The neurotrophin family consists of nerve growth factor (NGF), ()brain-derived neurotrophic factor (BDNF), neurotrophin-3, and neurotrophin-4(1, 2) . Receptors for these neurotrophins include the Trk family of membrane proteins and gp75 (also known as the low affinity NGF receptor). TrkA preferentially binds NGF, whereas, TrkB binds BDNF and neurotrophin-4, and TrkC binds neurotrophin-3. In addition, gp75 binds all four members of the neurotrophin family(3) .

For NGF, there have been extensive studies relating receptor expression with neurotrophin responsiveness. There are two classes of I-labeled NGF binding sites, low affinity (K 10M) and high affinity (K 10M) (4) . Cells which are nonresponsive to NGF express only low affinity binding sites, and cells which are responsive to NGF generally express both low affinity and high affinity NGF binding sites(5, 6) .

The identity of the functional high affinity NGF receptor is a source of continuing controversy. Jing et al. (7) reported that TrkA was sufficient for high affinity NGF binding, and Ibanez et al. (8) found that a mutated NGF which does not bind to gp75 was biologically active. In contrast, Hempstead et al.(9) reported that expression of both the gp75 low affinity NGF receptor and the TrkA NGF receptor was required for high affinity NGF binding. Transgenic mice which lack gp75 have neurological deficiencies, particularly in the sensory nervous system(10) . Furthermore, trigeminal neurons from these gp75 mice have a decreased sensitivity to NGF(11) . Gp75 appears to participate in the regulation of neurotrophic selectivity of TrkA and to enhance the response of TrkA to NGF(12, 13) . An anti-low affinity NGF receptor monoclonal antibody (mAb) inhibits responses of PC12 cells to NGF(14) . Furthermore, expression of gp75 in fibroblastic cells enhanced signal transduction by TrkA, TrkB and the TrkC(15) . However, direct biochemical or biophysical evidence for gp75-TrkA interaction and complexing has not been reported.

One approach to analysis of membrane structure is fluorescence recovery after photobleaching (FRAP)(16) . FRAP is a technique for measuring the lateral mobility of macromolecules in membranes and aqueous phases (16) . In FRAP, the molecule whose diffusion is to be measured is fluorescently tagged specifically in a non-cross-linking manner. In the current application, gp75 molecules were labeled with a Fab fragment of a mouse mAb against gp75 followed by a fluorescein conjugated Fab fragment of a goat anti-mouse IgG. A laser beam is focussed using a modified fluorescence microscope to a small (1 µm) spot on the sample. The fluorescence from this spot is monitored and found to be essentially constant with time, at the so called prebleach level. The incident light level is momentarily increased 10,000-fold so as to irreversibly photobleach a significant fraction of the fluorescence within the spot. Thus, when the laser returns to the monitoring intensity, the fluorescence intensity is significantly reduced. If the molecules are not free to move in and out of the spot, the fluorescence will remain at this level ad infinitum. This is the condition of no diffusibility or complete immobility. If, on the other hand there is complete mobility or freedom to diffuse, the bleached molecules diffuse out of the spot, the unbleached molecules diffuse in, and the fluorescence intensity recovers to the prebleach level. The diffusion coefficient, D, is obtained by fitting the recovery data to diffusion theory(16) . At the molecular level, D can be used to determine how long, , it takes a molecule to diffuse a distance x. = x/4D. A typical membrane protein with D = 10 cm/s will diffuse a distance of 1 µm in 2.5 s or 10 µm in 250 s. Most membrane proteins are only partially mobile, i.e. a fraction of these molecules are immobilized and do not diffuse. In this case, partial recovery is observed. The fractional recovery in a FRAP experiment is the mobile fraction. Thus FRAP curves yield two independent parameters. The percent recovery (R) or mobile fraction, is the fraction of molecules free to diffuse in the plane of the membrane into the bleached area. D is calculated from the rate of recovery and is a measure of the rate of diffusion for the mobile fraction of receptor proteins.

Motion of membrane proteins appears to be constrained by a complex set of protein specific interactions involving the extracellular domain, the transmembrane domain, or the intracellular domain. Using site-directed mutagenesis of glycosylation sites, Wier and Edidin (17) demonstrated that reducing the size of the extracellular domain of a major histocompatibility complex antigen enhanced D. Goncalves et al. (18) reported that mutation of the transmembrane domain of the insulin receptor affected diffusion, suggesting that there may be important interactions within the membrane. Studies in which the intracellular domains of membrane proteins have been deleted produced varied results. For some membrane proteins(19, 20, 21, 22) , the deletion has no effect, but for other proteins D is enhanced(23, 24) . The mechanisms by which these interactions affect diffusion are also varied. Wier and Edidin (17) suggest that interactions with the extracellular domain are nonspecific and caused by viscous drag. Sheetz et al. (25) have shown that the diffusion of band 3 in the erythrocyte plasma membrane is constrained due to interaction with or corralling by the spectrin cytoskeleton. Paccaud et al. (24) have suggested that interaction of intracellular domains with the clathrin of coated pits decreases lateral diffusion.

Our earlier studies (26, 27) of gp75 diffusion using FRAP are suggestive of a gp75-TrkA interaction. We demonstrated that gp75 is diffusely distributed and mobile on cell lines which lack TrkA and are nonresponsive to NGF. In contrast, gp75 is aggregated and relatively immobile on cell lines which express TrkA and are responsive to NGF (26, 27) . Significantly, while gp75 was immobilized on rat pheochromocytoma PC12 cells which express TrkA, it was relatively mobile on cells of the nonresponsive line nnr5, a PC12 variant which does not express significant levels of TrkA(5, 28) . Gp75 also was relatively immobilized on primary cultures of rat NGF-responsive sensory neurons, which express TrkA(27) .

In this report, we show that interaction with TrkA causes gp75 immobilization. The related receptor TrkB has a smaller effect. TrkA immobilization and high affinity binding requires an intact TrkA kinase region and gp75 cytoplasmic domains. Furthermore, analysis of diffusion coefficients suggests that mutated forms of gp75 and TrkA may still form complexes even in the absence of high affinity binding.

EXPERIMENTAL PROCEDURES

Antibodies and Fragments

()

Baculovirus Vectors

Cell Lines

Baculovirus Expression in Sf9 Cells

Assay of Baculovirus Expression in Sf9 Cells by Polyacrylamide Gel Electrophoresis

Assay of NGF Receptor Expression in Sf9 Cells by Indirect Immunofluorescence

Labeling of Cells for FRAP Measurements

FRAP measurements on Sf9 cells were carried out approximately 60 h postinfection. Cells (5 10) were incubated for 30 min at room temperature with 50 µl of a Fab fragment of mAb NGFR5 in Sf9 growth medium (0.1 mg/ml). The samples were centrifuged, and the cells were washed twice with 200 µl of medium. The cells were suspended in 25 µg/ml fluorescein Fab fragment of anti-mouse IgG and incubated for 30 min at room temperature. The cells were washed twice with medium, and FRAP measurements were performed as previously described(26, 27) . For measurements made in the presence of NGF, the cells were resuspended in medium containing 100 nM NGF immediately prior to measurements. All measurements were made within 30 min of labeling during which time no detectable internalization was observed.

FRAP Measurements

where I is the intensity at the center, x, y are the Cartesian coordinates in the plane of the object, and w is the beam radius = 0.9 µ. The monitoring intensity was 0.13 µW and the bleaching intensity was 1.3 mW for 25 ms. These conditions were chosen so that there would be no significant bleaching due to the monitoring beam. Samples were discarded if solution background intensities exceeded 10%. Data were fitted to the diffusion theory of Axelrod et al. (36) by a modification of the nonlinear least squares procedure of Bevington (16, 37, 38) . FRAP data is presented as averages (± S.E.) of n single bleach measurements made on n separate cells.

An example of a typical FRAP recovery curve is shown in Fig. 1. If F(t < 0) is the prebleach fluorescence intensity, F(0) the fluorescence intensity immediately following the bleach, and F() the fluorescence intensity after recovery is complete, then the percent R = (F() - F(0))/(F(t < 0) - F(0)). The diffusion coefficient can be calculated from the time for half recovery .

Figure 1: A typical FRAP recovery curve showing the diffusion of gp75 on gp75 + TrkA expressing Sf9 cells (see ``Results'' for details). Data have been normalized by dividing by the average prebleach intensity so that F(t < 0) = 1.0. The cell was prebleached for 25 ms so that the normalized intensity at time 0 was F(0) = 0.35. The recovery asymptotically approaches a value F() = 0.62. Therefore the percent R = 42%. The time at which the curve recovers half way to 0.62 (i.e. to a value of 0.49) occurs at = 1.2 s. This corresponds to a diffusion coefficient D = 2.05 10 cm/s.

where w is the exp(-2) beam radius and = / is a coefficient determined from diffusion theory (16) and dependent upon the fractional depth of bleach F(0)/F(t < 0)). Typically 1.3.

RESULTS

TrkA Immobilization of gp75

Figure 2: Histograms showing frequency of mobile fractions for diffusion of rat gp75 on (A) nnr5 (TrkA) (n = 170) and (B) T14 nnr5 (TrkA) (n = 171) cell lines which were derived from PC12 cells. Means, standard errors, and number of measurements are given in Table 1for both percent R and D.

Domains Required for Interaction

()

Figure 3: Coexpression of gp75 and TrkA. A, Western blot analysis of gp75 and TrkA in extracts of Sf9 cells infected with the baculovirus encoding gp75, infected with the baculovirus encoding TrkA or infected with both of these baculoviruses. The blots were probed either with anti-gp75 monoclonal antibody ME20.4 or with rabbit anti-TrkA antiserum 203. The molecular weight of TrkA in this insect cell system is 110,000, but the value reported in mammalian cells is 140,000. This difference is apparently due to decreased glycosylation in the insect cells and does not interfere with expression of high affinity NGF binding sites (R. M. Stephens, D. R. Kaplan, and& B. L. Hempstead, unpublished work). B and C, fluorescence photomicrographs showing coexpression of gp75 and TrkA on Sf9 cells infected with baculoviruses encoding gp75 and TrkA. Cells were labeled with mouse anti-gp75 (NGFR5) and rabbit anti-TrkA IA683 followed by fluorescein goat anti-mouse IgG and rhodamine anti-rabbit IgG. B shows fluorescein staining of gp75, and C shows rhodamine staining of TrkA on the same field of cells. (In some experiments like this one, co-patching of gp75 and TrkA was observed, which may be an additional indication of complexing.) No labeling was observed in the absence of primary antibody or of uninfected cells in the presence of primary (not shown).

We next tested whether, as in PC12 cells, TrkA would cause immobilization of gp75 expressed in Sf9 cells. When gp75 was expressed alone, it showed a relatively high mobile fraction (percent R = 62 ± 1) (Fig. 4A, see Table 1for tabulation of data). Coexpression with TrkA (Fig. 4B) resulted in immobilization of gp75 (percent R = 45 ± 1; p 0.001).

Figure 4: Histograms showing frequency of mobile fractions for diffusion of human gp75 expressed in Sf9 insect cells. Means, standard errors, and numbers of measurements are given in Table 1for both percent R and D. A, gp75 expressed alone (n = 603); B, gp75 coexpressed with TrkA (n = 435); C, gp75 coexpressed with kinase-deficient TrkA(K538N) (n = 193); D, gp75 coexpressed with TrkB (n = 166); E, gp75 truncation mutation gp75(Xba) expressed alone (n = 434); F, gp75(Xba) coexpressed with TrkA (n = 527).

To determine which domains of gp75 were responsible for immobilization and whether the tyrosine kinase activity of TrkA was involved in this interaction, baculoviruses encoding several NGF receptors were used (these are shown schematically in Fig. 5); mutant receptor TrkA(K538N) with lysine 538 replaced by asparagine; TrkB which includes the full-length BDNF receptor; gp75(Xba) which contains amino acids 1-248 but lacks the remaining cytoplasmic domain (amino acids 249-399); and gp75(PS) which has amino acids 249-305 deleted. The point mutation of TrkA(K538N) is in the ATP binding site of the kinase domain and significantly reduces high affinity binding of NGF and eliminates kinase activity(7) . Coexpression of gp75(Xba) or gp75(PS) with TrkA in PC12-derived cells does not result in high affinity binding of NGF(39) .

Figure 5: Schematic of NGF receptors wild type and mutated forms.

Coexpression with TrkA(K538N) (Fig. 4C) did not result in significant immobilization of gp75 (percent R = 59 ± 1). Coexpression of gp75 with TrkB caused a slight but significant immobilization of gp75 (percent R = 56 ± 1, p < 0.01) (Fig. 4D). As judged by Western blotting, TrkA(K538N) and TrkB levels were similar to those of TrkA (data not shown). Therefore, the lack of effect of TrkA(K538N) and reduced effect of TrkB on gp75 mobile fraction were not due to lower levels of expression. Truncated receptor gp75(Xba) showed similar mobility (percent R = 57 ± 1) (Fig. 4E) to gp75 expressed alone but was not immobilized by coexpression with TrkA (percent R = 57 ± 1) (Fig. 4F). Receptor gp75(PS) showed slightly higher mobility (percent R = 67 ± 1, p < 0.01) than intact gp75 but could not be detected on the surface of cells coinfected with TrkA. The deletion mutation in gp75(PS) results in a consensus sequence for interaction with coated pits(40) . This in turn may cause, in the presence of TrkA, accelerated internalization and therefore, a lack of gp75(PS) on the cell surface. Despite the difficulties in expressing gp75(PS) with TrkA, one can conclude that effective immobilization of gp75 requires both an active TrkA kinase and an intact gp75 cytoplasmic domain. These requirements are similar to those of high affinity NGF binding site formation and responsiveness(7, 39) .

Effect of NGF on Diffusion

DISCUSSION

These experiments confirm our hypothesis that immobilization (decreased percent R) of gp75 in NGF-responsive cells is a result of TrkA expression. Since TrkA-induced immobilization of gp75 occurs in both neuronal nnr5 cells and nonneuronal Sf9 cells, this interaction does not appear to require any other gene products expressed only in neuronal cell types. The simplest model for the interaction between gp75 and TrkA is that they form a physical complex, as proposed by Hempstead et al.(9) . An alternative model is that TrkA indirectly alters a third molecule which interacts with gp75 and results in immobilization. For example, TrkA is thought to activate a kinase which is non-covalently associated with the cytoplasmic domain of gp75(41) . However, the observation that NGF does not enhance immobilization of gp75 in the presence of TrkA argues against such indirect models and therefore favors the gp75-TrkA complex model.

TrkB induces a smaller immobilization of gp75. Our data detect an interaction. However, there is, in fact, little published biochemical or biological evidence for such a gp75-TrkB interaction. Loss of gp75 does not alter BDNF binding(11) , but recently it was reported that gp75 enhances the activity of TrkA, TrkB, and TrkC in fibroblastic cells(15) . Our results suggest the need for further studies to substantiate such an interaction.

Our results indicate that there is an immobile fraction of gp75 even in the absence of TrkA both on nnr5 and Sf9 cells. Thus, there are multiple factors controlling the mobility of gp75. Immobile fractions have been observed for the majority of membrane proteins (42) particularly those, which like gp75, have a significant extracellular domain and a single membrane spanning region. Indeed Wolf et al.(43) have shown that synthetic lipopolysaccharides have immmobile fractions similar to those of native membrane proteins and can interact with the cytoplasm, despite the fact that they do not span the bilayer. The base line immobile fraction for gp75 is higher on nnr5 cells than on Sf9 cells, which may reflect the more complete glycosylation of gp75 on mammalian cells or differences in the membrane or environment of mammalian versus insect cells.

Our results enable us to refine several aspects of the model of Hempstead et al.(9) . For the high affinity NGF receptor complex, the observation that immobilization of gp75 occurs in the absence of NGF indicates that the interaction of gp75 and TrkA exists prior to binding of NGF. Such a complex could enhance both the rate and affinity of NGF binding(9, 44) . These data rule out models in which NGF binding triggers formation of a gp75-TrkA heterodimer.

A second modification of the model is indicated by our finding that a gp75-TrkA interaction exists in the absence of high affinity NGF binding sites. Sf9 cells expressing (gp75(Xba) + TrkA) or (gp75 + TrkA(K538N)) have few high affinity NGF binding sites, but addition of NGF to cells expressing (gp75(Xba) + TrkA) or (gp75 + TrkA(K538N)) enhanced the rate of diffusion (D) for gp75, as it did when wild type receptors were expressed. Addition of NGF to cells expressing gp75 alone decreased D. Hence, there is a clear interaction between the two receptors which may represent a heterocomplex.

Since larger extracellular domains are thought to hinder diffusion rates(17) , an intriguing question is why binding of NGF to cells expressing both gp75 and TrkA enhances the diffusion of gp75. One possibility is that binding of NGF causes an ordering of the gp75-TrkA extracellular domain creating a more compact extracellular domain with a reduced Stokes' radius and frictional coefficient. Another possibility, is that binding of NGF blocks specific interactions of the receptors with other cell surface components.

Even in the absence of high affinity NGF binding sites, such gp75-TrkA complexes may still be of biological significance. In fibroblastic cells, TrkA receptor activated by NGF stimulates cell proliferation and survival at low serum levels(45) . This TrkA effect is enhanced by coexpression of gp75. A truncated gp75 similar to gp75(Xba) was shown to enhance the effects of TrkA to an even greater extent(15) .

A third refinement of the model is suggested by our finding that immobilization of gp75 requires an intact cytoplasmic domain of gp75 and a functional kinase domain on TrkA. Since these requirements are the same as those for high affinity NGF binding, these data indicate that immobilization and high affinity NGF binding may result from the same change in receptor structure. We conclude that the functional high affinity NGF binding site is not a simple consequence of physical proximity of gp75 and TrkA, but rather may involve a complex conformational change requiring intracellular and extracellular domains of both receptors.

FOOTNOTES

*

This work was supported in part by National Institutes of Health grants NS28760 (to D. E. W. and A. H. R.) and NS21716 (A. H. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom all correspondence should be addressed: The Worcester Foundation for Experimental Biology, 222 Maple Ave., Shrewsbury, MA 01545. Tel: 508-842-8921; Fax: 508-842-9632.

()

The abbreviations used are: NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; mAb, monoclonal antibody; FRAP, fluorescence recovery after photobleaching; m.o.i., multiplicity of infection; na, numerical aperture.

()

Loy, R., Lachyankar, M., Condon, P., Poluha, D., and Ross, A. H.(1994) Exp. Neurol., in press.

()

R. M. Stephens, D. R. Kaplan, and B. L. Hempstead, personal communication.

ACKNOWLEDGEMENTS

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