The clustering of acetylcholine receptors (AChRs) at the neuromuscular junction is mediated in part by the heparan-sulfate proteoglycan agrin. However, our previous studies have also suggested the role of heparin-binding growth-associated molecule (HB-GAM) in AChR clustering. Here the role of an agrin–HB-GAM interaction in this process was examined using culturedXenopusmuscle cells. Agrin-coated beads were found to be weakly effective while agrin-coated beads further treated with HB-GAM were highly effective in AChR cluster induction. Protein overlay assays showed specific binding of HB-GAM to agrin. In addition, agrin-enriched neuritic tracks bound HB-GAM in a manner that showed a high degree of colocalization between the neural agrin and the applied factor. Finally, the introduction of exogenous HB-GAM together with soluble agrin resulted in the appearance of AChR clusters on the dorsal surface of cells in an agrin isoform-dependent manner; a dramatic change from the characteristic ventral AChR clustering seen in response to agrin alone. These results suggest that agrin may mediate AChR clustering by interacting with muscle-bound heparin-binding growth factors such as HB-GAM.

Agrin is a large multidomain protein involved in the induction of postsynaptic differentiation of the neuromuscular junction. As a step toward further understanding the mechanisms by which agrin induces the aggregation of acetylcholine receptors (AChRs), we have characterized the activity of purified, full-length chick agrin isoforms on Xenopus muscle cells. Incubation with agrin isoforms led to the formation of numerous small AChR clusters primarily on the ventral surface of cells with differing efficacies: Y4Z8 > Y4Z11 = Y4Z19, with Y4B0 being ineffective. Agrin activity appeared to be tyrosine phosphorylation-dependent as the kinase inhibitor tyrphostin RG50864 (80 microM) completely abolished the effect. Initial binding sites for all agrin isoforms were evenly distributed in a punctate manner on the muscle cell surface. After a 14-h incubation, the active isoforms induced AChR clustering, and agrin was enriched at these sites of clustering. Agrin binding to the cell surface and induction of AChR clustering were Ca(2+)-dependent, as previously shown in other systems. This is the first quantitative characterization of agrin's effects using Xenopus cell culture, providing a basis for further elucidating agrin's role in synaptogenesis using this elegant system.

Neurotrophins are a family of highly conserved proteins that affect the development and maintenance of distinct neuronal populations. Neurotrophins exist in vivo as homodimers, but we show that neurotrophins can exist as heterodimers in vitro and are pluripotent, being able to bind and to activate different Trk tyrosine kinase receptors as well as promote neuronal differentiation in PC12 cells as effectively as wild type homodimers. These asymmetric neurotrophin dimers allow unique characterization of neurotrophin structure-function relationships with Trk receptors. The chimeric Trk activities of these heterodimers suggest an alternative model of neurotrophin-Trk receptor activation in which the critical Trk-interacting elements may be attributed to a single protomer.

The use of Schwann cell (SC) autotransplantation to influence neural repair in humans is dependent upon identifying mitogens that will effectively expand human Schwann cells (SCs) in culture. The recent purification and molecular cloning of glial growth factor (GGF), a potent mitogen for rat Schwann cells, has led to the recognition that a family of proteins (GGF/HRG/NDF/ARIA) are alternatively spliced products of a single gene. The heregulins (HRGs) have been characterized with respect to their influence on human breast cancer cell lines; here we examined whether the HRGs have mitogenic activity for human SCs. Using DNA synthesis assays and serial passaging of cells in culture, we demonstrate that HRG is an effective mitogen for human SCs and that, in the presence of agents that elevate cAMP, it is possible to expand these cells over multiple passages without overwhelming fibroblast contamination. One putative target for this family of proteins is p185erbB2, and EGF-like receptor tyrosine kinase that is encoded by the erbB2 protooncogene. In this report we also demonstrate that the erbB2/3/4 messages as well as the erbB2/3 receptor proteins are present within cultured human SCs. The addition of HRG to human SCs results in tyrosine phosphorylation of a 185 kDa protein. In the presence of stimulatory concentrations of HRG, a blocking monoclonal antibody (2C4) to p185erbB2 is capable of significantly inhibiting phosphorylation of a 185 kDa protein as well as the subsequent incorporation of 3H-thymidine within the human SC. These latter results implicate an important role for p185erbB2 in mediating the mitogenic response of human SCs to HRGs.

Members of the NGF family of proteins act as neurotrophic agents for defined populations of peripheral and central neurons during embryonic and postnatal development. We have studied the presence of receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 and - 4/5 (NT-3, NT-4/5) by cross-linking radioiodinated neurotrophins to specific cell surface receptors. We have identified neurotrophin receptors representing full-length TrkB and TrkC and their truncated forms (lacking a functional cytoplasmic kinase domain) in neuronal as well as in non-neuronal tissues. During chicken embryonic and early postnatal brain development, expression of full-length TrkB and TrkC proteins preceded the onset of the truncated forms of these receptors. A similar pattern was also observed in mouse embryonic and early postnatal brain. The relative levels of neurotrophin receptors in the basal forebrain and in the hippocampus did not change significantly with age in mice. High levels of receptors for the three neurotrophins were detected in the nigrostriatal system. Full-length TrkB and TrkC receptors were found in chicken and rat embryonic ventral spinal cord, as well as on purified motoneurons. Again, truncated TrkB appeared significantly later than the full-length form on spinal motoneurons. In chicken embryonic retina and optic tectum we detected full-length TrkB and TrkC; however, the optic tectum also expressed large amounts of the truncated form of TrkB. TrkC but not TrkB was detected in chicken embryonic skeletal muscle, suggesting that NT-3 may have a novel function in this tissue. The presence of neurotrophin receptors in a wide variety of embryonic and postnatal tissues underlines the significant role of BDNF, NT-3, and NT-4/5 in embryonic and postnatal development. The regulation of the ratio of full-length versus truncated neurotrophin receptors may play an important role in the development, maturation, and maintenance of various neuronal networks.

Cell division, cell death, and remodeling of connections are major features of the construction of the mammalian CNS. We have begun to address the role of neurotrophins in these events through characterization of the expression of their receptors in the developing ferret visual system. By use of chemical cross-linking of iodinated neurotrophins, proteins corresponding to trkB, trkC, and p75 were identified as receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) throughout development. BDNF was also cross- linked to a truncated form of trkB that lacks the tyrosine kinase domain (trkB. T1) in retinal target tissues and in cortex. At the earliest developmental age examined (E24), the ratio of full-length to truncated trkB is > > 1 in the retinal target tissues, LGN and superior colliculus. During the ensuing period of retinal ganglion cell death and segregation into eye-specific layers, the amount of truncated trkB increases markedly relative to full-length trkB. By P27, truncated trkB is the predominant receptor for BDNF in the retinal target tissues and this pattern is maintained into adulthood. Within all subdivisions of visual cortex including the ventricular zone (VZ), intermediate zone (IZ), and cortical plate (CP), similar profiles of bands are observed. The developmental increase in abundance of truncated trkB relative to full-length occurs earliest in the VZ, with a major increase between E30 and P3. In the IZ, this shift to a predominance of truncated trkB occurs between P15 and P30, while in the CP the shift is even further delayed, not occurring until after P30. Within each subdivision of cortex, the shift to a predominance of truncated trkB occurs at times that correlate with the onset of cell death and maturation of axonal connections. This study demonstrates that members of the trk family, previously identified in the CNS on the basis of mRNA transcripts, are present as receptors with specific binding affinities for BDNF and NT- 3. Moreover, the correspondence between the developmental shift from full-length to truncated trkB and the critical periods for cell fate determination, cell death, and axonal remodeling suggests an important role for neurotrophic factors in the development of the visual system.

Neurotrophic factors regulate the developmental survival and differentiation of specific neuronal populations. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are members of the nerve growth factor (NGF) protein family, also known as the neurotrophins. Insights into the different roles of neurotrophins can be gained by studying the expression of their functional receptors. Here we report the development of procedures for their radiolabeling and efficient crosslinking to specific cell-surface receptors. BDNF and NT-3 receptors in cell lines and tissue preparations expressing receptors for the 2 neurotrophins have been identified using this affinity crosslinking procedure. Like NGF, BDNF and NT-3 crosslinked to the low affinity NGF receptor (p75NGFR) on PC12 cells. BDNF and NT-3 also crosslinked to cells expressing p145trkB protein, producing an approximately 160 kD neurotrophin-receptor complex. Crosslinking of the 2 neurotrophins in vivo to specific trk family members in many areas of the central nervous system also produced a 160 kD receptor complex. However, in all brain regions a complex of approx. 100 kD could also be identified, all or most of which represents crosslinking to a truncated form of trkB. The broad distribution of BDNF and NT-3 receptors throughout the CNS suggests that neurotrophins may have yet unrecognized functions on specific neuronal populations. BDNF and NT-3 receptors were also found in brain areas in which the neurotrophins themselves are also synthesized, suggesting that beyond long-range trophic effects, these proteins may also act as autocrine or short-range paracrine regulators.

Members of the Trk tyrosine kinase family have recently been identified as functional receptors of the NGF family of neurotrophins. Here we show the rat trkC locus to be complex, encoding at least four distinct polypeptides. Three of the encoded polypeptides are full-length receptor tyrosine kinases that differ by novel amino acid insertions in the kinase domain. A fourth protein is a truncated receptor that lacks the catalytic domain. Tyrosine phosphorylation, cross-linking, and ligand binding assays indicate that TrkC receptors interact with NT-3 and not with the related neurotrophins NGF, BDNF, xNT-4, or hNT-5. Furthermore, high and low affinity NT-3-binding sites are associated with the TrkC receptors. Stable and transient expression of TrkC receptors in PC12 cells indicates that the neurite outgrowth response elicited by NT-3 is dramatic in receptors lacking the novel kinase insert (gp150trkC) but absent in receptors containing the 14 amino acid insert in the kinase domain (gp150trkC14). These data suggest that the trkC locus encodes receptors that may be capable of mediating different biological responses within the cell. This could have important implications in understanding the role of neurotrophins in the development of the vertebrate nervous system.

The expression of neurotrophin (NGF, BDNF, and NT-3) mRNAs in 24 cell lines derived from human malignant gliomas was studied by Northern analysis. Widespread expression of neurotrophin genes was found with BDNF being the most abundantly expressed. Nearly all cell lines expressed BDNF, and about two-thirds of the cell lines expressed NGF and NT-3. Half of the cell lines analyzed expressed all three neurotrophins. Secretion of NGF into the medium of several cell lines could be detected by ELISA and a PC12 neurite outgrowth assay. Immuno- and bioactive NGF was isolated from conditioned medium of one cell line. No evidence of expression of the neurotrophin receptors trk and trkB by Northern analysis was found. Receptor crosslinking with radiolabeled cognate ligands failed to detect functional receptors in all but one cell line. In this cell line a receptor complex for BDNF was found that corresponded to truncated trkB receptors that lack the signal transducing tyrosine kinase domain. Neurotrophins did not stimulate mitosis of the glioma cultures. The findings suggest that production of neurotrophins by glioma cells is a general phenomenon, although neurotrophins made by gliomas lacking their receptors may not play an autocrine but rather a paracrine role.

Agrin, a molecule produced by motoneurons that induces the aggregation of nicotinic acetylcholine receptors (nAChRs), has recently been structurally characterized. Agrin-related proteins (ARPs) that arise from differential splicing are synthesized by neurons and muscle. The C-terminal region of agrin that instructs muscle to aggregate nAChRs contains three laminin A modules separated by epidermal growth factor-like modules. Alternative splicing in the laminin A modules leads to the formation of at least three ARPs that are devoid of nAChR-aggregating activity. In their N-terminal regions, both agrin and ARPs contain nine follistatin-related modules that, like those in follistatin and in another related protein, osteonectin, may have the capability to bind members of the transforming growth factor beta (TGF-beta) or platelet-derived growth factor (PDGF) families. This review proposes that these follistatin-like regions of agrin and ARPs might bind and localize growth factors, and thus provide a matrix-bound concentration of them. Beyond agrin's role in inducing AChR aggregation, the function of agrin and ARPs to provide a localized reservoir of growth factors could contribute to the formation and maintenance of the long-lasting synaptic architecture by specifying and limiting the area of influence of these molecules.

Abstract: K‐252b, a protein kinase inhibitor, has been shown earlier to inhibit nerve growth factor actions on cholinergic neurons of the basal forebrain. In the present study, K‐252b was found to prevent trophic actions of two other neurotrophins, brain‐derived neurotrophic factor, and neurotrophin‐3, on central cholinergic and dopaminergic neurons, peripheral sensory neurons, and PC 12 pheochromocytoma cells, when used at >2 μM concentration. Comparable actions of nonneurotrophin growth factors were not affected. Surprisingly, at 0.1‐100 nM, K‐252b selectively enhanced the trophic action of neurotrophin‐3 on central cholinergic neurons, peripheral sensory neurons, and PC 12 cells. In PC 12 cells, K‐252b potentiated the neurotrophin‐3‐induced tyrosine phosphorylation of trk, a protein kinase responsible for transmitting neurotrophin signals. Of the three structurally related nerve growth factor inhibitors, K‐252a, K‐252b, and staurosporine, only the first two also mediated neurotrophin‐3 potentiation. These findings indicate that K‐252b generally and selectively potentiates the neurotrophic action of neurotrophin‐3 and suggest that this action involves trk‐type neurotrophin receptors.

Cell culture studies with dissociated primary cultures from embryonic rat brain revealed that brain-derived neurotrophic factor (BDNF) promotes the developmental differentiation of both basal forebrain cholinergic and mesencephalic dopaminergic neurons. These studies suggested that, in the adult brain, BDNF may be able to protect cholinergic and dopaminergic neurons from degenerative changes induced by axotomy, similar to the known protective action of NGF in cholinergic neurons. Testing this hypothesis, we found that intraventricular administration of recombinant human BDNF (rhBDNF) to adult rats with transections of the fimbria significantly reduces axotomy-induced degenerative changes of the cholinergic cells in the basal forebrain. No such effect was seen on the dopaminergic neurons of the ventral mesencephalon after transection of their axons ascending in the medial forebrain bundle. Injected in equal amounts, rhBDNF and recombinant human NGF had quantitatively different effects on the cholinergic neurons. BDNF sustained only part of the population of cholinergic neurons affected by the lesion, whereas the entire population was protected by NGF treatment.

Recombinant human brain-derived neurotrophic factor (rhBDNF) and neurotrophin 3 (rhNT-3), two recently cloned molecules closely related to nerve growth factor (NGF), were produced from human cDNA expressed in human embryonic kidney cells. The recombinant proteins were tested in cultures of dissociated fetal rat brain cells containing basal forebrain cholinergic neurons. rhBDNF stimulated the differentiation of the cholinergic neurons, similar to NGF, which is well established as a neurotrophic factor for these cells. However, rhBDNF was particularly effective during the first few days in vitro, whereas the stimulatory action of rhNGF was more pronounced later in the development of the cultures. This finding indicates the existence of different time periods of responsiveness of the cholinergic neurons to BDNF and NGF. To assess the selectivity of the effect of rhBDNF on cholinergic neurons, its actions were tested in cultures of ventral mesencephalon containing dopaminergic cells. In contrast to NGF, which does not affect central dopaminergic neurons, rhBDNF increased dopamine uptake activity. The findings suggest that BDNF stimulates survival or differentiation of other cells besides the cholinergic neurons.

Brain-derived neurotrophic factor (BDNF) is a 27-kDa basic protein of noncovalently linked 13.5-kD subunits related to nerve growth factor and is produced by the central nervous system (CNS). BDNF has been shown to promote the survival of neurons located in or directly connected with the CNS and is likely to function in adjusting the cell number within neuronal populations to the need of this projection field. Here we describe the primary structure of a human BDNF cDNA, the biological activities of pure recombinant human BDNF, and the tissue distribution of rat BDNF. BDNF mRNA can be found in some peripheral tissues as well as in the CNS, and recombinant human BDNF is a potent neurotrophic factor for primary peripheral sensory neurons.

Neurotrophic factors are essential for neuronal survival and function. Recent data have demonstrated that the product of the tyrosine kinase trk protooncogene binds NGF and is a component of the high affinity NGF receptor. Analysis of the trkB gene product, gp145trkB, in NIH 3T3 cells indicates that this tyrosine kinase receptor is rapidly phosphorylated on tyrosine residues upon exposure to the NGF-related neurotrophic factors BDNF and NT-3. Furthermore, gp145trkB specifically binds BDNF and NT-3 in NIH 3T3 cells and in hippocampal cells,but does not bind NGF. Thus, the trk family of receptors are likely to be important signal transducers of NGF-related trophic signals in the formation and maintenance of neuronal circuits.

The hypogonadal (hpg) mouse represents a unique animal model for hypogonadism. In this mutant the truncation of the gene encoding GnRH and its associated peptide GAP leads to drastically lowered gonadotropin levels and increased circulating prolactin. This deficiency in turn leads to a failure of testes and ovaries to develop normally. Using gene therapy we have restored the reproductive functions of the hpg mouse. The success of this therapy uniquely underscores the importance of the gene encoding the GnRH precursor and lends credence to the hypothesis that no other gene in mammals can replace it. As a consequence, defects in the control and/or structural properties of the human GnRH are expected to result in hypogonadism in humans.

Activin, which stimulates the secretion of FSH from anterior pituitary cells, is a dimer of the beta-subunits of inhibin. Two species of activin (A and AB) have been purified from ovarian follicular fluid and characterized. We have been able to biosynthetically produce recombinant human activin A by constructing stable cell lines expressing the mRNA for the beta A subunit of human inhibin. These cell lines secreted a 24 kilodalton beta A dimer which stimulated FSH secretion in cultured pituitary cells. The ability of this protein to stimulate FSH secretion was sensitive to reduction of disulfide bonds, exhibited a slow onset of action, and was blocked by actinomycin D. In addition, recombinant activin A stimulated hemoglobin accumulation in K562 cells. These data show that recombinant activin A has the biochemical properties and biological activities that have been ascribed to native activin A. In addition, these results provide an independent confirmation, and thereby a final proof, of the structure and function of activin.