Chapter 5. Trophic Factors and their Receptors in the CNS

Chapter 5. Trophic Factors and their Receptors in the CNS Steven Rosenberg Chiron Corporation 4560 Horton Street, Emet-yville, CA 94608

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Introduction Neurotrophicfactors are molecules produced by a variety of cell types which have been functionally defined by those activities which allow the growth and maintenance of neurons in and The last review in this series focused on nerve growth factor and 3 other neurotrophic factors (1). In the intervening 3 years the tools of molecular biology have led to an explosion in the identificationof novel neurotrophic factors and their receptors (2). This review will focus primarily on the neurotrophinfamily, of which nerve growth factor (NGF) is the progenitor. Other trophic factors, including members of the fibroblast growth factor family and ciliary neuronotrophicfactor, will also be covered. The focus of this review will be on the dissection of the interactions of the receptors and factors, and the signalling pathways they subsequently activate.

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Identification NGF, the prototypic neurotrophin, was first isolated from male mouse submaxillary gland as a factor which promoted the survival of neural crest derived neurons. The unique Occurrence of this rich source of the protein which exists in the mdllse as a complex of 3 subunits, a,p, and x has made NGF the best characterized member of the family. The active form of NGF is a non-covalent dimer of the 13 kD, 118 amino acid, p subunits. Each subunit contains 6 cysteines arranged in 3 disulfide bonds. This subunit is synthesized as a precursor containing a relatively long pro sequence, features of which are conserved in the neurotrophin family (3). More recently, brain derived neurotrophic factor (BDNF) was isolated from porcine brain

(43).This very rare protein is homologous to NGF in size and location of conserved cysteines.

Subsequent isolation of BDNFs from other species has been accomplished using PCR strategies, targetting common sequences between BDNF and NGF (6,7). The additional members of the neurotrophin family, as well as neurotrophins from other species have been identified as homologous proteins by similar cDNA and genomic cloning strategies using PCR (8). These strategies have been aided by the observation that the coding sequences for the neurotrophins all appear to be found on single exons. This has led to the identification of NT3 (human, mouse, rat (9,lO)).NT4 (Xenopus and viper (1 l), and NT5 (rat and human (8)) clones from cDNA and genomic sources.

An assessment of the roles of these new neurotrophins in neuronal development and survival has depended upon the expression of the proteins using recombinant DNA strategies. In particular, all of the neurotrophins discovered to date have been expressed using mammalian cell systems. Yeast or baculovirus have been successful in a few cases (6,12). In addition, the isolation of the cDNA clones has enabled workers to locate those cells that express the various hybridizationtechniques (6.8). neurotrophins using in Structure-Activitv RelationshiDs - The use of the PCR strategies defined above has led to the isolation and sequencing of a number of neurotrophins from a variety of species. An alignment of these sequences is presented in Figure 1.

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Copyright 0 1992 by Academic Prers, Inc. All rights (if reproduction in any torm reserved.

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1 10 20 ssthpvfhm[GE] fs[VCDlsvev[WVl g . . [DK] t ssshpifhr[GE] fs[VCD]svsv[WV] g.. [DK] t hsdparr [GEI Is[VCDIsise [WVI taa [DKI k hNT3 yaehkshr [GE] ys [VCD]see1 [WV] t. . [DK] s xNT4 asgsdsvslsrr [GEI Is[VCDIsvnv[WVI t . . [DK] r hNT5 gvsetapasrr[GE] la [VCDIaveg[WVl t . [DRI r mNGF hNGF hBDNJ?

.

mNGF hNGF hBDNF hNT3 xNT4 hNT5

IPNGF hNGF hBDNF

hNT3 m 4 hNT5

30 [TA] tDikGiev [TA] tDikGkev [TAI vIXnsGgtv [SA] iDirGhqv [TAI vDdrGkiv [TAI vDlrGrev

40 50 60 70 t [VLlaevninnsvf.RQYFFETKClrasnpves. . . . . . . [GCRGID] sKh m[VL]gevninnsvf.KQYFFETKC]rdpnpvds.. . . . . . [GCRGID] sth t[VL]ekvpvskgql.KQYFYETKC]npmgytke.......[GCRGID] kRh t [VLIgeiktgnspv.KQYFYETRC1kearpvkn.. . . . . [GCRGID] dKh t[VMlseiqtltgpl.KQYFFETKClnpsgsttr....... [GCRGVDI kKq e [VLIgevpaaggsplRQYFFETRC1 kadnaeeggpgaggg[GCRGVDI rRh

.

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WnsyCt t th WnsyCt tth WnSqCrt tq WnSqCkt sq WiSeCkakq WvSeCkakq

90 100 :TFVKALT]tDekqa. [AWRFIRIDTI a :TFVKALT] mDgkqa. [AWRFIRIDT] a :SYVRALT]mDskkri [ GWRFIRIDT] s :TYVRALT]sEnnklv [GWRWIRIM'] s :SYVRALT]iDanklv [ GWRWIRIDT] a :SYVRALTlaDaqgrv [GWRWIRIM'I a

110 [CVCIvLsrkatr. . ICVC1 vLsrkavrra iCVcjtltikrgr.. ICVCl aLsrkiart . [cvcjtLlsrtgrt. [CVCItLlsrtgra .

Figure 1 - Alignment of representative examples of the neurotrophin family. Conserved or identical amino acids are in upper case. The neurotrophins shown are mouse NGF, human NGF, human BDNF, human NT3, Xenopus NT4,and human NT5. Sequences in brackets are regions of 2 or more amino acid homology. The conserved cysteines and residues whose side chains are involved in tertiary interactions in mNGF (13) are in bold face. There are several noteworthyfeatures: 1) All of the cysteines are conserved; 2) Several regions show almost absolute conservation throughout the members of the family, especially the C terminal region from residues 99 to 110; 3) BDNF and NT3 are extremely conserved across species lines; 4) Dierences between the various neurotrophins appear to cluster in a few areas, which are likely of functional significance in determining high affinity receptor specificity (see below). These analyses have recently been given a rational structuralframework from the solution of the structure of the murine NGF dimer at 2.3 8, resolution using X-ray crystallographic techniques (13). The NGF dimer consists of primarily p-structure as predicted by Raman spectroscopy (14). There are 7 p strands, 6 of which form twisted antiparallel pairs bringing together the hydrophobic core of the monomer subunit. The 3 disulfide bonds appear to be clustered at one end of the molecule and are substantially buried, contributing as well to the hydrophobic core of the molecule. The overall geometry of the monomer is quite asymmetric consisting of a long, flat molecule. The subunit interface is substantially flat and hydrophobic. The most conserved residues near the C-terminus of the molecule are located at one end. The variable regions of the various neurotrophins cluster in p-hairpin loops at the other end of the molecule (residues 29-35, 43-48,and 92-98) suggesting this region is involved in the differential high affinity receptor binding of the factors. This topology is consistent with possible models for the multiple receptor interactions involved in neurotrophinaction, in which the p75 receptor binds the conserved regions and the selective frktyrosine kinases bind at or near the variable loops. A few studies have examined the role of a variety of residues in neurotrophin action by expression of mutant or chimeric proteins. In particular some conserved residues (V21, R99, R102) have been suggested as important in NGF structure and function, as judged by receptor binding and biological activity (15). However, these conclusions were drawn with crude preparations and without examinationof the intact nature of the protein structure. Further studies looking at the biological activity of NGFBDNF chimeras have suggested that no single region imparts selectivity in functional receptor binding and biological responses (16,17). Although these studies are not quantitative, they are consistent with the hypothesis that binding to the functional, high affinity receptor involves several regions of the neurotrophinmolecules.

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NGF has been shown to bind to both low and high affinity sites in neuronal cells or tissues, with only the latter correlating with biological activity. The low affinity NGF receptor protein (p75) and gene were isolated previously (18), but the components of the high affinity molecule(s) remained elusive. During the past year, a variety of workers have shown that members of the trkfamily of tyrosine kinase receptors are key components of the high affinity receptors for NGF and the other neurotrophins (2). It had been known that tyrosine phosphorylation occurred rapidly upon NGF addition to PC12 cells, but the kinase involved was unknown (19). Trk is a member of the tyrosine kinase receptor family, first identified as an oncogenic fusion with tropomyosin (3,20). The proto-oncogene was later shown to be expressed in tissues of neural crest origin, the principal targets of NGF action. Trk is rapidly phosphorylated upon addition of NGF (21) and binds NGF with relatively high affinity (22). It is controversial whether the high affinity, functional receptor involves both trk and the 75 kD NGF receptor, or whether trk itself is sufficient for the full range of NGF biologicalactivities (21-23). Two additional neurotrophins,NT3 and NT5, bind trk and activate tyrosine phosphorylation (8). Further members of the trk family of receptors have been cloned and their roles in neurotrophin receptor function have been investigated (24,25). Trk B, first identified as a gene related to trk, binds BDNF, NT3, and NT5, but not NGF (24,26-28). Most recently, the trk C gene has been shown to bind NT3, but not NGF and BDNF (25). The functional consequences of binding of the neurotrophins to the trk receptors have been measured using a variety of methods, including transfection of the trk genes into 3T3 cells yielding cell lines for which the cognate neurotrophins are mitogens (29). The complex pattern of binding of the neurotrophins to more than one receptor may reflect redundancy in the role of trophic factors or temporal regulation of expression, as has been suggested for BDNF and NT3 expression (28,30). With the identification of the role of the trk family in the high affinity neurotrophin receptors, the dissection of the subsequent signal transduction pathways becomes possible. The primary system used to investigate NGF signal transduction has been the rat phreochromocytomacell line, PC12, which differentiates to a neuronal phenotype specifically by the addition of exogenous NGF and bFGF. It has been shown that phospholipase C-y is phosphorylated by frk or a closely associated kinase within one minute of NGF addition in this system (31). In addition, a series of tyrosine phosphorylation events occurs, leading to modification of a cascade of protein serine/threonine kinases, known as MAP or ERK kinases (32). Work done using PC12 cells transfected with a & v-src protein, showed a & inducible neuritic phenotype, which is relatedto but distinguishable from that imposed by NGF (33). Finally, recent experiments with neutralizing antibodies suggest that both src and RAS, in that order, are necessary for PC12 cell differentiation(34). The role of the low affinity, p75 NGF receptor in neurotrophin signalling is unresolved. All of the neurotrophins examined to date bind this molecule with moderate affinity. A series of experiments suggest that the cytoplasmic domain of this molecule is capable of playing a role in signal transduction (35); this is most dramatically seen in experiments where a hybrid receptor with the EGF receptor extracellular domain and p75 transmembrane and cytoplasmic domains, causes PC12 cells to respond to EGF by forming neurites (36). It is not clear, however, whether this represents mimicry of the normal signalling pathway, or activation of an alternative route, since the pathways of activation by NGF and EGF appear to be convergent in PC12 cells (37). It has also been postulated that the p75 receptor is involved in embryonic development of the inner ear, via a signalling pathway using glycosyl-phosphatidylinositol hydrolysis (38). and S m a l l c u l e T V - A number of studies have shown that treatment of both rodents and primates with NGF prevents degeneration of basal forebrain cholinergic neurons after fimbria-fornixtransection (39). lntraventricularadministrationof NGF in rats and primates leads to receptor-mediated retrograde transport and reduces cholinergic neuronal degeneration, suggesting that such an approach might be useful in degenerative neurological disorders such as Alzheimer's disease(40-42). This is somewhat controversial, as NGF has also been implicated in increasing the toxicity of 0-amyloid (43). NGF has also been shown to prevent toxic neuropathy in mice after treatment with chemotherapeutic agents such as taxol (44). BDNF has been shown to be a neurotrophic factor for dopaminergic neurons of the substantia nigra, and blocks the toxic effects of MPTP (1-methyl-4-phenyl-l,2,3,6tetrahydropyridine) on these cells, when administered prior to MPTP treatment (45). This suggests that BDNF may have utility in Parkinsonism.

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No small molecules have been identified which act as agonists for neurotrophin activity. This is not surprising given the very recent identification of the trk receptors. However, two related molecules, K252a (JJ and K252b have been isolated which specifically block NGF action (46).

(a,

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K252a has recently been shown to directly inhibit NGF induced tyrosine phosphorylation by the frk receptor (47). BI AST GROWTH FACTORS

. . . - The fibroblast growth factors, or heparin binding growth factors, are a family of ldentlflcatlon

seven molecules first identified as mitogens or oncogenes (48)). The first two molecules recognized in this family, acidic and basic FGF, are approximately 150 amino acid proteins with ca. 55% sequence identity. They contain no intramolecular disulfide bonds and are quite basic proteins with a characteristic ability to bind heparin and related cell surface proteoglycans (49). The other members of the group (int-2, kFGFlhst, FGF5, FGF6, and kFGF), have been identified as proto-oncogenes (int-2, hsVkFGF, FGF5), mitogens (KGF), or homologues of other members of the family (FGFG); see reference (50) for a recent review. This section will focus on aFGF and bFGF, which are the proteins whose role in the CNS has been investigatedin some detail(51).

- The synthesis of aFGF and bFGF is unusual in that the molecules are found on the cell surface but do not contain a signal sequence (52). Other members of the family including kFGF, int-2, FGF5, and KGF are secreted by a standard mechanism, via an encoded hydrophobic signal sequence (50). In addition, a number of variant forms of aFGF and bFGF are synthesized, with differing amino termini due to translational initiation with an alternate CUG codon, leading to some molecules which are retained in the nucleus (53). Several groups recently reported the high resolution X-ray crystallographic analysis of bFGF (54-56). The structure of aFGF was also reported by one of these groups (54). The most striking feature of the structures is their similarity to each other and identical overall topology with interleukin-1-beta,the structure of which has been determined by both X-ray and NMR methods (57-59). The bFGF molecule shows overall approximate 3-fold symmetry and consists of all p structure, with 12 anti-parallel p-strands defining the core of the molecule. A peptide sequence implicated in receptor binding in bFGF is shown to exist in an irregular loop on the surface of the molecule (55,60). Two sites which bind sulfate ion in the crystal have been suggested as possible sites for binding of heparin (56). Relatively few studies have addressed structure-function relationships within the FGF family using either synthetic peptides or site-directed mutagenesis. The most extensive work has been done showing that two of the cysteines are not required for bFGF or aFGF activity (49,61,62). Additional mutagenesis experiments have focused on trying to define the heparin binding region and to determine the nature of the receptor binding sites (63,64). These studies have yielded complex results, in part due to a lack of determinationof the effects of mutations on the overall structure of the molecule. Two regions of the bFGF molecule have been implicated in biological activity using synthetic peptides. Peptides from the region 103-120 have been shown to have weak agonist activity; part of this sequence is on the surface of bFGF (60). A larger region

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nearer the N-terminus of the molecule (residues 24-68) has also been shown to have weak mitogenic activity. Interestingly, a mutation in this region (deletion of residues 27-32) eliminates the induction of plasminogen activator synthesis by bFGF (65).

- The FGF family has a complex set of receptors and binding molecules. Binding studies implicated two types of receptors of high (1 0-1 M) and low (1 O-9M) affinity which have subsequently been identified as a set of tyrosine kinase receptors and heparan sulfated proteoglycans, respectively (66,67). At least 4 different gene products have been identified as tyrosine kinase receptorsfor the FGF family; these are currently referred to as FGFRI-4, although the nomenclature in the literature is complex (68). These molecules are characterized by extracellular domains which contain between 1 and 3 immunoglobulin-like domains, and, in some proteins, a characteristic acidic sequence in the interdomain region between regions 1 and 2 (69,70). Several variants of these species are present in many cell types, primarily due to alternate splicing, yielding molecules with 2 Ig domains, without the acidic sequence, and in some cases without a transmembrane and intracellular domain (71). The cytoplasmic domain is a "split'' tyrosine kinase, as has been found for the PDGF receptors (72). There appears to be a great deal of redundancy in the receptor system; several members of the FGF family bind to most of the receptor species studied, and the mechanism by which ligand specificity is regulated in this system is just beginning to be understood (73-75). Recent work shows that splice variants in which alternate exons are expressed in the third lg domain affect the affinity of FGFRI for bFGF (73). Recent studies have implicated the role of heparan sulfate proteoglycans (HSPGs) in a variety of biological processes, especially the functional binding of the FGFs to their receptors (66,76). Studies with cell lines deficient in proteoglycansshowed a requirement for them for FGF activity (77). The exact mechanism by which heparan sulfate proteoglycans are involved in FGF binding to the high affinity receptor is not known; two models being considered are formation of a ternary complex of HSP:FGF:FGFR or stabilization of a receptor active conformationof FGF by the HSP (66). Soluble heparin stimulates the activity of aFGF and stabilizes it to degradation (78). Binding of FGFs to the high affinity receptors leads to receptor transphosphorylation and activation of the tyrosine kinases, even between receptor subtypes (79). This initial step is linked to a variety of secondary signalling pathways, including phosphorylation of phospholipase C-y, phosphoinositide release and activation of protein kinase C, and activation of adenylate cyclase (51). The relative importance of these pathways in signal transduction in the CNS has not been evaluated in detail, although recent work suggests that activation of both ras and src are involved, at least in PC12 cells (34). The actions of bFGF and NGF in PC12 cells appear quite similar, although they can be distinguished by the inhibition of the latter by K252a (I), a specific inhibitor of the trktyrosine kinase (47,80).

- The FGFs implicated in the CNS are Bales in the CNS a n d a l Ther predominantly bFGF, with somewhat lesser roles for aFGF and FGF5 (51,81). bFGF is the only factor besides NGF which will induce neuriie outgrowth in the PC12 cell line (82). Yiyn studies have shown that bFGF and to a lesser extent aFGF bind to specific, overlapping but distinct sites in the brain, and that this binding is to high affinity receptors (83). In addition, as for NGF, bFGF is retrogradely transpoaed to the neuronal cell body in a receptor dependent manner, presumably as a receptor:ligand complex (83). The key receptor in neurons appears to be FGFR-1, as judged by inm hybridization (83,84). Both bFGF and aFGF can stimulate the synthesis of NGF by astrocytes, hm,providing a second indirect mechanism for the action of the FGFs as trophic factors (85). This also suggests a role for the FGFs in response to neuronal injury. Interestingly, bFGF is accumulated by neurons of the substantia nigra and is a trophic factor for dopaminergic neurons in culture (86). These observations suggest that bFGF might play a role in treatment of Parkinsonism. rv N e u r o n o w Factor - CNTF is a unique protein with no known homologues, unlike the FGFs and neurotrophins. CNTF is a 27 kDa protein with a single cysteine and no signal sequence; rat, rabbit, and human proteins have been identified based upon cDNA and genomic clones, and overexpressed at high levels in E.coli (87-90). Recently, a receptor for CNTF has been identified using an expression cloning strategy involving a specifically tagged ligand (91). The receptor is most closely homologous to the IL-6 receptor, but it lacks a cytoplasmic domain and is linked to the membrane via a glycosylphosphoinositol linkage. These factors suggests that

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a second component is required for CNTF mediated signal transduction, as is the case with the gp130 signal transducer component of the IL-6 receptor (92). Although first identified based o n trophic activity for peripheral neurons, specifically, the chick ciliary ganglion, m r e recent results also indicate CNTF is a survival factor for hippocampal neurons (93). Moreover, studies now suggest that CNTF is a survival factor for motor neurons (94,95), and the suggestion has been made that it might be useful in treatment of ALS (Lou Gehrig's disease).

Conclusion - There has been a great deal of progress in the molecular biology of the neurotrophic

factors and their receptors during the past year. A number of new factors (NT4, NT5) and new complex receptor families (the trk tyrosine kinases and p75; the HSPGs and FGFR1-4) have been discovered. Structural studies have suggested possible mechanisms of receptor activation at the molecular level, although signal transduction pathways are just beginning to b e understood. Finally, the pharmacology of these receptors and their roles as targets for medicinal chemists, particularly in neurodegenerative disorders, will b e elucidated as receptor subtype specific agonists and antagonists are discovered.

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