The amino terminus of nerve growth factor is involved in the interaction with the receptor tyrosine kinase p140trkA.

The amino terminus of nerve growth factor (NGF) is susceptible to proteolytic cleavage. A comparison of the bioactivity of highly purified full-length recombinant human (1-118)rhNGF and NH2-terminal truncated (10-118)rhNGF revealed lower potency of (10-118)rhNGF with regard to early NGF responses in neuron-like PC12 cells. Approximately 50 times higher concentrations of (10-118)rhNGF than (1-118)rhNGF were required to elicit the same extent of tyrosine phosphorylation of key enzymes in different second messenger pathways, i.e. the NGF receptor tyrosine kinase p140trkA, phospholipase C gamma-1, and the extracellular signal-regulated kinase ERK1. A similar reduced potency for induction of the transcription factor c-Fos was observed with (10-118)rhNGF compared to (1-118)rhNGF. The lower potency of (10-118)rhNGF in triggering early responses correlated with its 40-fold lower affinity for PC12 cells. Whereas (10-118)rhNGF had a more than 300-fold lower affinity for the high affinity receptor p140trkA than (1-118)rhNGF, amino-terminal truncation of NGF changed its affinity for the low affinity receptor p75NGFR only slightly (5-10-fold). These observations suggest that amino acids 1-9 of NGF are important for binding to the signal transducing receptor p140trkA. Proteolytic cleavage of the NGF amino terminus, therefore, reduces its potency in starting several second messenger pathways leading to neuronal differentiation of PC12 cells.

Binding of NGF to its two receptors seems to be determined by distinct regions of the NGF molecule. The solvent-accessible loop made up by residues 29-35 ( 5 ) was identified to be crucial for recognition by ~7 5~"~~ (15). On the other hand, amino acids Trp-21 and Val-22 residing in the @-pleated sheet of NGF appear to be critically involved in eliciting biological activity in neurons (16). The amino-terminal 8 (murine NGF, mNGF) or 9 (human NGF, hNGF) amino acids, which are susceptible to proteolytic cleavage, were considered not to be involved in interactions with NGFRs (17). Full-length and amino-truncated NGF were considered equal in biological activity based on the initial experiments performed with mNGF. Recently, highly purified recombinant human fulllength rhNGF and amino-truncated (10-118)rhNGF became available (18). Using these polypeptides, a lower potency of (10-118)rhNGF compared to rhNGF with regard to survival of sensory and sympathetic neurons, as well as neurite outgrowth from PC12 cells, was observed by these authors.
We report that ~7 5~"~~ binds (1-118)rhNGF with slightly higher affinity than (10-118)rhNGF. In contrast, p140trkA binds (1-118)rhNGF with over 300-fold higher affinity than (10-118)rhNGF. In PC12 cells expressing both ~7 5~"~' and p140trkA, a 40-fold difference in receptor binding affinity of the two polypeptides was observed, which correlated well with the difference in their potency for inducing early NGF responses, namely autophosphorylation of p140trkA, as well as tyrosine phosphorylation of phospholipase CT.l, and the extracellular signal-regulated kinase ERK1. Likewise the potency for induction of the transcription factor c-Fos differed between (1-118)rhNGF and (10-118)rhNGF. These results suggest that the NH2 terminus of NGF is important for functional binding to p140trkA.
Cell Culture-Cells were maintained in the presence of 1% penicillin/streptomycin. Rat 33B glioma cells (European Collection of Animal Cell Cultures) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (19) NGF Binding-Binding of "'I-mNGF (specific activity -1500 Ci/ mmol) was competed with increasing concentrations of unlabeled NGF in the suspension assay described by Vale and Shooter (23). To prevent Ca'+-dependent cell clumping (24), 33B cells were assayed in phosphate-buffered saline instead of Krebs-Ringer solution. For PC12 cells similar results were obtained in both buffers. Competition isotherms were fitted according to Rodbard and Frazier (25).
However, the ICs0 values differed between NGF species (Table I). In A875 and 33B cells expressing p75NGFR as only NGFR, a slight difference in ICs0 values was observed compared to a more than 300-fold decrease in affinity of (10-118)rhNGF in K562 cells expressing p140trkA. In the PC12 cells expressing both ~7 5~'~~ and p140trkA the difference was approximately 40-fold.
Induction of Tyrosine Phosphorylation-To determine whether the observed differences between (1-118)rhNGF and (10-118)rhNGF in binding to p140trkA correlate with induction of signal transducing pathways, NGF induction of tyrosine phosphorylation of p14OLrkA, PLC,.l, and ERKl was measured.
These immediate responses are thought to be mediated by p140trkA. All three enzymes were phosphorylated after 3 min of stimulation with either NGF form (Fig. 3). The phosphorylations were concentration-dependent in a manner that paralleled the competition binding curves. Densitometric scanning (not shown) revealed that approximately 50 times more (10-118)rhNGF than rhNGF was required to elicit the same phosphorylation response. D. Hartman and C. Hertel, manuscript in preparation. Induction of c-Fos-Like other growth factors, NGF induces transcription factors encoded by immediate early genes. One of these, the product of the c-fos protooncogene, was induced by (1-118)rhNGF as well as (10-118)rhNGF in a concentration-dependent manner (Fig. 4). Similar to the results obtained for the phosphorylation responses, densitometric scanning (not shown) revealed that an about 50-fold higher concentration of (10-118)rhNGF compared to (1-118)rhNGF was required for the same extent of c-Fos induction. The lower molecular weight band which cross-reacted with the anti-Fos antiserum (probably c-Jun), was apparently not induced by NGF.

DISCUSSION
PNGF is the end product of proteolytic processing of preproNGF. During purification of PNGF from mouse submaxillary glands further proteolytic degradation at both ends of the polypeptide occurs (17). These proteolytic products of pNGF had been considered equal in biological activity. Recently, recombinant human NGF became available, which made it feasible to produce large quantities of the purified proteolytic forms and to determine their biological activity. While truncation at the COOH terminus did not affect the biological activity of NGF (28), truncation at the NH2 terminus resulted in 50-100-fold reduced bioactivity in neuronal survival and differentiation assays (18). These functional responses to NGF are thought to be mediated by the high

TARLE I ICco values of NGF variants for binding to NGFR expressed by different cell lines
Two to six independent competition isotherms were pooled and analyzed by nonlinear least-squares curve-fitting (25). The resulting ICs0 values are given in nmol/liter f standard deviation of the calculated IC,,". affinity NGFR ( K d = 10 pM). Whether pl4OCrkA is a high affinity NGFR on its own or as a complex with ~7 5~'~' is still unclear (9,13). We have investigated whether the reduced bioactivity of amino-terminal truncated NGF is correlated to a reduction in the affinity to one or both NGFR. It is demonstrated that rhNGF has a more than 300-fold higher affinity for pl40lrkA than (10-118)rhNGF. In contrast, (10-118)rhNGF bound to ~7 5~"~~ with only slightly lower affinity than rhNGF. Based on the three-dimensional model of mNGF (5), these results suggest that the binding sites of NGF for p140trkA and ~7 5~'~~ are located in two distinct regions of the NGF molecule, i.e. p140trkA binds to the NH2 terminus and ~7 5~'~~ to the solvent-accessible loop containing residues 29-35. In PC12 cells, which express both receptors, the difference in affinity is approximately 40-fold. The affinity of 2.5 S  4 ) and (10-118)rhNGF (lanes 5-7) in N2. Anti-phosphotyrosine immunoprecipitates were prepared from PC12 lysates as described under "Experimental Procedures" and subjected to SDS-PAGE (7.5% acrylamide:bisacrylamide, 37.5:l). The corresponding nitrocellulose blot was probed with anti-TrkA, stripped and reprobed with anti-ERKI, and stripped and reprobed with anti-PLC,.,. The experiment was repeated resulting in the same relative band intensities.  5-7) at the concentrations indicated, or nothing (lane 1 ) was added to sister PC12 cultures. After stimulation, crude nuclear extracts were prepared and separated by SDS-PAGE (7.5% acrylamide:bisacrylamide, 201). The corresponding nitrocellulose blot was probed with anti-Fos (see "Experimental Procedures"). The experiment was repeated giving similar results. mNGF was always slightly lower than that of (1-118)rhNGF. The K d of 2.5 S mNGF for p140trkA varied between preparations (data not shown), a finding that is most likely due to varying amounts of NHZ-terminal truncated desocta-mNGF in each preparation. The presence of contaminating desocta-mNGF may therefore account for at least a part of the reported lower potency of mNGF compared to rhNGF in eliciting neurite outgrowth from chick dorsal root ganglion cells and induction of choline acetyltransferase activity in rat septal cells (29).
In the absence of p75NGFR, p140trkA was found to bind (1-118)rhNGF with a K d of approximately 0.3 nM, whereas 2.5 s mNGF had a K d varying between 0.6 and 3.5 nM depending on the quality of the preparation. This suggests that p140trkA is the high affinity receptor for full-length NGF. Slight differences in the composition of 2.5 S mNGF may explain why some groups find high as well as low affinity NGF binding sites in cells expressing p140trkA (9), while others find only low affinity sites (13). Species differences are unlikely to account for the differences in affinity of murine 2.5 S NGF and human rhNGF to p140trkA, given the high species homology of NGF (3). Moreover, while no differences in bioactivity for the full-length forms of mNGF and rhNGF were seen, differences in potency of purified full-length and desocta-mNGF analogous to those observed with (1-118)rhNGF and (10-118)rhNGF, have been shown (18, 30). However, differential affinities of NGF for rat p140trkA studied by Hempstead et al. (13) and its human homolog employed by Klein et al. (9) and in our study can presently

Involvement of NGF NH2 Terminus in Interaction Withp140trkA
not be ruled out, despite the high homology between rat and human trkA (14). The drastically smaller binding affinity of (10-118)rhNGF to the receptor tyrosine kinase p140trkA was correlated to immediate early NGF responses in PC12 cells. The concentrations required to obtain an equal extent of tyrosine phosphorylation of key enzymes involved in signal transduction (p140trkA, PLC,.l, ERK1) and induction of the transcription factor c-Fos were approximately 50 times higher for (10-118)rhNGF than for rhNGF. These values coincide with the dose dependence of the late response neurite outgrowth (18). Thus, the lower binding affinity of aminotruncated NGF to p140trkA appears to cause a reduced potency with regard to induction of the signal transduction pathways leading to neuronal survival and differentiation.