stabilizes interactions between FGF molecules, allowing multiple ligands to associate and effect receptor dirnerizati~n'"~~~'.Altematively the receptor may require an additional factor in order to bind its For instance, a ligand for the Ret receptor has recently been identified as glial ce11 line derived neurotrophic factor (GDNF)~*?This is thought to activate Ret indirectly through the intemediary protein GDNFR-a,a glycosyl- phosphatidyl inositol (GPI)-linked ceil surface protein2). Finally, some receptors such as the ErbB2 receptor (frorn the EGF receptor subfamily) form a heterodimer with the EGF receptor, rather than a homodimer with another ErbB2 receptor, upon ligand binding2. Dimerization of RTKs brings the inmicellular domains of the receptors into close apposition, and is thought to induce a conformational change in the kinase d~rnain~~"~~~.This in tum allows trans-phosphorylation of specific tyrosine residues in the intracellular region of the receptors to ~ccurl*~~.Phosphorylation of tyrosine residues has two key effects on the activity of the receptor. The first is that it increases the kinase activity of the receptor, which cm lead either to additional autophosphorylation, or to phosphorylation of receptor s~bstrates'*~*'.The mechanism whereby thp kinase activity is increased foilowing autophosphorylation has been explained for the insulin receptor, based on its crystal These studies have shown that the mphosphorylated tyrosine residues are positioned such that they fom part of a Ioop that physically blocks the active site of the kinase. Phosphorylation is thought to induce a conformational change which moves the loop out of position, thus exposing the active There are many substrates that RTKs act onL4. These include other enzymes whose activities are regulated through phosphoryiation by RTKs, and structural protehs, the phosphorylation of which can lead to changes in ce1 shape2*'. A second effect of tyrosine autophosphorylation is that the phosphorylated tyrosines residues can contribute to the formation of sites that are recognized by proteins containing Src Homology-2 (SH2), and phosphotyrosine binding (PTB) d~rnaios'~"~~~".SH2 domains are 100 arnino acid motifs that occur in a variety of proteins, includhg Src, phospholipase Cy, and the p85 subunit of phosphoinositol-3 (PI3) kinase2A2031a25.SH2 domains interact with phosphorylated tyrosines that have specific 3-6 amino acid motifs on their C-terminai side2? PTE3 domains are also 100 amino acid motifs, and similarly recognize phosphorylated tyrosines, but in the context of specific arnino acid motifs on their N-terminal side2'. PTB domains are found in the Shc signaling rnolecule, and in insulin receptor substrate-1 (IRS-I)~'.There are a variety of SH2 and PTB domains, each recognieng a specific phosphotyrosine/arnino acid target, allowing for a diversity of specific signais to be gex~erated~~~'.Activation of these downstream signaling pathways ultimately leads to changes in cellular activity (discussed below). Any particuiar RTK may have a number of potential autophosphorylation sites, some of which are maidy responsible for aitering kinase activity when phosphorylated, whereas others prirnarily serve as binding sites for SH2 and PTB domain containing rnolec~les~~~~~.In some cases the signaling molecules are themselves phosphorylated by RTKs, creating binding sites for additional signahg rnoiec~ieJ"~~.The first signaiing molecuie can therefore serve as an adaptor molecule between the RTK, and other factors with which the RTK cannot directly intera~t?'~'. For exarnple, SH2 and PTB containing molecules ofien have other signalllig domains as well, such as SH3 domains (which bind proline-rich regions in other proteins) and pleckstrin homology domains (which bind to G-protein subunits and phospholipids)2~4J021.The RTK may thereby activate signaluig pathways through these molecules as well. The insulin receptor for instance phosphorylates a number of sites on IRS-I , which has several signal transduction domains, including binding sites for SH2, SH3, and PTE3 domaid? Another possibility is that the SH2 domain containing molecule may be a subunit of larger molecule that is activated when the SH2 subunit is bound to phosphorylated tyrosine on the RTK~.For instance, the activity of PI3 kinase is increased when its regulatory subunit is bound to phosphorylated Binding of molecules with SH2 domains may also be used as a mechanism of localking other proteins that are bound to the SH2 dornain to the plasma membrane region, where they can interact with membrane associated factors2. Many specific sigrÿiliag pathways have been shown to be initiated following RTK activation, each of which is thought to lead to specific changes in cellular activity2. One pathway is ded the Ras pathway, and involves activation of Ras, a mal1 guanosine trinucleotide phosphate (GTP) binding protein2*4Jo.The pathway is activated when the SH2 domain containhg Grb2 molecule bhds to target sites on RTKS~"~'.Grb2 is constitutively associated with another factor SOS, which cm catalyze exchange of GDP for GTP to Ras, thus activating a as^*^*^^. Ras ultimately leads to activation of MAP kinase, which is known to phosphorylate various transcription factors, leading to changes in gene expre~sion~*~~'.It is thought that this activation of the Ras pathway leads to stimulation of cell growthJ. Another factor activated by RTKs is the PI3 kinase pathway2"*20.Activation of this pathway can occur via binduig of the PI3 kinase regulatory subunit to RTK phosphotyrosine residues via its SH2 domain as described aboveL4.PI3 kinase activation is thought to be important in chernotavis and cell shape changes4. RTKs can aiso activate phospholipase-Cy, which catdyzes the conversion of phosphoinositol-4,5- bisphosphate (PIP2) to inositol-3,4,5-triphosphate (IP3) and diacylglycerol (DAG)*". DAG is an activator of protein kinase C (PKC), IP3 lads to release of intmcellular calcium stores, which is also important for activation of calcium-dependent isoforms of PKC! PKC subsequently mediates changes in a number of cellular activities4.

RTKs Have Diverse Roles in Manv Orwismg RTKs are known to play roles in many cellular processes. FGFs, for instance, are capable of inducing celi proliferation and chemotaxis, as well as angiogenesis in mad6.The FGFR has also been shown to be important in the formation of the mesoderm in ~enopd'. Many RTKs also have important roles in the nervous system. For instance FGF receptors are involved in the formation of the central nervous system (CNS) in a number of organisms, such as mouse and ~roso~hifa'~~~~'.In particular, the mouse FGF receptor has been show to be expressed in neuronal tissues fiom developing mouse embryos, and the FGFR in Drosophifa has also been shown to be expressed in the CNS where it is thought to play a role in the formation of axon c~mmisures~~~~. The neurotrophin Trk receptors also have important roles in the functioning and development of the nervous ~~stern~'~~~'.The Trk receptors have ken shown to be particularly important in the bctioning of several aspects of the mammalian nervous system, including synaptic plasticity, neurod dnerentiation, su~val, and regeneration following injury29J013'.For instance, it has ken show that the Ligand of the RTK Trk 8, brain derived neurotrophic factor (BDNF), can induce enhanced synaptic activity in the hippocampus, and that this can be blocked by inhibitors of tran~lation~~.Similarly, it has been shown that induction of long term potentiation (LTP), a process thought to be central to memory formation in humans, is also blocked by inhibitors of RTKS~~.The neurotrophins BDNF, nerve growth factor (NGF), and neurotrophin-3 (NT3), ali of which act through RTKs have also been shown to cause dendritic gmwth and arborization in the developing visuai cortex, suggesting their RTK receptors are important in this developmental process as wei134*35.It has also been show that neurotrophins can stimulate proliferation, differentiation, and survival of neuroblasts31*36*37. For example mice expressing defectively mutated TrkA display extensive neuronal loss in trigeminal, dorsal root, and sympathetic gangiia38. Behaviorally these mice have severe sensory defects, including los of nociceptive activity, and deficits in sensation of temperature3'. Another RTK that has been show to have an important role in the development of the mdannervous sy stem is muscle specific kinase (MUSK).'~-~*. A muscle specific kinase receptor was originally cloned hmTorpedo californica4',and human and rat homologues have now been cloned as They aii have a hi& level of amino acid identity to members of the Trk subfamily on theû intracellula. portion, and to members of the Rot subfamily on their exîraceîiuiar portiodgAO? However, MuSK receptors are different enough hmthe 0th receptor abfamilies that they are thought to form a distinct s~bfamil~~~~~*~'.Human MuSK is expressed primanly in myoblasts at the neuromuscular junction (NMJ) and contributes to clustering of acetylcholine receptors (AchR) and other synaptic cornponent~~~*~'".It also modulates synapse specific transcription, and presynaptic growth and differentiati~n~~~'~~.The mechanism whereby activation of MuSK leads to AchR clustering is beginning to be elucidated, and is similar to that between Ret and GDNF where an intermediary molecule is important in ligand binding. Specifically, MuSK is thought to be indirectly activated by the protein agrh, a nerve cell derived factor that becomes embedded in the synaptic basal la~nina~~~'.It has ken suggested that agrin may bind some other unidentified factor(s) refened to as myotube-associated specificity component (MASC), which then binds to MuSK and activates it, leading to the effects descnbed ab~ve''~?In addition, the MuSK and Ror receptors both have cysteine rich domains in their extracellular portion that are homologous to each other and to cysteine rich domains from an unrelated family of receptors cded fiitrled3". These are seven- transmembrane receptors that are known to have molecules fiom the Wnt farnily of glycoproteins as ligands43w44.In particular, the Wnt ligands are known to bind to the fnzzled cysteine rich domains, and because of this it has been suggested that the Ror and MuSK receptors may have related ligands43". Receptors fiom the Ret subfamily have been shown to be important in the development of some human diseases, in particular in the formation of type-2 neoplasias, and in the development of the hereditary disorder Hirschprung's diseaseJ5. In the development of neoplasias, Ret is altered by a gain of bction mutation, while a loss of function mutation is thought to occur in the development of Hirschprung's diseaseJ5. Each of these diseases is charactenzed by abnormal migration, proliferation and survival of neural crest cells, suggesting a role for Ret receptors in these activities? Ret receptors have been shown to be expressed in cells of the adrenal medulla, and thyroid, as well as Ui Schwann cells, sympathetic neurons, sensory neurons, motor neurons, and doparninergic ne~rons~~.The Ret ligand GDNF has been shown to play an important role in suMval and Werentiation in these neuronsgu? RTKs in Invertebrates A number of RTKs have also been recently cloned fiom non-rnammalian organisms such as the fitfly Drosophila melanogaster and the mollusks Ly»inaea stagncrlis and Apfysia caliji7rnica. Three RTKs, cded ~trk~~,~ror", and ~nrk~',have been recently cloned fiom Drosophila that are related to the Trk and Ror subfamilies. Dtrk is homologous to members of the Trk family of RTKs, is expressed maidy in neuronal embryonic tissue, and may regdate neuronal celi adhesion and axonai guidance during development of the Drosophila nervous ~ystern~~.Dror, is a Drosophila homologue of human Rorl and ~or2~'.Human Rorl and Ror2 are expressed in both neuronal and non-neuronal tissue, while Dror is expressed mdy in late embryonic stages in nervous system tissue, and may be involved in regulating neuronal de~elo~rnent'~~~'.Finally, Dnrk has strong identity with members of the Ror subfamily, and is also expressed specifically in the nervous system of Drosophila during neuronal development. It is thought to play a role in neuronal differentiation and synapse formationJ8.A Trk-like receptor has also ken recently cloned fkom the snail Lymnaea stagnafis, and is designated ~trk~~.That receptor is expressed mainîy in the adult brain of that animai, including expression in neurons and in associated endocrine ti~sue''~.Ltrk is also expressed during embryonic development in Lpnaea, though a specific function has yet to be a~si~ned~~.A homologue of the insulin receptor has been cloned frorn ~~f~sia~~.It has been shown to have a dein modulating synaptic currents in neurons involved in reproductive behaviors in that animals0. Other RTKs are likely present in Aplysia. For instance, prelirninary results suggest BDNF causes a similar enhancement of synaptic activity in Aplysia as it does in the hippocampus, suggesting neurotrophin- like receptors are present in Aplysia as well, and are involved in neuronal activities5l.

RTKs and Memory Formation in Aplwia califwnicg One important role RTKs may have in Aplysia is in the formation of memory. Memory is believed to be created through the formation dorstrengthening of specific syaaptic connections between ne~cons~~~?Memory is also believed to consist of 3 stages: short term memory, intermediate memory, and long term memorysz5s56*57.Each of these stages of memory is associated with molecular changes that have effects on the strength of synaptic connections between neuronss2. Sensitization is one form of memory that is observable in Apijda. ApZysia norrnaily withdraw their gill, siphon and made shelf in response to a light touch to the siphons2. This reflexive response is considered analogous to defensive withdrawal stimuli observed in vertebrates. However, if a noxious stimulus is delivered to the animal prior to the touch stimulus then the length of time of the withdrawal to the light touch will be greatiy increased. The animal is then said to be sensitized to physical stimuli. A single noxious stimulus has been shown to lead to short term sensitization lasting for minutes, while several repeated noxious stimuli cm lead to long term sensitization lasting for up to 24 hours or moresz~ss~s8~sg.The neural circuitry that is responsible for mediating the gll withdrawal reflex has been determined and consists of sensory cells in the siphon and made shelf that synapse either directly to motor neurons in the gill and siphon, or indirectly to them through intemeurons"? The motor neurons control the muscles involved in the withdrawal movement. Alterations in the activity of these cells have been shown to be invo lved in the behavioral ~ensitization~~.In particular, monitoring neuronal activity of these cells has show that the magnitude of monosynaptic excitatory postsynaptic potentials (EPSPs) in the rnotor cells is greatly increased in response to sensitizing stimuli. This increase in response is due to hcreased release of transmitter containing vesicles by the presynaptic sensory neuron (or intemeuron) to the motor neuron involved in the movement? This process whereby sensitizing stimuli lead to increased transmitter release, and stronger EPSPs is referred to as facilitation of synaptic activitys2. Facilitation accompanies sensitization, with a single stimulus producing facilitation lasting for minutes, while a train of stimuli produces facilitation lasting for up to hours 52.55.58.62

Serotonin Induces Translation and Intermediate Memory in ApZvsia Serotonin (SHT) is a modulatory transmitter that has ken shown to be released in intact Aplysia in response to sensitizing stimuli6'. 5HT is also capable of stimuiating synaptic facilitatiod3*" when the neurai circuit involved in the withdrawal reflex of Aplysia is ncoostnicted in ce11 cultures6? As with physical stimuli there are short- and long-term effects of administration of 5HT. A brief pulse results in facilitation in the cultures lasting for minutes, while several pulses mult in facilitation lasting for up to hourss2. An intermediate phase of facilitation has also been recently show in sensory- motor ceil cultures that is stimulated by 5HT, and which is dependent on translation but not on transcriptionss. Furthemore, administration of 5HT to isolated clusters of sensory cells fkom Aplysia, as well as isolated pleural ganglia, has been shown to lead to changes in the rate of translation in the cells during the same tirne intermediate facilitation de~elo~s*~*~~*~~.Based on this it has been proposed that activation of translation by 5HT is a critical step in the formation of intermediate memory in ~~lysia~'.

RTKs and Intermediate Memory in A~lvsia Some of the second messagers which 5HT acts through h Aplysia, and which are believed to lead to the formation of intemediate memory have aiready been detemined. It has been shown that the 5HT mediated increase in translation at intermediate time points can be blocked by administration of the dmg rapamycid7. In ce11 lines, there is a growth factor stimulated pathway that leads to increases in translation and which is also sensitive to rapamycin6'. Induction of this pathway occurs through RTKs and leads to the activation of a number of other factors which al1 have effects on translation. In particular, activation of RTKs are thought to lead to induction of PI3 kinase activity, and through this to activation of a kinase called FRAP. FRAP can modulate the activity of a number of other factors that lead to changes in the rate of translation. In support of there king a sWar pathway in Aplysia it has been show that the 5HT mediated increase in translation can be inhibited not only by rapamycin, but also by herbimycin A, an inhibitor of tyrosine kinases. It is possible therefore that 5HT also acts through an RTK in the Aplysa pathway. If RTKs are involved in the Apiysia rapamycin sensitive pathway in pleural ganglia, they could prove to be important factors in the development of intermediate memory5'.

. . Rationale and Ob!eca ves The aim of this project was to clone receptor tyrosine kinases hmAplysia. These clones could be investigated in the hture as to their precise role, such as in memory formation. In order to clone RTKs f5om Aplysiu we took advantage of the high degree of sequence identity between RTKs. In particular, we designed primers aga& well conserved regions of RTKs that are known to be important in neuronal activities, such as the Trk recepton. We originally amplified fragments of RTKs by polymerase chah reaction (PCR), and subsequently designed new primers against those fragments to screen an Aplysia nervous system library by hybridization. This process was employed to obtain full length clones.

Clonine of Rn

~~Discoidln 1-Il& domin ECF-llh dodn 0 C3dhuln Cysteinc rtch regions 0 domain lIrrrllIilllll l\M Lrucfne rich motifs 0mde~~

. . . . mm:Van Der Gcer P. and Hunter T. 1994. +wavs. Annual Review of Cc11 Biotogy, Vol 10: 251-3371. Fieure 2 - Phvlo~eneticRelationships of Various RTKs Molecular phylogeny of 52 RTKs. Abbreviations are: IGF-IR, insulin like growth factor- 1 receptor; CSF-I R, colony stimulating growth factor4 receptor; SCFR, stem ce11 factor receptor. Other abbreviations are as defined in figure 1. Taken nom reference 2. Figure 2: Molecular Phvlogeny of Receptor Tvrosine

. . . . Fmm: Van Der Gcer P, and Huntcr T. f 994. 9W. Annual Review of Cell Biology, Vol 10: 251-3371. Bure 3 - Ali-nt of RTK base Domains Amino acid sequence alignrnent of the catalytic domains of 14 RTKs. Highly conserved residues arnong aü protein kinase sequences are indicated by black boxes with white lettering. Arnim acid numbering is for human PDGFRP. Roman numerals indicate the kinase sub-domain. These I 1 sub-domains are regions of higher conservation amongst the kinases. Taken from reference 2. Figure 3: Sequence Alignment of Receptor Tvrosine Kinase Catalytic Domains

600 LVLGRT UTAHCLSHSQ...... LV '&ln KATAFHUCRA...... DlFEDL RAnIKKOC,...... RURL OCMARüIIWE...... ,. IVtKWE UECHNUPEQ...... VHFNW HGTLLDNDC...... VAUUT ECQLlUQOO...... fUKIKV XCLWKPEG&K.*...... o*,. WN RG'tLRLPSO...... LQTftF LAKAKGAUME...... :Y 'Al@ LAfXZC*~rnKFN...... RWtOV HCILSDEKDPN...... VRFHÇC KCHLYtPCE1D...... HHPIIVCU LRFKCK LCEVûSPQDLVSLûFPraVRffi ..ICD?Y LIRLL 1 1I III

~~669 E1 1~~

CLCRU...... CACT W...... CPLWIVEYASUGblLRtYLQCLRAOPCtMCrW?SHW?EEQ...... WSKOLVSCIIYQVA HVCIEEG.....CKPWVIU~GHLKtttAQcirt~OA*...... ~...... ISCgDL~IgU GAWQE...... QPVCXtFEYtNOCOWlCFLIWPHSDVGCsSDtOGNICSS...... UHCDP LHUIQIA CVCVOD...... ~PIL)~I~~~~~~~,u~QF*JMQ~E~~uAE~QwcP~...... ISIPMU(YIUOIA

~~Vtb VI1 VXXI 1X~~

. . . . From: Van Der Gccr P. and Hunter T. 1994. gekina~cs -. -. Annual Revicw of CeIl Biology, Vol 10: 25 1-3371. Fipure 4 - Crystal Structure of the lnsulin Recmtor Crystal Structure of the Insuiin Receptor. A) Stereo view of a Ca trace of the insulin receptor. Every tenth residue is marked with a Wed cucle, and every twentieth residue is labelled. Taken hmreference 19. B) Activation loop of the phosphory lated insulin receptor. Taken fkorn reference 18. Figure 4: Crvstal Structure of the Insulin Receptor

[Figure 4A from: Hubbard SR. Wei L, Ellis L, and Hendnckson WA, Nature, Vol 372: 746-754, 1994. Figurc 4B from: Hubbard SR, EMBO Journiil, Vol 16: 5572-5581, 19971. Materials and Methods Construction of cDNA Librarv

Apiysia califmica (75- 125 grams) were O btained nom Marine Specimens Unlimited (Pacific Palisades, CA, USA), and maintained in an aquarium for at least 3 days before experimentation. The animals were fmt placed in a bath of isotonic MgClz/artificial sea water (1: 1, voi/vol) and then anaesthetized by injection with isotonic MgCI2 solution. Pleural, pedal, and abdominal ganglia were dissected fiom the animais and immediately frozen in liquid nitrogen, then processed using Qiagen RNEasy Minikit (Qiagen, Santa Clarita, CA, USA) to obtain total RNA, accorâing to manufacturers specifications. Complementary DNA (cDNA) template was made using Superscript II reverse transcriptase (Gibco BRL, Gaithersburg, MD, USA), according to manufacturers specifications. Finally, cDNA product was used as a template in polymerase chab reactions (PCR)to ampli@ RTK fragments as described below.

PCR Based Screen and Design of Oligodegenerate Primers In order to ampli@ fragments of RTKs, oligodegenerate primers were designed that would hybndize to well conserved regions of RTKs. To do ths the arnino acid sequences of a nurnber of RTKs were aligned using Geneworks alignrnent sofiware (Intelligenetics Inc.. Mountain View, CA, USA), and highly conserved regions were identified (Figure 5). Those regions which were the least degenerate were chosen as target sites against which primers were designed. Aplysia codon usage tables were employed to Mer lirnit degeneracy by choosing primer sequences that would anneal mainly to those sequences most likely to be represented in the Aplysia gemme. Seven prirners were designed in dl, and are indicated in figure 5 by arrowheads with conespondhg primer names above the regions against which they were designed.

The forward primer narnes and their sequences (5' to 3') are ('N' indicates all four nucleotides used in that position): FO- GA(A/G)GGNGCNTT(C/T)GGNAA n- ATG(G/ C/T)TNTT(T/C)GA(A/G)TA(T/C)ATG The reverse primea are (5' to 3'): RO- TANGT(A/G)(A/T)A(A/G/T)AT(T/C)TCCCA R02- AGCCA(C/G)A(A/G)(A/G/C)A(T/C)(A/G/C)AC(AfG/T)CC FU- TCN(G/C)(A/T/G)NGGCATCCA Ru- GC(AlGlT)GC(CIG)A(NG)(A/G)TC(T/C)C(G/T)(G/A)TG

PCR reactions were performed using ber reaction): 1 unit Taq DNA polymerase (Promega, Madison, WC, USA), 1X Taq DNA polymerase bufFer (Promega), various concentrations of MgCl2 (varied to optimize amplification) mging from 1 to 2 mM, 1 pM of each primer, and 8 pl of cDNA template obtained fiom Superscript II reverse transcriptase reaction described above. Controls included no template, and reactions where only a singie of each primer was used. Products fiorn each PCR reaction were separated on a 1% agarose gel that was incorporated with 0.5 @ml ethidiurn brornide, and visualized over ultraviolet light. Bands in the expected range of size were excised using sterile technique and soaked in Iml of water to elute amplified DNA into solution. 8 pi of this was then used as template in additional PCR reactions where bands were re-amplified using primers internai to those used in the original reaction (i.e.- a 'nesting' strategy) to determine whether the interna1 primer sites were present in the amplified hgment as well, as would be expected only in achial RTK fragments. In cases where nesting strategies were successful both the PCR product that contained the origuial amplified band, and the product that contained the srnalier 'nested' band were cloned into TOP-IO celis using a TOPO TA cloning kit (uivitrogen, Carlsbad, CA, USA) according to manufacturers specifications. Sarnple DNA was then extracted fiom clones using Miniprep Kit (Qiagen) and digested with 5 units EcoRI, Ix EcoRl reaction bder (both hmPromega), supplemented with 10 pgpi acetylated Bovine senim albumin, to ensure the presence of the cloned insert. Finally DNA was sent for sequencing using the services of The W.M. Keck Biotechwlogy Resource Labonitory (Yale University, New Haven, CT, USA), and Bio S&T Inc. (Montreal, PQ, Canada).

Screening for Full Length Clones As discussed in 'Results' the PCR reactions produced fbgments of putative RTKs nom which it was necessary to obtain full length clones. To obtain full length clones probes were designed against those hgments already obtained and wdin a hybridization screen of phage plaques. Specifically, probes were designed by randomly priming the PCR Fragments for each clone in the presence of radioactive deoxycitidine 5'- triphosphate (dCTP) [ao3*p;6000Ci~mmol] (NEN, Boston, MA, USA) and cold dATP. dGTP and dTTP, using a Random Primer Labeling Kit from Gibco BRL, according to manufacturers specifications. For Aror. a partial clone extending to the end of the ORF was initially obtained (see Results), from which it was necessary to construct a new probe targeting the 5' end of that clone. To do this primers were designed targeting the 5' end of the clone, and used in a PCR reaction employing DNA fiom the clone as templaie, and radiolabelled dCTP (as above) in addition to 'cold' (IATP, dTTP, and dGTP. The procedure for the PCR reaction was otherwise followed as described above.

~~The primers used were (5' to 3'): Forward primer: TACTGCTACACCGGAAGAGG Reverse primer: TACATCAGCTTGTTGGAGCC~~

Phage library plates were made using XL 1Blues cens as hosts. Specificaily, 10 pl of 5000 phageipl lambda zap-II library phage were inoculated in 600~1of XL l Blues cells grown to O.D. 600 of 1.O in Luria-Bertani (LB) medium (IO@ NaCl, IO& tryptone, 5g/L yeast extract), supplemented with the aatibiotic tetracycline (0.5 mglml), 1OmM MgCl*, and 0.2% maltose solution. Celis were then incubated 15 min at 37OC, and subsequently mixed with 9 ml of 4g°C top agarose (0.7% agarose, LB [as above], supplemented with lOmM MgC12, 0.2% maltose) and poured over 150x15 mm agar plates (Li3 [as above], 1.4% agar). Plates were allowed to harden for 15 min at room temperature, and then incubated ovemight (ON) at 37OC. The number of phage plaques were counted on a section hma number of plates to ensure a useful number of phage were present to be screened (roughly 1 million phage were screened in d). Phage were then lifted off the plates by overlaying them with 0.45 micron nitroceilulose membranes (Micron Separations Inc., Westborough, MA, USA) cut to fit the surface of the plate. Membranes were left in contact with the plate for 60 seconds, and then irnmersed successively in denaturation solution (1SM NaCl, 0.5N NaOH) for 3 min, neutralization solution (1 SM NaCl, 0.5M Tris [pH 7.51) for 5 min, and 2X SSC (20X stock: 175.38 NaCI, 88.2g sodium citrate, pH 7.0) for 30 sec. Two such filters were 'lifted' for each plate so they could be aligned later for determination of background levels of radioactivity. Filters were then air dried for 30 min and baked in a vacuum oven at 80°C for 2 hrs. Prior to hybridization nitrocellulose filters were pre-washed in SOmM Tris (pH &O), 1M NaCl, 1mM EDTA, 0.1% SDS for 1 hr at 50°C to remove ioose surface particles , residue, and dust. This wash solution was changed after 30 min of wash time and replaced with pre-warmed, fresh wash solution. Filters were then prehybridized in 6X SSC, 0.1% SDS, 1mgtml tRNA, 5X Denhardt's reagent (50X stock: 5g Ficoll Type 400 [Pharmacia, Uppsala, Sweden], 5g polyvinylpyrrolidone, 5g fraction V BSA, 500ml H20)for 2 hrs at 50°C. The radiolabelled DNA probe (see above) was denatured immediately before hybridization by boiling at l OO°C for 5- 10 min. Hybridization was carried out in prehybridization solution supplemented with the a-"P dCTP probe at 65OC ON using probe concentration of at least 1 million counts per minute (cpm) per ml of hybridization solution, and 2ml hybridization solution per nitrocellulose filter. After hybridization, fdters were air dried for 30 min, and exposed to X-OMAT blue XB-1 füm from Eastman Kodak (New Haven, CT, USA) for 36 hrs. Positive signals were taken to be only those that appeared in equivalent positions on films correspondhg to both filtea lifted off a single plate. Cores of agar around phage that rehimed positive signals were then removed off the plates and soaked ON at 4OC in 1 ml SM (5.8g NaCl, 2g MgS04, 5Oml 1M Tris [pH7.5], 5ml 2% gelatin solution [2g gelatin, 1OOml H20],fil1 to 1L with &O) supplemented with 2 drops of chloroform (Fisher Scientifïc). The titer of these new stocks of phage in SM were detennined by pourhg new plates (as described above for screening, except using SM fiom the cored phage instead of the phage library), and counting the number of phage plaques obtained for various dilutions. Appropriate amounts of SM from the cored phage were then used to pour plates that were used in a secondary screen, such that approxirnately 500-1000 plaques would be screened per clone. A third screen was sirnilarly carried out, where the titer was chosen so that approximately 50-100 phage plaques would be obtained. From this screen a single phage plaque (well separated from other plaques on the same plate) was removed for each clone, and a final screen was performed to ensure all phage plaques were rehiming a positive signal (controls included phage that did not previously return a signal). Phage that retmed mostly positive signal kom the final screen were 'cored', and cloned as descnbed below. To obtain clones, excision reactions were performed accordhg to the method of Russel et aL6* on the cored phage from the final screen. A number of colonies from each clone were then grown OM and DNA was purified by 'rapid miniprep'. 'Rapid minipreps' were performed as follows. 1.5 ml cultures were grown oveniight in LB medium supplemented with ampicillin. Samples were ceneifuged 1 minute in a rnicrofuge at lOOOOg and supernatant was aspirated and discarded. Pellets were resuspended in 100 pl of solution of 5 mM sucrose, 10 mM EDTA, and 25 mM Tris, pH 8.0. Subsequently

200 pl of solution of 0.2 N NaOH, and 1% (wlv) SDS was gently mked into this. This was incubated at room temperature for 3 min and then 150 11 of solution of 3 M sodium acetate, pH 4.8 was gently mixed into this. This was then incubated on ice for 20 min, and centrifuged for 5 minutes at 10000g. The supernatant was then removed to a clean microfige tube and 1 mi of 95% ice-cold ethanol wad added and mixed. The sample was then centrifuged for 5 min, and the pellet was washed with 0.5 ml ice-cold 70% ethanol. Finally the pellet was ait-dried for 15 minutes, and resuspended in 100~1of water. Clones were subjected to digestion with a number of restriction enzymes to detennine theu size, whether my clones overlapped with each other, and the approximate position of the regions correspondhg to the original PCR fragments within the clone. As discussed in the redts a number of clones had to be sequenced to obtain the full Iength of the RTK coâing region. Sequencing was again performed by the Keck Biotechnology Resource Laboratory, and Bio S&T. Fi~ure5 - Clonintz of Ap[vsia RTKs Strategy for cloning RTKs fiom Aplysia. The alignment includes the kinase domains of Drosophila Trk (Dtrk), human TrkA, and human Rorl. The consensus sequence is inàicated above the other sequences. Highly conserved residues arnong aii the sequences are indicated by black boxes with white lettering, dashes indicate gaps, and dots indicate no consensus. Above the aiigment, degenerate primer sites are displayed by name (FO, FI,. ..), and arrowheads indicate the direction in which the primer amplifies template. Fie. 5 Strategv for clonhg RTKs fiom Aplvsia

~~FO---w Consensus WGmKVYK G.-L.LP...... LmImAm PE.AS.... Q D~RMAE~~..~~

~~Consensus Humrorl Drotrk Humtrka~~

~~Consensus Humror 1 Drotrk Humtrka~~

~~<--- R 1 Consensus ~SMF~L~Dm .---r# Q s K s L~I= mPMSILOGO UT T EmVm~~

Humrorl ~s~L~L~€=A- s K s ~mImmPmAfl)iIP~m OS s DWI~ Drotrk ~sPF~LWD~S- Q s K s LQPIv~~s~~s~L~~ PTT EMV~ Humtrka ~GPF~I~I~DmTmG 6 R T MmIm ~PUSBLORHMT T EmVm Results Clonine of Aplysia RTKs d rn~lificationof PCR- fiamnents To identify RTK genes that are expressed in the Aplysa nervous system, cDNA template was constructed hm Aplysu nexvous system RNA, and screened using oiigodegenerate prirnen designed against well conserved regions of known RTKs. PCR proilucts were obtained using severai of the primer pairs [Table il. Specificaily a 500 base pair (bp) fragment using primers 'FO' and 'RI', a 550 bp fiagrnent using the same primers, as well as a 450 bp fiagrnent using primers 'FO' and 'Ri2'. These fragments were then subjected to MerPCR employing a nesting strategy, where intemal pnmers were used to amplify smaller Fragments that would be expected only if the original fragments were from genuine RTKs. Nesting the 550 bp fragment with pnmers 'FO' and RI2' produced a 420 bp fragment, while nesting the 450 bp and 500 bp products produced no new fragments [Table 11. Al1 four Fragments were cloned into pcr-2.1 vector, and DNA fiom several colonies of each clone was sequenced. The clones obtained hm the 500 bp 'F0R.i' fiagrnent were found to have slight sirnilarity to a number of E. coZi genes. Using a BLAST search6', some of the clones obtained fiom the 550 bp çdgment were found to have strong similarities to the kinase domain of the RTK ~nrk~'(and to a lesser extent to human ~orl/2'~,~ror", and human MUSIC'?. This fiagrnent wouid ultirnately lead to identification of a fidl length clone that we named Aror (for Aplysia Ror). Other colonies fiom this clone were found to have DNA encoding non-RTK DNA that had weak similarity to E. coli genes, and were not investigated Mer.DNA from the colonies cloned with the nested product were found to be identical in sequence to the region from the 550 bp fiagrnent comsponding to where the intemal primers were expected to ampli@ the DNA. The other 450 bp fiagment however was fond to be distinct from the first clone, and had strong identity to the kinase domains of chicken et", Drosophila et", mouse FGFR~', and Drosophila FGFR'~. This hgment also led to identification of a NI length clone that was named Ad[Table 11. Aror HvbridizafionScreen A hybndization screen was perfomied to obtain full length clones using randomly primed, radiolabelled probes for each clone. S~reeningwith the Aror probe led to the isolation of 3 clones, the sizes of which were 4 kilobases (kb), 1.5 kb and 0.8 kb. Each of these clones was subjected to a number of restriction digests to ensure they contained restriction sites present in the original 550 bp PCR fragment, and to determine what additional regions of sequence they would encode. The restriction maps of these clones (labeled as clones lA, 1B and lC, respectively) are shown in figure 6. These results suggested the 4 kb clone (1A) would encode the entire 3' end of the RTK open reading frame (ORF)and would likely extend into the 3' untranslated region of the gene. Al1 three clones were sequenced [Figure 61. As expected, the 4 kb clone extended to the end of the ORF, contained an in frame stop codon, included some 3' untranslated region, but based on the deduced amino acid similarity to other RTKs did not extend to the beginning of the ORF. There were a few discrepancies between the sequences obtained from each of the clones, however these were all codined to the third nucleotide positions of codons, and therefore do not affect the deduced amino acid sequence of the protein. These discrepancies may reflect polymorphisms present in Aplysia. To obtain the rest of the ORF a new probe was designed using prhers targeting segments near the 5' end of the 4 kb clone, and performing PCR reactions in the presence of radiolabelled nucleotide (see Methods). A new hybridization screen was then performed using this probe, which resulted in identification of 4 new clones. These are indicated as clones 2A through 2D in figure 6. Initially clones ZB, ZC, and 2D were obtained, and restriction analysis suggested 2B and 2C were equivalent and extended fùrther towards the 5' end than 2D. Based on this clone 2B was sequenced [Figure 61. Clone 2A was later obtained as well and restriction analysis indicated it would extend the furthest towards the 5' end of the ORF, and a portion of it was therefore sequenced as well Figure 61. This clone was found to include an initiating methionine, and when ail the sequenced DNA was assembled (using DNAstar software; Intelligenetics Inc.), a complete open reading fnune was observed Figure 61. Amr encodes a putative RTK reluted to the Ror subfàmilyy The nucleotide sequence of Aror [Figure 71 contains a 3435 bp open reading he with an in hune stop codon at the 3' end. The sequence beyond this stop codon is highly repetitive, characteristic of 3' untranslated region. There are five putative hitiating methionines near the 5' end of the ORF, the fust of which is preceded by an in frame stop codon. It is unclear which of these will be the one that is used in vivo. The second, and fourth methionines are associated with sequence that most closely matches the eukaryotic consensus for translation initiation, while the fïrst methionine has the weakest. Specifically, the most important residue in the consensus sequences are an adenosine or guanosine at the -3 position72,as occurs at the second, and fourth methionines. Also important is the presence of a guanosine at the +1 po~ition'~,which does not occur at either of these methionines. The first, and fifth methionine in contrast does not have either of the consensus sequence residues, while the third has a guanosine at the +1 position. The length of the signal sequence and its cleavage site were detemiined using PSORT-II ~oftware'~.Sequence starting from each of the possible initiating methionines was evaluated using the software. Using the first methionine the software predicted there would be no signal sequence, and that the sequence fiom a valine which occurs six residues prior to the second methionine through 1-1 1 (d residue numbering is from second methionine) would correspond to a transmembrane domain. The presence of a second transmembrane domain such as this is unusual in RTKs, although the Drosophila sevenless RTK is similarly arra~~~ed'~.Using any of the other methionines predicted the signal sequence extending from the methionine until a cleavage site between S-49 and Q-50 Figure 71. Based on this the signal sequence associated with the second methionine would be 49 amino acids, which is unusually long, whereas that for the fourth methionine would be 22 amino acids, which is a more common length? There are no other methionines in or out of frame prior to the 5' stop codon that wouid make better initiating methionines, nor 3 ' to the fiAh methionine discussed. The deduced 1 145 amino acid (a.&) protein resembles a typical Rn< Figure 71. It contains a 507 a.a extracelidair domain, a highly hydrophobie 25 aa trmsmembraae domah, and a 613 a.a intraceiiular domain. The sequence includes a number of residues, which lie in the kinase domain, that generally distinguish between tyrosine, and serine/threonine kinases. In particular, the sequence includes the motifs 713- HRDLAARN-720, and 755-PVRWMPPE-762,which in serinelthreonine kinases are HRDLKPEN, and G-[TISI-xx-[Y/F]-xAPE, respectivelysPp7.A BLAST search indicated Aror has the strongest identity to human Rorl and ~or2'~,and ~ror'~.It also has strong similarity with ~nrk'*,and to a lesser extent MUSK~~.Overail, Aror has 25-33% identity with the Rom, which is similar to the level of identity between hurnan Rorl and ~rorl'". In contrast Aror has only 15-20% identity with ~trk~~and human ~rk~?Domain-by- domain comparison revealed that Aror is closest to Human Rorl and Ror2 in ali regions, except in the kinase domain where it is closer to Dror [Figure 81. The sequence alignment of Aror, with Rorl, Ror2, Dror, Dnrk, and MuSK is shown in figure 9, and its phylogenetic relationship to the other receptors is show schematically in figure 10. The extracellular domain of Aror Uicludes a cysteine rich domain, an Ig-like domain, and a kringle domain Figures 7,8]. The cysteine rich domain consists of a number of cysteines occurring at regular intervals, and is homologous to those present in the Rors, Dnrk and MUSK'~? The spacing of the cysteine residues is particularly well conserved, though a number of other residues in this region are also conserved. To a lesser extent the cysteine rich domain of Aror is also conserved with those from the friaed family, pnmarily in the spacing of the cysteine resid~es'~*".The consensus sequence for the IG-like domain is Vx(VL)xC(S- 12x)W(ZO-SOx)DxGxYxC, w here 'x' indicates any amino acid". There is some similarity between the Aror and other Ror Ig- like domains Figure 81. The knngle domain also is homologous to those from the other Rors [Figure 81, as well as fiom MuSK. Each of the conseived regions occurs in similar locations to those in the other receptors Figure 8,9]. The extracellular domain of Aror also appears to include a 45 a.a. insert between the cysteine rich region and the kringle domain that is not present in the other Rors [Figure 91. This region is not homologous to any known sequence. Dror also has a 55 a.& insert however it is placed 5' to the cysteine nch region, and is not homologous to the Aror insert Figure 91. The extracellular domain of Aror also contains four potential N-linked glycosylation sites. Two of these occur at corresponding regions in Human Rorl and Ror2, one is not present in the other Rot receptors, and one occurs in the Aror exüaceiiular insen region Fig- 71- The location of the transmembrane domain of Aror was also detennined using PSORT-II software Figure 71. The domain is equal in size to those of human Rorl, Ror2 and Dror, each being 25 amino acids. The Aror transmembrane dornain shares 40- 50% identity with the vertebrate Ror domains, but is distinct fiom that of Dror Figures 8.91. The j uxtarnembrane region in Amr is also similas in size to those of the other Rors (46 amino acids), but is not homologous to them (1 0025% identity ) Figure 81. Based on sequence similarity to other RTKS'*~*~,the kinase domain of Aror likely begins at a hydrophobie residue (1-571) 7 amino acids upstream of the motif 578- GxGxxG-583. This motif dong with K-605 forms an ATP and ~g'+binding motif present in kinases in general. Also as in moa other RTKs the kinase domain likely ends at L-847. There is significantly less identity to other RTKs outside the boundaries defmed by these residuessl6*'.The kinase domain includes 39 of the 40 amino acids that are nearly invariant amongst tyrosine kinases (and are also present in most serindthreonine kina~es)~*~~'.The one substitution is R-809 which is usually a glycine (G), and this change is conserved amongst al1 the Roa as well as in Ltrk. The kinase domain of Aror is closest to that of Dror, sharing approximately 65% identity with it4' [Figure 101. However it also has strong similarity to hurnan Rorl and Ror2, with which it shares approximately 63%, and 58% identity, respectively. In contrast it has 51% identity with human TrkA, and only 3 1% identity wi th the kinase domain of Dtrk. Like the other Trk and Ror family RTKs, Aror also contains the YdYY sequence motif (amino acids 847-852), conesponding to the autophosphorylation site of the insulin re~e~to?"*~*~.A 16 amino acid insert is also present fiom residues G-682 through T-697. Dror has a gap in this region, while the human Rors have a similarly shed but different insert. Finally, the motif 689-YSEM-692, which occurs within this insert region Figure 71, is a potential binding site for the SH2 domains of Shc, and PI3 kina~e''~'*'~~. The 298 a.a C-terminal domain of Aror is longer than that of any of the other Rors, Dnrk, Dtrk, or Ltrk. It has a number of specific amino acid-rich regions, some of which are present in the other RTKs. The first occurs immediately following the kinase domain, where there is a 60 a.a serine/threonine nch region. Over 48% of the residws are either serine or tbreonine in this region. A similar region with over 50% serine/thteonine residues is also present in human Rorl and RoR, though at 30 aa. it is significantiy shorter thaa the Amr segment. Ako it not well conserved with the Aror segment, sharing only 8 residues in common with it (Figure 8,9). This region is not present in Dror, which has only 15 a.a. in the C-terminal domain4'. Aror dso possesses a large proportion of glutamines (Q) in this region (IO%), which does not occur with the Rorl and Ror2 segments. Once the serines and threonines are factored out, glutamines account for 20% of the remaining residues. Folîowing the serine/threoninelglutamine rich region is a proline rich region that is again present in Rorl and Ror2. In the vertebrate Rors 25% of the residues in this region are proline, as compared to 2 1% in Aror. Once agah there is little overall sequence identity in this region between the RTKs Figue 91. Within this proline rich region there is a potential binding site (932-SPPPY-936)for molecules containhg WW domains Figure 71. WW domains are motifs present in a variety of proteins, and bind the sequence motif XPPX[LIYJ~*'~.They are thought to be involved in intracellular signal transduction"*85.Shortly after the proline nch region in Aror there is an extremely glutamine rich region, where glutamine (Q) residues account for 22 out of 25 positions (88%), the remaining 3 king leucine (L). There is also a 36 a.a. stretch beyond the glutamine rich region where the motif GQ repeats 5 times approximately every 7 residues. Next there is another serinelthreonine rich region, which also occurs in Ror 1 and Ror2. In this 34 a.a. segment 41% of the residues are serine or threonine, compared to 50% in Rorl and Ror2. As with the previous serinelthreonine rich segment there is little identity to the corresponding region in the Rors. Finally there is a PDZ binding domain present at the carboxy-terminus. PD2 domains are also motifs present in various proteins, and are thought to be important in signal transd~ction'~~'~.The recognition site for this sequence is the motif x[S/T]xpA] at the carbxy-terminus of a sequences6-", which in Aror is the motif 1 142-TSNI-1 145.

The hybridization screen for Adclones produced seven clones, ranging h size hmapproximately 1 kb to 4 kb. A number of restriction digests were performed once again to determine what regions of the putative RTK the clones encoded. The restriction maps deduced from thex digests is shown in figure 11. Clone 1 was obtained fist and sequenced. It was found to encode the entire 3' end of the ORF, as weli as 200 bp of 3' untnuislated region, and extends to within 100 bp of the 5' end. Clone 3 was later obtained and sequenced, it was found to encode a full length clone. From these two clones the full length sequence was assembled using DNAstar software.

Aruf- encodes a outative RTK related to the RetIFGF receoior subhmi[ies The nucleotide sequence of Aruf (Figure 12) contains a 2974 bp open reading frame with an in fnime stop codon at the 3' end. As in Aror, the sequence beyond this stop codon is highiy repetitive, characteristic of 3' untranslated region. There is an initiating methionine near the 5' end, preceded by an in fiame stop codon, and associated with a favorable consensus sequence for translation initiation at both the -3 (adenosine) and the + 1 position (guanosine). There is only one other methionine (M- 13) that occun within 100 a.a., and it is not associated with a favorable Kozak sequence. The deduced 991 a.a. Aruf protein also resembles a typical RTK. It contains a 5 14 a.a. putative extracellular domain, 21 a.a. putative transmembrane domain, and a 455 a.a. putative intracellular domain Figure 121. The N-terminal portion of the protein contains a potential hydrophobic signal peptide (a.a. 2-29) that based on cornparisons to other signal peptides would be cleaved between A-29 and T-30, resdting in a mature protein of 962 a.a. The extracellular domain of Adcontains five potentid N-linked glycosylation sites. The kinase domah of Ad includes the characteristic tyrosine kinase motif 733- HRDLAARN-740. It also contains a motif similar to the tyrosine kinase characteristic P- [I NI-[KR]-W-[Tm-APE sequence. Specifically , the Amf sequence (77 5- PLK WMAI E- 782) has substitutions of [Wl to L-776 and P to 1-781. Despite these substitutions the motif remains distinct from the serine/threonine specific motif G-[T/S]-xx-[WF] -xAPE. A BLAST search indicates Aruf has strong sequence identity to chicken c et", mouse FGFR'~~~',~ret", and DFGFR~~.However despite these homologies, it seems likely that Aruf is not a true member of either the Ret or FGFR subfamilies. Anif has only 18-23% identity with each of the receptors listed above overall, which is more similar to the degree of identity between members of diffennt subfdlies than between members of the same subfamily. For example DFGFR and Dret share approxirnately 16% identity, compared to 30.35% betweea the two FGF receptors, or the two Ret receptors. Similarly within the kinase domain there is 53060% identity between the two FGF recepton or the two Ret receptors, as compared to 38% between DFGFR and Dret, and 41 -47% between Anif and any of chicken Ret, Dret, mouse FGFR, or DFGFR Figure 131. The sequence of Aruf was also compared to that of the relatively unrelated human TrkA, and was found to have 21% identity to it overall, and 37% within the kinase domain. Once again this suggests Aruf does not belong to either of the Ret of FGF receptor subfamilies. The ttansmembrane and j-embrane domain of Anif are roughly equal in size to those of the Ret and the FGF receptors, but are not homologous to them. The sequence alignment of Anif with each of these is show in figure 14, and its phylogenetic relationship to the receptors is show schematically in figure 15. The extracellular dornain of Aruf is not homologous with those of the Ret and FGF receptor subfamilies. It shares 17% of residues in common with the mouse FGF receptor, and just 7% with Dret. From examination of the sequence alignment of these receptors Figure 141 it seems likely that most of the matches which do occur are merely by chance. There appear to be two sequence motifs in the extracellular domain of Anif. One is a cysteine rich domain near the N-terminal end of the sequence (C-40 through C-80; Figure 12). This dornain is not homologous to cysteine rich domains fiom other molecules. Another motif is the sequence 270-LRE-272. This LRE motif has also been reported in one other RTK, ~trk~,and was originally reported in s-laminin, where it has been shown to encode an adhesion site between s-laminin and motor neuron~~~*~'. Based on sequence similarity to other RTKs, the kinase domain of Aruf likely begins at the hydrophobic residue L-586,which is 7 arnino acids upstrearn of the motif GxGxxG, as in most RTKs Figure 121. Also as in moa other RTKs the domain likely ends at L-866. As was the case for Aror, the kinase domain includes 39 of the 40 amino acids that are nearly invariant arnongst tyrosine kinases. The only substitution is A-722 which is usually a glycine. This is the only type of substitution that occurs at this position, and it only occurs in a few other tyrosine kinase such as MET,and c-abl, both non-recep tor tyrosine kinase^^*^*'. Motifs present in the kinase domain of Anif include the ATP binding motif (593-GxGxxG-598K-613; Figure 12) witb its kinase domain, characteristic of most RTKs. Aruf also possesses a short kinase insertion sequence (amino acids 679-706), that is characteristic of FGF receptors. Kinase insertion aquences also occur in the PDGF receptors, however they are significantly longe?. There is little conservation between the Aruf insertion and that of the FGF receptors, however they are similar in size (Anif: 27 a.a., DFGFR: 19 aa., moue FGFR: 14 a.&), and in location. The Ret recepton do not have these kinase inserts. Aruf has a 125 a.a. C-temiioal domain, making it longer than most of the Ret and FGF receptor C-tenninai domains. The Aruf domain bears has little identity to the domains fiom the other receptors. An exception to this however is in the region immediately following the kinase domain, where Aruf has residues conserved with the correspondhg region in FGF receptors. Within this region in FGF receptors the motif YL,DL~~~~~~is a binding site for the N-temiinal SH2 domain of phospholipase Cy

(PLC~)~~~'**'~+~~.This motif is replaced by the motif 876-YLVL-879, which would not be as strong a binàing site for the domain, but would fom a binding site for the C- terminai SH2 domain of PLC~'"'"'~. Aside fiom this, as with Aror there are a number of specific amino acid-rich regions in the C-terminal region of Anif. The fiat such region is a 33 a.a. segment that is nch in serinelthreonines (39%) and in prolines ( 18%) [Figure 121. Finally, a second serine/threonine rich segment occurs, starting fiom amino acid 937 to the end of the rnolecule, where 29% of residues are serine or threonine. Neither of these two regions have ken identified in either the Ret or FGF receptors. Table 1 - Summary of Clones ObtaineQ Table of PCR products and clones obtained from screening cDNA template. Primer pair indicates the fonvard-reverse primary combination employed. Nested bands were obtained using DNA purified nom first proceeding non-nested reaction as template in additional PCRs. The expected size indicates the sue of the band that primer pair would amplify if used on a number of other RTKs. Actual band size indicates size of PCR product obtained, as determined by running the PCR products on an agarose gel. Other PCR products obtained outside this range are not listed. In some cases where it was dificuit to visualize a band, DNA was purified frorn an amof gel which covered the expected size range for that primer pair. Table 1: Summay of Clones Obtained by PCR EJuwMA= - EQmhY FOW 500-660 500 Yes Yes E.Coli

~~FOR1 500-460 550 Yes Yes Amr~~

~~Nested FOW 380-420 420 Yes Yes &or~~

FORi2 380-420 400 Y~s Yes Ad EIpure 6 - Aror Hvbridization Scmq Restriction map of Aror clones obtained hm hybridization screen. The white bar indicates the hi11 length of DNA that was sequenced. Enqmes used to construct the map, and the region they cut are indicated by positioning dong the white bar. Clones are labeled as 1A through 1C (fiat screen), and 2A through 2D (second screen). The region corresponding to the open reading frame (ORF), and the origuial PCR product are indicated by black bars. Other black bars: sequenced portions of clones, white mows: unsequenced portions of clones. Clone 1 A extends approximately 1.5 kb beyond the nght end of the figure.

Fiaire 7 - Nucleotide and Amino Acid Seauence of Aror Nucleotide and deduced amino acid sequence of Aror. 5' and 3' untranslated region is indicated at beginning and end of the sequence, respectively. Small black arrows, potential initiating methionines; double-headed white arrow, predicted signal sequence; small white arro ws, gly cosy lation sites; underlines (in order), CRD, Ig-like domain, kringle domain; thick underline, trazlsmembrane domain; large black arrows, edges of kinase domain; double underline, SHZ domain binding site; dotted underline, serine/threonine-rich regions; dash-dot-dot underline, proline nch region; thin white bar, WW binding domain; oval circle, glutamine-rich region; dashed underline, glycine-glutamine repeats; thick white bar, PDZ binding domain; large white arrow, stop codon at end of ORF. EigUle 7: cDNA Sequew and Deduced Amino Acid Sequence of Aror

TGACTTGTCTTCGGTCGAAGTGAGCTTCAAATCACAGCCCATGGTCTATCTTATGATATCATAGATTCCTTATGC TAATCAACGAGTGTAATTCTGTTCTGTATGACCTGTAAGTAGATCTAGCAGGAACCTCTCTTCATATTTAGTTCA CATCATCCGTTAACAATTATAATCGGTATTCGCAAATATTACTATGGGCATTGCTTAGCGCGCCCAGAGTATCAA CTCAGCCACAAAGTGTTCCTCGCTGAACTGTAGAATTGTGCACTTACTATACATTATTTCCGAGTAATTGAATTG TCAAGTCACATATTTCCGACAGCAAACGGTCGTCGAGGTTTGAACCCCAGTCTACAGAGTAGATGTCCCAGCTAC CCTCCTGCCCACGACGCCTCGTTGATGAGGTGGATTAGTCTGTTATTGGAATAACTGGATTACTTTCGTGTGTGT

~~GAGCCGGGAGATATGCTGGACCCATTGATGACCTTTCTCAAGGAAAGCAGCGGCACTTTCGTTGGCAACGGCTAC 300 EPGDMLDPLMTFLKESSGTFVGNGY~~

GGACACAAACCCACGGCCTGGGGTTCTAGACTGAAGATCAACGATGTACGACCTTCCGACTCTGCAGTTAAT 525 GHKPTAWGSRLKINOVRPSDSAVYT TGCAAGGCTGAGAACGACTTTGGCAACGAGGAGACAACTGGCTCTCTCACTGTCCTCAACGAAAATCCTCCGCCC- 600 CKAENDFGNEETSGSLTVLNENPPP

~~TCCAAGTCTCAAGGTGGCAGCAACAACAACGATGACGACTACCCCACTGACACTGACGTGGTGGAAGGTGGAGAG 675 SKSOGGSNNNDODYPTDTOVVEGGE~~

~~FCQIYRGSTCAKFVGNNSIYVTSKL~~

~~-- CQQYGIOSLCYHAFPLCOKTADRPT~~

~~CCGAGGAAGATCTGTAGGGATGAGTGTCTGGCCCTGGAGAATGACATCTGCAGGACGGAGTACTTGATGGCCAAA 1050 AACTGTATCCGTATTGGGATGCCCCCTGGCTCCACGTCCGGACGCGGCCGCCCCAAAGGGGGCAACCCGAGCTGG- 1200 NCIR~GMPPGSTSGRGRPKGGNPSW~~

~~AACAATCCGGGAACCCGCCGTGACCCTCCCAGGGGGTCAAAGCAGCGAATAAACGACCACGTCTATAA 1275 NNPGTRRDPPRGSKGSGSKRPTSOK~~

~~SATAC?SS?tSCSSCCACCAASSAI:fAACASATSTSTACTACCGGTGAG 1350 DTGRGQOGPTOVYCYTGRGTNYRGE~~

~~YCRNPNGREDAPWCFTNORKMPKEL~~

~~TGCGCAGTGCCCAAGTGTAG~GACTACGACGAGGGTCACCCAAGCGAGGCTGACGAAGGCTCAAAACTGAT 1575 I CAVPKCSDYDEGHPSEAOEGSNKLH~~

~~TACATCCTCATCCCGTCCTTGACCGTGCCGCTGGCTCTGGGTATTCTGCTGGCGCTGATCTGCTTCTGTCAGAAG 1650~~

~~TCTCACAACACCAGAGCCTCCAGGCCTAACAACAAGCAGGCCCAGCCGGTAGAGATGAGTCCGCTCAACCCCAAG SHNTRASRPNNKQAQPVEHSPLNPK~~

~~AAGGTCTACAAGGGGGAACTGGTCGGCTTGTACGGAGAGAGCTCTGTTACGACAGTGGCAATCAAGACGCTGAA 1875 KVYKGELVGLYGESSVTTVAIKTLK~~

~~TTGCACGAGTACTTGCTCTCCCACTCACCACACTCTGACGTCACGGCGGCTGAAGACGACAGCGGTACCGGAGGA 2 LHEYLLSHSPHSOVTAAEODSGTGG~~

~~CACCATTTCG~TCACAGAGATCTGGCCGCGAGGAACATCCTCGTGCTGACGGCTAACTGTGAAATCTCCGAC2250 HHFVHROLAARNILVADGLTVKISO~~

TTTGGrTTGTCCAGAGATGTCTACTCTTCTGATTACrACAGAGGCAGAGCAAGTCTTGCTCCCCGTCAGATGG 2325 FGLSROVYSSOYYRVQSKSLLPVRW ATGCCCCCGGAAGCGATCTTGTACGGAAAGTTCACCACAGACAGTGATGTGTGGGCTTTTGGCGTTGTCCTCTGG 2400 MPPEAILYGKFTTOSDVWAFGVVLW GAGGTCTTCAGCTATGGACTGCAGCCGTATTACGGTTTCTCCAATCAAGAGGTCATTGAGATGAACGCTCCAGG 2475 EVFSYGLQPYYGFSNOEVIEMIRSR

~~CAGATTCTGGGCTGTCCTGAAGAATGCCCGGCTCGTATTTACGGTCTGATGGTGGAGTGTTGGCACGAGATG 2550 OlLGCPEECPARIYGLMVECWHEnP~~

~~GOPHYTPHYMPYNNHGVAGGSLSPP t.----..-~~

GGCCAGCCACCTGTCTTGAATCTCCAGTCTGGCCAGGTTCAAATCCCGCGCAACGTGGACAAGCCATGCCCCC 3150 GOPAVLNLOSGOVQlPRNVGOAMPP 0-0 -00 GTCAACCCGACCACAGCAGGGCTGGCCAACAATGGTCCCAGCAAGTTCGCCGCTGTTCTTGCCACTCC 3225 VNPTTAGLANNGPSKVSPAGSVASS...... AAGTCTTCAAACAGCGCTTCCTCCACCCACAACAGCGGGGGTGTTGGGGGTGCCTCCCCGACAGCATG 3300 ...... KSSNSASSTHNSGGVGGVPPRQGMA CACGCCGG~CAGAACACGAGCGGACAGCCAATGATGAACGCTAACTACAAGCTCCAACAATTTCAAA3375 HAGONTSGQPHMNANYKLQPOAFNG

~~TCTGCTTACAGCCCTGATCAGAGAACATCGAACATCTAAAGTACTCCTCACATACACACTATTACATACAT 3489~~

SAYSPDORTSNI. ACATACACACACACTAACATACGCAGACAGTACACACAGACAGACAGTACACACAGGCAAACACACACACACACA CACACACACACACACACACACACATCACACACACACGCACACATCACACACACACACATCATACGCACACACGCA- CAAACACTCACGCACGTGTGCGCACAAATAHGTWCRCRTACACTCGCGTATCCTAATGCATGTAAANGAAATAAT TGTATACTTTTTGATGCTATAATTTTGATGAATTACAACTTTTAGTTATCAATGTTTGTTTGTTCACTTGAAGAA AAACAGAACTACAACCGTGATATTTATACGAATTTTCATACATGATAACACCATGCCAAATTAAAATGTTGTTTT bure 8 - Homolopv of Aror to other Ror Rece~tors Percentage of amino acids shared, by domain, of Aplysa Ror to human Rorl and Drosophila Ror (percentages for human Ror2 are similar to those for Rorl and are discussed in the text). Percentages calculated by aligning sequences and counting number of residues chat match between Aror and the other sequences. Figure 8:- 1Ali Rors fiom Human and Drosophila

~~Kringle Domain~~

~~8% 48% 4-b Transmembrane - Domain Juxtamembrane Oomain~~

~~Tyrosine Kinase \ Domain~~

Dmsophila Ror Human Rorl Figure 9 - Seauence Alignent of Aror to Other RTKs Alignment of ApZysia Ror (Aror) amho acid sequence with those of Drosophila neurospecifïc kinase (Dnrk), Drosophila Ror (Dror), human muscle specific kinase (MuSK), and human RorlIRor2. Highly conserveci residues are indicated by white lettering on biack background. Dashes Uidicate gaps introduced to perfonn alignment. Amino acid position br each sequence indicated at left. Position der above sequences starts from position 1 and includes al1 gaps in numbe~g.DNAstar software was used to construct this alignment.

~~Figure 9: Continued~~

~~I 1~~

~~taniw- Hriw Rorl Hnaan Ror2~~

~~1 1 I 8 I I 430 440 450 460 470 480~~

~~NVAl'NAKQLKNVSIRRKRTKSKDIKNISIPKKKSTIYEDVFSTDI ...... ------*------D------maman Rorl ......~~

~~I IKSRG YNASNRRPNC UV------QHK RSE~H - - - SMHWDPT msK HR - - - --QPESPEAA Rorl nR--- - - HPESPDAA Ror2 DN--- - - GPGTREGD~~

~~I I~~

~~TVS T TASTT BVS~~

~~1 D-ESVRW DYv~PNAVDLNTPI SSRERXX 0--KXWXAXVGTTA ------TY----SHTVIISI nls D-LSNPKS ç-----KBKN Ror l N-KNVRn - s Ror2 D-RKHPK Figure 9: Continued~~

730 740 750 760 770 780 320 TLHLHILLVYKLSKH DYsQPAGAATAECSVSHRGGGDCG-GNLNTqRETLGVNGNHtJTLnuk 337 LPKRRTInt~yGnRNI~NxNTpsAnKNIyGNsQLNNAQDAGRGNLGNLsD~vALNsuLI-- 510 - - - NKKRESAAV- -TLTTL HiaranWK KHVRGQNVEH-SHLN- - tbniianR0~1 -nns~s~,~nen-PLIN--mna~~or2 -AQPVEHSP--L--Mar

~~GD1 QDV NNI SAV SAV PNI~~

940 ------A-..- TEGK Q------~TVC HSDLSUR iDV s a E - --- n R SDV --- PVHL SDV AAEDD- - - * nnv 1 WUU I mcns I l SZY I mmm l S',& I cr)&W 1 YYZ I I tZ I I I< I I Id I I la I I I4 I I lb 1 I I& I I I& I I I2 I I I> 1 &P.L I I >PI I I EX 1 10s I PnbO l &LO ou>> xxaz eaaa m I && 3b44w w>xa d&U> I LZO l PILO IIIll< I I 1 UW', IIIll~ 1 1 I truo IIIIIb I I I &*a IIIII* 4 I lOLZ ''1'14 I I i **a *#*I I I I tb 8 I r Ib> - I I l L&Pl I I r 424 1 I I a&& 1 1 13&& I I I UP4 r1111g I I IWSb rrlt10 I t I &EM IllIlO I I I uao I t I &a& 1 I I *s* I 1 I lrr* I I I wnO I 1 l CCO 0 I I '000 1 I t*WS I t I -g. 1 I I POP) )rr* * I I l osa 1 1 1030 $1IlXO Ill110 I I l &Pl& Illll0 I I l clwa IIIIIS Figure 10 - Phylo~eneticRelationshi~ of Aror to Other RTKs Evolutionary relationship of Aplysa Ror (Aror) to Drosophila Ror, human Rorl and Ror2, human muscle speci fie kinase (MuSK), and Drosophifa neurospecific kinase (Dnrk). The length of each pair of branches represents the distance between sequence pairs. The units at the bottom of the tree indicate the number of substitution events. Number at left is largest unit. Tree constnicted using DNAstar software. Figure 10: Phylogenetic Relationship of Aror to Other RTKs

Dror Aror Human Rorl Human Ror2 Human MuSK Dnrk 1 1 - Anif Hvbridization Screen Restriction map of Anif clones obtained hm hybridization screen. The white bar indicates the full length of DNA that was sequenced. Enzymes used to construct the map, and the region they cut are indicated by positionhg dong the white bar. Clones are labeled as 1 through 7. The region corresponding to the open reading frame (ORF), and the original PCR product are indicated by black bars. Other black bars: sequenced portions of clones, white arrows: unsequenced portions of clones. 111l11111 1 'mcrmmawt~mamstmtmiz~&&mnmFz081z&di&1&i&i&i di&iaéi$i&i oh m( oh + oh oh & & h I 11 I 1 1 1- 1 1 1 1 l 1 JW I JW 116s t I'JS l MX l Jan II i/\1nv l l iav I Jbn II l0V II lay lJW Ilos Fiwe 12 - Nucleotide and Amino Acid Seauence of Aruf Nucleotide and deduced amino adsequence of Anif. 5' and 3' untranslated region indicated at beginning and end of sequence, respectively. Sdblack arrows, initiating methionine; double-headed white arrow, predicted signal sequence; small white arrows, glycosylation sites; underline, CRD; white bar, LRE motif; thick underline, trammembrane domain; large black arrows, edges of kinase domain; double underline, kinase insert; oval circle, SH2 domain binding site (YLVL); dotted underline, serine/threonine-richregions; large white arrow, stop codon at end of ORF. CCGGACTTGTTTGCTTTCAAAGGGCACTTTGGATTTATTGAATTTCTGTAAATGTGTGGACAAGACGTGTGG GATTTATTGACGATTTTGAATCGACATTCTGTTCCCATCGTGTGAGGGATCTCTGAAAATAATATCTTCAAA

~~ATGGCTGTTCGTGTATGGAAAGTGGGCGCCTACTTGATGTGTCTGGTTCTTTGGGAGTTATTACTGGTGACATCA 75~~

~~MAVRVWKVGAYLMCLVLWELLLVTS~~

GCGGTGTACGCCACAGAGGAAGTGTTTATCACCTACGCCAGATGCCGGGTGCAGTGTGTCACACAGTTTA 150 -AV'~ATEEVF~T'~ARC~JQCV~OC~D CTGTGCAAGAAGTTTGCGGAGAGCAGTGCCCTACGGAAACACGGATGTGACCGCAAGATTCTTGCGGCATGGC 225 LCKKFAESSALRKHGCDRKlRCGDG

~~TGCCAGACTGCTTGTTCATTCCTCACAAAGAAACCAAAAGCCCCAGCACTGACGGGCGAGTGGCGTTTCCCGAG 300 CQTACSFLTKKPKAPALTGEWRFPE~~

~~GATGTCAACTCGGATTCGTCCAATCCACGCGAGCTGATGATAGAGTGGCAGGCTCCGAACCTGGTGTCCGAC 375 DVNSOSSNPRELMIEWOAPNLVSGD~~

~~ACGGATGACGTCACCAGTCCAACGGAAGCCGATTACCGGTCGGAAGTTGGCCCCGTTATCTACGTGCTCACC 450 TDOVTSPTEAOYRSEVGPVIYVLAT~~

~GGAACTTCAACCAGCCGCAGGACGGATGGAAGATTGTTCAGCAGCTGGTATCCACTCGTGTTCGGATAGAATG525 RNFNOPQDGWKIVQOLVSTRVRIDM GACGCTGTCCCTTTCTCCCCGGAGTTCCTGCTACTGGCTGTCCGACCGTCTCATGACGCAGATAGTTC 600 DAVPFSPEFLLLAVSEAGLMTOIEF

AAGACAAACTCTTTGGTTCAGACTGACCTGAACCAGCCTAAAGGGGCGGAGTTCCGCAAACTTCTGGACTCGGAC 675 KTNSLVOTDLNQPKGAEFRKLLDSD GGAGCCGGCTGGTACGTGAACAAGTACGACGGGGTCAACGCCACCCTTACCCTGTCCACTCACGTGTCAAAA 750 GAGWYVNKYOGVNATLTLSTHVVNT- GACTCCGGTTCGTACTCGGTCCAGAGAGCTGAGGTCAAATTCCACGGACCTGATTGGCTGAGGGAAGATACCGGG 825 DSGSYSVORAEVKFHGPDWLREDTG-

AAAGACCATTTCGCCGCCACGGTGACGAATAGAGGCACGGAGGAGGCGGCCTACACCGTGGACGACTCCTTC 975 KDHFAATVTNRGTEEAAYTVOELRF AACAGCATTTACCAGCTGGTCATTCACCTGGGCGAAGACTACCGCGTGAAGACGGAACTCGTGCTGAAGACGTCA 1050 NSIYQLVfHLGEOYRVKTELVLKTS

~~CCGTGCCAGGAGCCGGACAAACTCAAACTGAGCCACTGCCTGGACAAGATAGAGGAGAGTCGAGACCATCGCG 1125 PCQEPDKLKLSHCLDKIEEESRPSA~~

CCCATACGGACGAAGGAGGGCGAGGATTACGATCCTGACGTCCTGCGACTGTTTGTCAACGTGTCCTCGATGGTT 1200 PIRTKEGEOYOPDVLRLFVNVSSllV- CTTCCACCGGACTCCGATCTCGTTCTGGTGAACATCACGTGGTCTCCCATCTGAACCAATCGGTAGCCTTTAC 1275 LPPDSDLVLVNITWSPIVNOSVAFY- - AACGTGACGACATTCGTGGAGAACTACCCG~TCAACGTTCAGCAGACGTCTACATCTCTGTCRCATGTGTTGCT 1350 -NVTTFVENYPFNVQOTSTSLSHVLL CGGTTACAACAGAACACACAGTACCACGTCCAGGTGGAGGCCCTGGTCCCCCCCAGCCCGTGAGTGCCCCGC 1425 RLQQNTQYHVQVEALVRPQPGEVPR

~~CTGCTTCCTGTGGTGGACCGTCTACAGTTCAACACCTCCGAGGTCAACCTGGCCATCTACCAGGGGCCGCAACCC 1500 LLPVVDRLQFNTSEVNLAIYQGPOP~~

~~CTCACGCTCGGCACAGATCACGAGATGGGTCGGGTGAACAGTATCATCATCGGAGTACTATTCATCCT 1575~~

~~LTLGTOHEMGRVNSIllGVTlVlVt~~

~~CTCTTCCTCTCCATTCTCCTCGGCCTACTGTACGAAAAGAGAAAGAGCTTCAAAGACATCATCGTTACCAAGGCG 1650 I~~

~~SOEWELDPHSLKFSTLLGQGAFGKV~~

~~GACCTGGTGGCCGAGATCAACTTGATGAAGCGTATCGGCAGCCATCCGAACATCGTGGCCTCATCGAGCCTGC 1950 OLVAEINLMKRIGSHPNIVCLIGAC~~

~~CGCATGGACGGAGATGTGCGCAAGCGCTGTGACGGCCCCAGCGAGATAACGTACACGGTGATCCAGGACAATGGC 2100 RMDGDVRKRCDGPSEITYTVlODNG~~

~~AGTATGGAGAGCGGAGTGGTCACCCCAGTCGACATGCTGTCCTTCGCCAGACAAGTGGCCATGGCAATGGGTAC 2175 SflESGVVTPVOMLSFAROVAMAMEY~~

~~CTAGCGGAGAAGAAGTACGTCCACCGAGACTTGGCGGCTCGCAACGTGCTCATCGACTACAACAAAGTGGTCAAA 2250 1AEKKYVHRDLAARNVLIOYNKVVK~~

~~GTCTGCGATTTCGGCCTGTCACGTGACATCTTCAACGACAACCACTACAAGAAGTTACCAATGGCAAGCTGCCG 2325 VCDFGLSRDIFNDNHYKKLTNGKLP~~

~~CTCAAGTGGATGGCGATCGAGTCTCTGAGAGACCGGATGTTCACCACTCAGTCGGACGTGTGGTCCTTCGGCATT 2400 LKWMAlESLRDRflFTTOSDVWSFGI~~

TCCAACGGCTACCGCATGGACAGACCCAGCAACTGCTCACAGGAACTGTACGCCATCATGCGAGCGTGCTGAA 2550 SNGYRfiDRPSNCSOELYAtHRACWE AGTGGCGGGGACTTGAGACCTTACGCTGGTCGCTCCGGCCCCACCTCCACCTCCACCAGCCCGTCCCAGGCCATG 2050 SGGDLRPYAGRSGPTSTSTSPSOAM...... CTGAAGGGCGTGAAGAAGGTGTCCCCACGACGGGACAACATGCGAATCAACGTTTGCATCCACCAGAAGTCAC 2925 ...... LKGVKKVSPRRDNMRINVCIHOKST GATCGGTTGATACGGCCAAGTGAGAGCGACTCCAGTCCTTCTTCGTGTTAACGGAGAGAGAAAGAAA 2976 0 ...... ORLIRPSESOSSPSSC.

GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAACAGAGAGAGAACAGAGAGAG AGAGAGCGACAGCAACGGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGC TCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTT mure 13 - Homoloev of Aruf to Ret/FGF Receoton Percentage of amino acids shared by domain of Aplysia Ruf to moue and Drosophila FGF receptor, and to chicken and hsophila Ret receptors. Domains where no nurnber is indicated had little sequence identity (CS%) and are therefore left blank. For Anif* nurnbers above mws are cornparison to neighboring receptor in figure, nurnbers below arrow are comparison to receptor at edge of figure. Cornparison between Ret and FGF receptors is also show at bottom. Number above arrow is between Drosophila Ret and FGF receptors. Number below arrow is between Drosophila and mouse FGF receptors. Percentages calculated by aligning sequences and counting number of residues that match between Aruf and the other sequences. Figure 13: The novel4plvsia RTK. Aruf. is in the FRET RTK farnilvs

~~Mouse Orosophila Chicken Diosophila~~

FGF Receptors Figure 14 - Sequence Aliment of Aruf to ReVFGF Rece~tors Alignment of Aplysa Ruf amino acid sequence with those of Drosophila Ret (Dret), Drosophila FGFR (DFGFR), chicken Ret, and mouse FGFR. Highly conserved residues are indicated by white lettering on black background. Dashes hdicate gaps introduced to perform aiignrnent. Arnino acid position for each sequence indicated at lefi. Position der above sequences starts fiom position 1 and includes al1 gaps in numbering. DNAstar software was used to construct this alignment.

~~Figure 14: Continued~~

1 5jo 620 630 ------VNV TTP-VENYPPN------430 SRYCPDHV-----CDPLEE 174 ------FVP--GGLIPI------313 IR- - - -NLLQUATCDIVES 567 ------IIYCTGAFLIS------Mg Figure 14: Continued Figure 15 - Phvloeenetic Relationship of Aruf to RetFGF Receotors Evolutionary relationship of Aplysiu Ruf to Drosophila Ret (Dret), Drosophila FGFR (DFGFR), chicken Ret, and moue FGFR The length of each pair of branches represents the distance between xquence pain. The units at the bottom of the tree indicate the number of substitution events. Number at lefi is largest unit. Tree constnicted using DNAstar software. Figure 15: Phylogenetic Relationship of Aruf to RetIFGF

DFGFR Mouse FGFR Dret Chicken Ret Aruf Discussion SummarY Using a PCR based approach, we have cloned cDNAs of two putative receptor tyrosine kinases fiom Aplysia caffornica. The first of these, designateci Aror, appears to encode an Aplysia homologue of the vertebrate Ror (Ror l, ~or2)'~and Drosophila Ror (~ror)~'receptors. The second is sirnilar to receptors fiom the ~et'~*~'*~'and FGF rc~e~tor'~'~~~subfamilies of RTKs, but the Izvel of squrnce identity to those receptors is more like that between members of different subfamilies than between members of the same subfarnily .

Additional RTK Fragments Arnplified Aside from the Aror and Aruf clones, several other PCR products were also originally obtained, and some of them were found to produce new fragments when 'nested' reactions were performed. It is possible that some of these encode fragments of different RTKs fiom Aplysia. There are three general ways clones fiom these fragments could be screened to determine whether they encode portions of the two receptors already obtained, or whether they encode hgments of novel receptors. One is to design new, specific pnmen to areas of low sequence similarity in the kinase domains of the Aror and Aruf clones, within the region that the origd primers were designed against. These can then be used in PCR reactions with the original fragments as template. If the primers ampli@ a fragments they wifl likely be portions Aror, or Anif. The second way to screen the original PCR products is to clone the PCR products of interest and perform restriction digests of DNA purified fiom this, using enzymes that are known to cut in Aror and Aruf'. Although one might expect a novel receptor that is highly homologous to Aror or Aruf to ampli@ fiom the new primers (ex- a fictitious 'AroR') it is unlikely a novei receptor would both amplify with a specific set of primers, and contain al1 the same restriction sites as Aror and M. Finally, southem blots could be performed using radioactive probes designeâ fiom regions of Aror and Adintemal to the area the original primers were targeted against. It would be interesting in the fùture to investigate these clones and detemine if additional Rn< hgments are present. Features of Aror

The extracelular domain of Aror shares a nurnber of motifs in cornmon with the other Ror receptors, including an Ig-like domain that is present in the human Rors, and kringle and cysteine nch domains that are present in ail the ors'^*^'. The positioning of these domains within the various receptors is also sllnilar. The most N-terminal extracellular motif in Aror is the immunoglobulin-like domain, that is also present in the human Rors, the Drosophila Trk-like receptor (Dtrk), and in ali FGF receptors identified so far, but not in Dror. It remains unclear what function these domains have in the Rors, although some studies have suggested roles for them in ligand binding in other receptors. In the FGF receptors for instance, it has been shown that the third Ig-like dornain of FGF2 is involved in ligand binding8. Similady, the second Ig-like domain of the vascular endothelial growth factor (VEGF)receptor, Flt 1, has been shown to be the ligand binding domain for that receptor8'. It is possible therefore that the Ig-üke domain in Aror performs a similar function, although this has not been investigated. Alternatively, Ig-like domains are also found in a diversity of cell adhesion molecules, such as neural ce11 adhesion molecule (NCAM), and Ll, where they contribute to ce11 adhesiod6. In addition Dtrk, which has Ig-like domains homologous to those of L 1, has been shown to cause ceU aggregation w hen its expression is induced in non-expressing cells4! Furthemore, this aggregation led to phosphorylation of the receptor4! Thus a possible role for this domain in Aror is that it foms part of a ce11 adhesion site. &or also contains a cysteine-rich domain (CRD)in its extracellular portion, and this has been identified in a diversity of molecules as well. The CRD has been identified in a number of RTKs, includlig aii the Rors, the Drosophila neurospecific tyrosine kinase (Dnrk), and the muscle specific kinases, and has also ken identified in the fibled family of molecules, secretedfiizzled-related proteins (sFRPs), mouse collagen al (XVIII) chah, and human carboxypeptidase z~~*~.Genes of the fiizzled fiunily encode seven-pass transmembrane proteins which act as receptors for secreted Wnt g~ycoproteins~~".nie Wnt molecules are important in a number of processes such as ceU proliferation, and d~erentiation~~~~'!For exarnple, some of the Wnt ligands can induce differentiation neutal crest ce~ls'~.The CRDs of molecules hmthese various families were fint related based on the rigid spacing of cysteine residues that is observed in these d~rnains~~?In some cases the spacing is not ngidly adhered to, however it is still clear that these domains are homologous to each ~ther~~".Based on the example of the fiialed receptors, it has ken suggested that these CRDs fom ligand binding domains, with different CRDs binding to different types of ligands43". Finally, Aror also contains a kringle domain within its ex&acellular region, nearby the transmembrane domain. This is a conserved feature amongst aU the Rors, Dnrk, and Torpedo MuSK, and occurs in similar positions in those rnole~ules'~*~~~~~~.Kringle domaind have also been identified in apolipoprotein, in proteins involved in blood coagulation, and in hepatocyte growth factor (HGF)'~*"*~~*".In these other proteins there are generally clusters of the kringle domains, and they are usually associated with a senne protease-like domaintO*''*12. The Ror receptoa (including Aror), and MuSK have only the single domain, and no associated serine protease-like d~rnainl~~'"'.These domains are thought to be important in mediating protein-protein interactions, other than ligand binding ' '* 12? For instance they may be important in associating with other membrane bound, or exttaceliular matrix proteins. It remains unclear however what role the luingle domain will have in the Rors. Although some of the above domains may be important in ligand binding, no ligands have been identified yet for any of the Ror receptoa. A ligand has been identified however for the related MuSK re~epto?~*'*.Specifically it has been show that agrin and MASC act together to activate the recept~?~*~~.Since MuSK shares many of the same extracellular domains as the Rors, they may also have a related ligand.

Intraceilular Domain The inûaceUular domain of Aror contains a number of motifs that may be important in signal transduction. One of these sites occurs in the insert region of the kinase domain, whereas the other two are in the C-terminal domain. The kinase site is a potential binding site for the SH2 domains of Shc, and PI3 kinase. The SH2 domains of both of these molecules recognize the motif YXXM~~'*'~~",and the Aror sequence correspondhg to this is 689-YSEM-692. This motif is not present in the other Ror receptors, although binàing sites for both these domains are present in various RTKs. Binchg of PI3 kinase to the target site is thought to lead to phosphorylation of PI3 kinase, and an increase in its catalytic activity2. Activation of PI3 kinase is thought to lead to activation of FRAP, the kinase involved in translational regdation in ceU lines6'. PI3 kinase also catdyzes the generation of phosphoinositides such as PIP2, and PIP~~.While PIPl is a substrate for hydrolysis by PKC, PIP3 is not, and it has therefore been suggested that generation of PIPl by PI3 kinase may be a form of signal transduction, where PIP, activates other signahg molecules2. For instance it has been shown that phosphoinositide-dependent protein kinase- 1 (PDK 1) becomes active in the presence of p1pja4.Activated PDKl can phosphorylaie and activate c-Akt, an intracellular protein kinase that provides a survival signal protecting cells fiom apoptosis induced by various stresses. PDKl has also been shown to activate P70 S6 kinase, the enzyme involved in translational regulations5. Another effect binding of PI3 kinase to RTKs is thought to have is that it localizes PI3 kinase to the membrane where it has access to membrane- associated substrate molecule8. Shc is another SH2 domain containhg molecule that has also been shown to associate with a number of RTKS~.Phosphorylated Shc has ken shown to associate with the SHî domain of Grb2 and may therefore act in this capacity as an adaptor molecule2. It is possible that Shc activates the Ras pathway via its association with ~rb2~.Overexpression of Shc in fibroblasts has been show to lead to transformation of those cells2. The two potential signaiing domains in the C-terminal region are binding sites for WW domains and PDZ domains. WW domains are 3 8 amino acid motifs found in a nurnber of proteins, including YAP (Yes-associated protein; yes is an intracellular tyrosine kinase), and dystrophin, the protein involved in the development of muscular dystrophyf16*". The WW binâing dornain is the sequence motif XPPX[Y/L]'~*~'.This site is present in Aror as 932-SPPPY-936. WW binding domains are also present in the same region in human Rorl and RoR [Figure 91. PDZ domains are also signahg domains, which recognize the sequence motif x[Smx[Vn] when it is present at the carboxy temiinus of the tatget protein88e89.This binding site is also present at the carboxy-terminus of Amr, as TSNI. None of the other Ror recepton have a PDZ binàing domain. In some cases PDZ domains are thought to participate in the clustering of molecules. The synaptic protein PSD-95 for instance contains a PDZ dornain that can be bound by a recognition site in the enzyme neuronal niûic oxide synthase NOS)? PSD- 95 also binds to N-methyl-D-aspartate (NMDA) receptors, and it is thought that PSD-95 clusters nNOS and the NMDA receptors together forming a signaling cornplexgo. Nitric oxide produced by nNOS is important in NMDA-dependent neurotransmitter release, and neurotoxicityW.Sinilarly the molecule CRIPT contains a PD2 binding domain, and is thought to mediate interactions between PSD-95 and micro tubule^^^. In addition to these sites there are a number of specific amino acid rich regions, including serine-threonine-, and glutamine-nch regions. Neither of these regions has any specific known function, although the serine and threonine residues could be phosphorylation sites for regulation by serine/threonine kinases, such as PKC.The glutamine rich region is dso identifiable as a CAG trinucleotide repeat. These are observed in various proteins, some of which are involved in the development of human disease. Huntingtins protein for instance contains a CAG trinucleotide repeat that is thought to expand in the development of Huntington's Disease. There is no evidence however that the CAG repeat here corresponds to the same type of expanciable repeat. It is unclear what, if any significance the GQ repeat in the C- terminal domain has. Taking ail these facts together, there are several ways in which Aror may activate intracellular signaling pathways following activation by ligand.

Features of Anif Exrracellular Domain Aruf also possesses two extracellular motifs that may have important functional roles in the molecule. The motifs are a cysteine rich domain, and the LEmotif. Although the CRD in Anif was lound not to be homologous to other CRDs examine4 including al1 of those discussed above for Aror, it may still have a similar function. Altematively it may be important for maintaining the structural integrity of the receptor. CRDs present in the EGF receptor for instance are thought to be important for maintainhg its structure2. The LRE motif was originally reported in s-laminin, a recently cloned lamirlin-like glycoprotein that is concentrated in the synaptic basai lamina of muscle fibed! It is thought that s-laminin plays a mle either in the selective re-innervation of original synaptic sites following denervation, or in the differentiation of axons into nerve tennuiais at these sitesg'. The LRE motif is thought to fonthe primary binding site for adhesion of s-laminin to motor neurons innervating muscle fibers at the neuromuscular junction8'. In support of this it has been show that motor neurons adhere to an immobilized LRE containing peptide, and that LRE peptides interfere in the association of motor neurons with s-laminin fragments8'. Although one would expect the LRE motif to occur by chance once in every 4713 a.a.", there are a number of reasons why the motif may still be functional in this instance. First, it occurs in the extracellular domain of the receptor where it would have access to its target site. Second, it is located in an area of the protein that has a high surface probability. It is therefore not expected to be buried within the protein folds, which might otherwise render it non-functional. Finally, Aruf is derived fiom a neuronal template, and since the LRE motif functions in the nervous system, it is reasonable for a functional LRE motif to be present. It is unclear whether there are any additional domains in the extracellular region of Anif that may be important in Ligand binding, or in other functional aspects such as protein-protein interactions. In this regard it will be important in the future to identify homologues of the receptor so that associations between their respective sequences can be made.

One intracellular domain observed in Anif that may be important in signal transduction is the potential binding site (YLVL) for the C-terminai SH2 domain of PLC~"*~~-~~.~~.In the FGF receptors this site is usually YLDL, which is a site for the N-

~~terminal SH2 domain of PLCy. It has been show that PLCy associates with several~~

~~RTKS'. Binding of PLCy to the RTK is thought to lead to phosphorylation, and~~

activation of PLCy. PLCy may then catalyze the hydrolysis of PIPl to IP3 and DAG. DAG is the physiological activator of PKC,which has been implicated in a many cellular processes, such as growth control and tumorigenesis. IP3 mobilizes ca2+fiom intracellular stores, thereby affecthg ca2+regulateâ processes in the ceil, such as activation of ca2+- dependent enzymes. The fact that Aruf has a different SH2 domain binding site than the FGF receptors would suggest that Anif may activate diffemnt signahg pathways than they do. Aruf also has serineithreonine nch regions in its C-temllnai domain, and as for Aror these may form phosphorylation sites for regulation by serinehhreonine kinases.

Future Directions It wiil be interesting in the future to examine the tissue expression of both the Aror and Amf clones. The other Ror receptors are expressed predominantly in embryonic nervous sy stem tissue, and based on this have been postulated to have a developmental role""'. It would not be surprishg if Aror has a similar expression profile. For the Amf receptor it is less clear when and where it will be expressed, as there are no known homologues of the receptor to compare to. nie Ret and FGF receptors are expressed in a diversity of tissues in the adult, including in the nervous system, but since Anif probably belongs to a different subfarnily the expression of these receptors wiil probably not reflect the expression of the Amf clone. Antibodies can now be designed agalist each of the clones, and used io study activation of the RTKs. Phosphopeptide antibodies can also be designed that will recognize the phosphorylated foms of each of the RTKs. These will be particularly usefid in measuring the activation of the RTK under various stimuli, and will therefore assist in determining a huictional role for the receptor. For instance it will be interesting to examine whether administration of SHT Ieads to activation of the receptor in Aplysia. Another important project for the fùture will be to identify ligands for the receptors. Extracellular ligand binding constructs can be generated and used to purify factors fiom Aplysia hemolymph that bind to the receptor. An amdogous procedure has been used to identify cysteine-rich neurotrophic factor (CRNF), a neurotrophic-like factor purified fiom Lymnaea using the p75 neurotrophin receptor in a binding assayg'. In light of the presence of the Kringle and Ig-üke domains, and the ceil adhesion properties of Dtrk, it will also be important to allow for the possibility that the Aror ligand may be a membrane bound factor. The observation that there are a nurnber of potential signal transduction domains, such as Shc and PI3 kinase SH2 binding domaias for Aror, and a PLCy SH2 binding domain for Anif, aiso raise the possibility that the receptors may interact with these signaluig molecules through those motifs. Deteminhg which of these signaüng pathways are induced following receptor activation will provide additional clues as to the functional roles of the receptors. Finally, it wiii be interesthg to express mutant forms of Aror and Anif, and examine what functional deficits they have, which will again illuminate their hctional characteristics. Although a precise function has not yet ken determined for either of the two receptors cloned in this project, it is possible to speculate as to their potential roles in Aplysia. The Ror receptor would be expected to have a similar role to that of it's homologues from human, rat, and Drosophila. 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