nerve growth factor receptors

THE JOURNAL OP BIOLOGICAL CHEMISTRY Vol. 254, No. 13, Issue of July 10, pp. 5972-5982, 1979 Prrnted m U.S.A.

Nerve Growth Factor Receptors CHARACTERIZATION OF TWO DISTINCT CLASSES OF BINDING SITES ON CHICK EMBRYO SENSORY GANGLIA CELLS*

(Received for publication, October 24, 1978, and in revised form, February 8,1979)

Arne Sutter,$ Richard J. Riopelle,g Ronald M. Harris-War&k,1 and Eric M. Shooter

From the Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305

Steady state and kinetic studies on the binding of lz51- p nerve growth factor (NGF) to single cells from sensory ganglia of g-day-old chick embryos show two distinct, saturable binding sites with dissociation constants of &(I) = 2.3 X 10-l’ M and &(II) = 1.7 X lo-’ M.

The difference in the affinities is due to different rate constants of dissociation (kl(I) = 10m3 s-l, k-1(11) = 2 x 10-l s-l). The association to both sites is apparently diffusion controlled (k+l(I) = 4.8 x lo7 M-’ s-‘, k+z(II) = lo7 to lo8 M-’ s-l). The binding of PNGF to both sites is specific, since none of a number of hormones or pro- teins tested compete for the binding of ‘251-PNGF to either of those two sites.

The heterogeneity of the binding of ‘251-/?NGF is not due to heterogeneity of the 12?-/3NGF preparation nor to a negatively cooperative binding. In experiments where the dissociation of ‘251-fiNGF is induced by the addition of saturating amounts of unlabeled BNGF, the ratio of the ‘251-j3NGF released with either of the two dissociation rate constants is solely dependent on the occupancy of the two sites before dissociation is started and is independent of the total occupancy of the sites during dissociation. The rate of dissociation of 1251- /?NGF from the higher affinity binding site I is accelerated by unlabeled PNGF under conditions where the occupancy is both increased and decreased.

Although the dissociation characteristics of 1251- PNGF change with increasing times of exposure of the cells to the ligand, and ‘251-fiNGF is degraded after it binds to the cells, these secondary processes do not interfere with the analysis of the binding data. At the lowest concentration of ‘251-fiNGF used for the analysis less than 10% of the ‘251-PNGF is degraded. Both kinetic and steady state binding data reveal the two NGF binding sites at 2°C as well as at 37°C.

Relatively little is known so far about the regulatory mechanisms by which peptide hormones control the metabolic activities of their target cells. There is general agreement, however, that the action of peptide hormones is mediated by

* This research was supported by National Institutes of Health Grant NS 04270 and by a grant from the Sloan Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Fellow of the Deutsche Forschungsgemeinschaft. 5 Supported by a Fellowship from the Canadian Medical Research

Council. Present address, Department of Neurology, Queens Univer- sity, Kingston, Ontario K7L 3N6.

1 Recipient of a National Research Service Award NS 05192. Present address, Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115.

specific cell surface receptors. The analysis of hormone-receptor interaction can, therefore, provide a basis for further experiments designed to probe the chain of molecular events following the formation of the hormone *receptor complex and which finally leads to a change in the phenotype of the target cell.

Nerve growth factor is a peptide hormone (1) which pro- motes the morphological and biochemical differentiation of sensory and sympathetic neurons (2). It is known that sensory and sympathetic cells carry specific cell surface receptors for NGF’ (3-5). However, the published data on the binding characteristics of these receptors are not entirely consistent. Both heterogeneous binding affinities with negative cooperativity of binding (5, 6), as well as Michaelis-Menten type binding to one receptor (3, 4), have been reported. Although heterogeneous binding characteristics have been noted for a wide range of ligands (5, 7-12) there is no general agreement on the reasons for such behavior (13, 14). Heterogeneity of binding might, for example, stem from the dynamics of the cell membrane itself and the ligand-induced reorganization of cell surface receptors which occurs as a prerequisite for the internalization of the ligand. receptor complex. This process was first described for lymphocyte cell surface immunoglob- ulins (15) and for receptors of multivalent lectins on fibroblasts (16). A ligand-induced clustering of cell surface receptors has been proposed (17) as one of several models to account for the apparently negative cooperative binding behavior of several peptide hormones (5,7,9,10) as well as neurotransmitters (11, 12). The physiological significance of the cooperative binding of ligands has been widely discussed (18-21). On the other hand, the heterogeneity of binding may reflect the presence of two sets of receptors with the binding of the ligand to each receptor being noncooperative and serving separate functions.

The present study was undertaken to explore the factors which could lead to the reported differences in the binding of NGF to its responsive cells and, in particular, to determine whether the NGF-binding sites on sensory ganglia cells are heterogeneous and whether the interaction of NGF with these sites occurs in a cooperative or noncooperative manner. One key to the study was the development of a method for obtain- ing an improved preparation of lz51-NGF. In addition, special attention was given to the identification of binding sites characterized by readily reversible binding equilibria with short half-lives for the ligand. receptor complex. These binding sites may not be detected in filtration or centrifugation procedures which include washing steps to reduce nonspecific binding. The criteria previously used to establish the cooperative binding of NGF (5, 6) were found to be insufficient. Additional experimental procedures were used for the analysis of the dissociation behavior of NGF at various receptor occupancies.

’ The abbreviations used are: NGF, nerve growth factor; EGF, epidermal growth factor; PBG, phosphate buffered Gey’s salt solution.

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Nerve Growth Factor Receptors

The data presented here confnm that the binding of NGF to at least sensory ganglia cells is heterogeneous, but support a model of two distinct NGF-binding sites rather than cooperativity induced by NGF within a homogeneous class of receptors. A preliminary account of this work has already been given (22).

MATERIALS AND METHODS’

RESULTS

Characterization of the lz51-/3NGF Preparation-In order to avoid complications in the analysis of binding data, the binding characteristics of a radiolabeled hormone should be the same as those of the native hormone. Also, for maximal sensitivity in the binding assays, the backgrounds due to nonspecific binding or receptor-independent precipitation of radioactivity should be minimal. The ‘251-PNGF preparation used in these studies met both criteria.

The trichloroacetic acid and the specific immune precipi- tabilities of the ““I-PNGF preparation were 298% and ?90%, respectively. To obtain minimal backgrounds in the binding assays the ““I-PNGF preparations were filtered through Cen- triflo CF 50A fnters. This filtration step reduced the nonspecific binding by a factor of 10. For example, at a ‘251-PNGF concentration of 2 X lo-’ M and a cell concentration of >2 X

106/ml, the nonspecific binding was reduced from 20 to 50% to less than 5%. A major part of the nonspecific binding is due to label which sediments even in the absence of cells. As a consequence the ratio of specific to nonspecific bound label will be smaller at low compared to high cell concentrations. Fig. 1 shows the analysis of a “?-fiNGF preparation on a Bio- Gel P-60 column after Centriflo filtration. In addition to small amounts of free iodine (Peak III) the different preparations contained 2 to 6% of radioactive impurities of higher molecular weight (Peak I). Peaks I and II were further analyzed on acrylamide gels under denaturing and reducing conditions (Fig. 1, insets). The radioactive material of Peak I (Fig. 1, left inset) contained several components with high molecular weights, indicating that it was partly composed of aggregates of ‘251-/?NGF, whereas the radioactive material of Peak II (Fig. 1, right inset) corn&rated with the monomer (98%) and dimer (2%) of PNGF. The immunoprecipitability of material from Peaks I and II with rabbit-ant$NGF antiserum was 30% and 95%, respectively. The binding characteristics of the material from Peak II were compared with that of the 1251-/3NGF preparation before the chromatographic purification on Bio- Gel P-60. The binding curves were indistinguishable, showing that the radioactive impurities did not interfere with the binding properties of purified 1251-PNGF (data not shown). The affinities of the ‘251-/?NGF preparation and of native PNGF for binding sites on sensory ganglia cells were practically identical. Fig. 2 shows how the ratio of bound 1251-PNGF to total iZ51-/3NGF decreased to the same extent with increasing concentrations of either iz51-PNGF alone or of unlabeled PNGF. Different preparations of ‘251-/-?NGF tested in this way had 80 (*20%) of the binding affinity of native PNGF.

Steady State Binding-Viable cell dissociates rather than cell membrane preparations of sensory ganglia of &day-old chick embryos were used in this study in order to preserve

’ Portions of this paper (including “Materials and Methods” and Figs. 1, 7, 8, 10, 11, and 12) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magni- fying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Maryland 20014. Request Document No. 78M-1899, cite author(s), and include a check or money order for $1.95 per set of photocopies.

(i25i-) BNGF TOTAL (Ml

FIG. 2. Comparison of the binding affinities of ““I-PNGF and unlabeled PNGF. The cells (2.8 x lO”/ml) were incubated at 37°C for 45 min with various concentrations of ““I-PNGF (0) (specific activity 58 cpm/pg), or with 0.3 rig/ml of ‘““I-PNGF and increasing concentrations of unlabeled PNGF (0). Triplicates of IOO-~1 aliquots of the incubations were processed for each point as described under “Ma- terials and Methods.” The bars indicate the standard deviation of the mean.

cellular cytoskeletal elements which might modulate the binding of NGF to its receptors. For maximal cell yields and cell viability trypsin was used in the dissociation procedure. The ganglia were exposed to minimal amounts of trypsin, and the trypsin was inactivated by the addition of trypsin inhibitor before fetal calf serum was added and the mechanical dissociation started. The cell yield per ganglion was 40,000 under these conditions.

In control experiments with cell suspensions from ganglia which were dissociated without the aid of trypsin the same two specific binding sites for NGF were found. Although the affinities were the same the site numbers for both sites were approximately 40% higher on the cells from nontrypsinized ganglia. This is a consequence of either the trypsin lability of the two sites” (23) or the presence of considerable cell debris in the mechanically dissociated ganglia (or both) which results in an upward bias of the site numbers when calculated on the per cell basis.

The concentration dependence of the binding of ‘““I-PNGF to sensory ganglion cells under steady state conditions is shown in Fig. 3a over a wide range of ‘251-PNGF concentrations (3 PM to 3.7 nM). Fig. 3b depicts the analysis of this binding data according to Scatchard (24). The Scatchard analysis revealed two saturable binding components (the component with the lower affinity is shown in the inset of Fig. 3b with an expanded ordinate) with equilibrium dissociation constants (&) which differ by 2 orders of magnitude (&(I) = 2.3 x lo-‘* M, and K&I) = 1.7 X 10m9 M, see Table I). The apparent site numbers per cell corresponding to these two affinities are 3,000 for Site I and 45,000 for Site II (Table I). The ratio of the numbers of the two binding sites depends on the type of centrifugation procedure which is used in the binding assay.

It will be shown later that the dissociation of ‘““I-PNGF from binding site II is very rapid, sufficiently rapid for a significant fraction of the bound 12”I+NGF to dissociate from binding site II during the centrifugation of the cells through the sucrose gradient where no ‘““I-/3NGF is present. No loss of bound lZ51-PNGF from Site II should occur if the cells are collected by centrifugation of the original incubation mixture where the free i2”I-/?NGF concentration is maintained throughout. The data shown in Fig. 4, which compare the binding curves for ““I-/?NGF obtained by the two different

” A. Sutter, unpublished data.


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‘2%/3NGF FREE (nM)

FIG. 3. Binding of ““I-PNGF to sensory ganglion cells of &day-old chick embryos. a, binding of ‘““I-,8NGF as a function of concentration; b, Scatchard plot of specific “’ I-,f?NGF binding with low affinity region expanded (inset). Cells (0.6 x lO”/ml) were incubated at 37’C for 45 min with various concentrations (3 PM to 3.7 nM) of ‘*%PNGF. Bound

TABLE I

Kinetic and equilibrium constants for the specific binding of ““I- PNGF to sensory cells of R-day-old chick embryos at 37’C.

The apparent dissociation constants for both sites and site numbers per cell are average values calculated from Scatchard plots of 15 different experiments with 10 different ‘““I-PNGF preparations. The rate constants of association and dissociation (chase conditions) are average values of five experiments. For the calculation of the rate constant of dissociation from binding site II the value for the average sedimentation time of bound ‘““I-BNGF in the gradient centrifugation assay (3 s) was used, as well as the observation that one-half of the ‘““I-PNGF specifically bound to binding site II is lost while the cells sediment through the ‘2”I-/3NGF-free sucrose gradient.

Bindina constant Site I site II

k+l, Mu’ s-’ 4.8 (f1.7) x 10’ lo’-10” km,, S-I 1.03 (kO.08) x lo-” 2 x 10-l

Concentration for 2.3 (H.4) x lo-“” 1.7 (kO.74) x lo-“” one-half satura- 2.1 x 10m”h -1p tion, M

Maximum binding, 23 (+ll) x 10”’ molecules per cell 3 (fl.1) x 10” -45 x lo””

(1 From steady state data. ’ From kinetic data. ’ Gradient centrifugation. ” Centrifugation in the presence of ‘““I-PNGF.

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I

0 0.2 0.4 0.6 0.8

‘251-/9NGF FREE inM)

Fro. 4. Concentration dependence of specific binding of ““I-PNGF measured with different centrifugation assays. Cells (lOh/ml) were incubated at 37°C for 45 min with various concentrations of I’?- PNGF. Five aliquots of 100 ~1 for the gradient centrifugation (0) and of 500 al for the centrifugation in the presence of “‘I-PNGF (a) were processed for each point as described under “Materials and Methods.”

0 0 - .I 0 10 20 30

‘251-,9NGF BOUND (fmoles/106 cells)

““1-PNGF was separated from free as described under “Materials and Methods.” For each point, 3 aliquots of 100 1.11 each were processed. Nonspecific binding of ‘““I-PNGF was measured in the presence of 10 pg/ml of unlabeled /3NGF. 0, total binding, A, specific binding, 0, nonspecific binding.

centrifugation procedures, illustrate this point. The ‘s51+NGF concentration range in this experiment was chosen to empha-

size binding to Site II (Kd = 1.7 nM). The extent of specific binding was significantly greater when the cells were centri- fuged through the ““I-PNGF-containing medium rather than the r2”I-PNGF-free sucrose gradient. The ratio of the site numbers given above is the maximum ratio determined by centrifugation in the presence of “‘1-/3NGF. The Site II numbers determined by the two procedures are given in Table I.

Association and Dissociation Kinetics-The kinetic rate constants for the association and dissociation of ?-/3NGF from the higher affinity Site I have been determined independently of the binding to the lower affinity Site II by using ‘“51-/?NGF concentrations below the apparent Kd of Site I. Fig. 5a shows the data from an experiment in which the time course of the binding was measured at an ““I+NGF concentration of 1.6 X 10-l’ M, a concentration at which >85% of the total binding occurs at Site I. The association was complete after 45 min. For the analysis of the association data (Fig. 5b) the integrated rate equation of a reversible second order reaction was used (25). The high value of the association rate constant k+l (I) = 4.8 X lo7 M-’ s-l suggests that the association is diffusion controlled. The rate constant of association for the lower affinity Site II is similar to the association rate constant for the high affinity Site I. It is difficult to study the association behavior of ‘251-/?NGF to Site II independently of Site I. The estimate for K+, (II) (Table I) stems from experiments in which the cells were equilibrated with 2 X 10-l” M ,L?NGF in order to saturate 90% of Site I before the association of ?-PNGF to Site II was initiated at a concentration of 1.2 X lOmy M. At this concentration 50% of the equilibrium binding is already reached in 25 s compared to 7 min at 1.6 X lo-” M (Fig. 5a). Since the fist measurement cannot be made before 20 s after the addition of ‘ZSI-/3NGF, the differences between the equilibrium binding and the binding at different times during the association phase, which form the basis of the analysis of the binding data, are small and subject to large variation. The lack of accuracy in the estimate for the association rate constant for Site II is reflected in the large standard deviation of the value for h+l(II) in Table I. That K+l(II) is similar to k+l(I) is also apparent from experiments in which the concentration dependence of the initial association rates were studied over a wide range of ‘?-PNGF concentrations (5 X 10-l’ M to 2 X lo-” M). No concentration dependence of the association rate constant was seen. This method would have distinguished


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FIG. 5. a, time course of association of ‘251-PNGF to binding site I on sensory ganglion cells of S-day-old chick embryos. Cells (1.5 x 106/ ml) were incubated at 37°C with 1.7 x lo-” M ‘*“I+NGF. At the times indicated 3 lOO+l aliquots were subjected to sucrose gradient centrifugation as described under “Materials and Methods.” The data are corrected for nonspecific binding. b, analysis of the association data of a. For the analysis the integrated rate equation for a reversible second order reaction was used (20):

k,l t = I/& ln (/3(2x + y)/y(Zx + p,, = w

withQ=D’-4a&p=D+Jg;y=D-&&D=(-a-b-c);a = initial concentration of ‘*?-/3NGF (17 fmol/ml); b = initial concentration of binding sites I (7.5 fmol/ml); c = (a - n,)(b - x,)/x,; x, = ““I-PNGF bound at equilibrium (2 fmol/ml) t = time (min); x = ‘?- BNGF specifically bound at time t (fmol/ml). The slope yields k+,(I).

a 5-fold difference in the association rate constants of Site I and Site II.

In order to study the dissociation behavior of ‘251-PNGF from the two sites, dissociation was induced by the addition of excess amounts of unlabeled PNGF (3.8 x 10m7 M). The slight difference which was observed in the dissociation rates from Site I when dissociation was induced by dilution rather than by addition of unlabeled ,8NGF will be discussed in the following section. The dissociation of r2?-PNGF from Site I was determined independently of dissociation from Site II by pre-equilibrating the cells at low 12?-/?NGF concentrations at which binding was predominantly (290%) to Site I. Fig. 6 (top curue) shows the dissociation of ‘251-PNGF from the higher affinity site. The release of PNGF followed fist order kinetics with a half-life of approximately 10 min (Table I). The dissociation of ?-pNGF from Site II was determined by pre- equilibrating the cells at higher concentrations (0.16 nM and

1.3 nM in Fig. 6). The dissociation from cells loaded under these conditions was biphasic. Part of the bound ‘251-PNGF was released very rapidly and the remainder at a rate which was close to that observed for dissociation from Site I (Fig. 6). The proportion of the bound ““I-/?NGF which was released rapidly increased with increasing concentration of ‘2”I-/3NGF used to pre-equilibrate the cells and, therefore, with increasing occupancy of the binding site II. The rapid phase represents dissociation of the labeled NGF from Site II and is too fast to be followed by the techniques used here. The data in Fig. 4 show that 50% of the lzSI-PNGF bound to Site II dissociates during the time it takes to sediment the cells through the sucrose gradient. Since the average sedimentation time for the cellular-bound iz51-PNGF in this gradient centrifugation step is approximately 3 s, it follows that the halftime of dissociation from site II is approximately 3 s, leading to an approximate value of K-1(11) of 2 X 10-l s-‘. While the association rate constants of ‘251-/?NGF to sites I and II are similar and seem to be diffusion controlled, the rate constants of dissociation from the two sites are 2 orders of magnitude apart (Table I). The higher affinity binding site I is characterized by the slower rate of dissociation with the reverse being true for the lower affinity binding site II.

Cooperatiuity-For an increasing number of polypeptide hormones experimental data have been presented suggesting that the site heterogeneity observed in the Scatchard analysis of steady state binding data is not due to the presence of two independent binding sites but rather to negative cooperativity of the hormone-receptor interaction (5,9,10,19). The negative cooperativity is assumed to arise from ligand-induced interactions of receptors or receptor subunits or to the interaction of the ligand with itself and is characterized by a decreased affinity of the receptor for the ligand as the receptor occupancy is increased (20). A further corollary is that the decreased affinity is reflected, at least in part, by an increased rate of dissociation of the hormone from the receptor. Exper- imentally, a system is usually described as showing negative cooperativity when the rate of dissociation of the labeled hormone is increased in the presence of an excess of unlabeled

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FIG. 6. Dissociation of “51-/3NGF after pre-equilibration with different ‘*‘I-/?NGF concentrations at 37°C. Cells (3 X 106/ml) were preincubated for 30 min with 0.6 X 10-l’ M (O), 1.6 X lo-” M (A), and 1.36 x lo-” M (A) ‘Z”I-/3NGF. The dissociation of ““I-/?NGF was induced by the addition of 3.8 X 1O-7 M unlabeled PNGF. The specific binding at to was measured in sextuplets and after different times of dissociation in triplicates of 106 ~1 each. The data are corrected for nonspecific binding.


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5976 Nerve Growth Factor Receptors

hormone (a chase experiment where the receptor occupancy is increased in the dissociation step) compared to its absence (a dilution experiment where receptor occupancy decreases) (20).

An alternative way to test for negatively cooperative binding is to determine if the dissociation behavior of the labeled hormone in the presence of an excess of unlabeled hormone (chase conditions) is affected by the extent of receptor occupancy before dissociation is initiated. If the hormone-receptor interaction displays negative cooperativity, then the rate of dissociation under these conditions should be independent of the prior receptor occupancy, because the receptors will be transformed into the low affinity state by the addition of the excess unlabeled hormone. On the other hand, if the hormone binds to two independent sites, dissociation should occur at rates characteristic for these sites, and the overall dissociation behavior will depend on the relative occupancy of the two sites prior to the chase.

The time courses of the dissociation of ““I-PNGF shown in Fig. 6 were obtained under chase conditions by the addition of 3.8 x lo-’ M unlabeled ,L?NGF. The unoccupied specific binding sites for PNGF are saturated within seconds under these conditions. In spite of this, the pattern of dissociation was dependent on the concentration of ‘*“I-PNGF used to pre- equilibrate the cells and, therefore, on the relative occupancy of Sites I and II prior to dissociation. Moreover, the ratio of the occupancy of Site I to that of Site II determined from the intercept of the dissociation curves on the ordinate of Fig. 6, was close to that predicted from t.he equilibrium-binding data. For the three concentrations chosen in the experiment depicted in Fig. 6, the percentage of “‘I-PNGF bound to the higher affinity Site I calculated from the steady state binding data was 90% (0.6 X lO-‘i M), 58% (0.16 X lo-” M), and 24% (1.36 x lo-!’ M) compared to the experimental values of lOO%, 688, and 35%, respectively. Qualitatively, the same dissociation pattern was seen when the experiments were carried out at low temperatures (Fig. 8). This dissociation behavior is consistent with the existence of two independent binding sites and not with a cooperative hormone-receptor interaction.

The effect of the addition of excess unlabeled /3NGF on the dissociation rate of lZ51-/3NGF from Site I was determined by diluting prelabeled cells into medium alone or into medium containing unlabeled PNGF and following the release of the label. The choice of the appropriate dilution required to prevent rebinding of the dissociated ““I-PNGF was determined in control experiments. After pre-equilibration at 1.6 x 10-l’ M ““I-PNGF, for example, the dissociation curves were identical when the dilution was varied from 25- to 150-fold, indicating that no measurable rebinding of dissociated 12’1- ,L?NGF occurred provided the dilution was at least 25-fold. At higher initial ‘““I-PNGF concentrations greater dilutions were required. The time course of the dissociation of ““I#NGF after dilution of the cell suspension into medium alone and medium containing excess unlabeled /?NGF are compared in Fig. 9a. At both 37°C (Fig. 9a) and 24°C (data not shown) the rate of dissociation was accelerated by the presence of unlabeled ,L?NGF. These data are suggestive of a negatively cooperative interaction. However, it was found that the rate of dissociation was also accelerated when the concentration of unlabeled PNGF was lower, rather than higher, than that of the ‘““I-PNGF used to pre-equilibrate the cells. In Fig. 9b the concentration of unlabeled PNGF was only 25% of the 12’1- PNGF used in the pre-equilibration, yet the dissociation was still accelerated compared to that observed in the absence of unlabeled PNGF. The dissociation also followed first order kinetics under these conditions. The dissociation behavior shown in Fig. 9b is not the one expected for a negative

cooperative binding behavior of PNGF to its receptors. It should be reiterated here that the binding affinities of labeled and unlabeled PNGF were identical (Fig. 2).

The dissociation kinetics of ‘251-PNGF from binding site II cannot be analyzed in the same way since the assay technique is not sufficiently rapid. However, the linearity of the Scat- chard plot in the low affinity range (Fig. 3b, inset) suggests that ““I-/3NGF also binds to Site II in a noncooperative fashion.

Processes Following the Interaction of NGF with Its Re- ceptor-The agreement between the equilibrium dissociation constants determined from the equilibrium and kinetic data, respectively (Table I), suggests that the interaction of PNGF with its binding sites is, at least in the time scale used in these experiments, a reversible interaction and that irreversible processes such as degradation or internalization do not con- tribute in a major way to the measured binding characteristics. This has been confirmed directly for a number of possibilities, Degradation of ‘251-PNGF, for example, occurs only if low concentrations of labeled PNGF are incubated at high cell densities over long periods of time (Fig. 10). The amount of degraded /3NGF in the cell supernatants was measured by two different methods by determining (a) the difference in the trichloroacetic acid-precipitable 125I before and after exposure to the cells; or (b) the ratio of radioactive material of high and low molecular weight resolved by electrophoresis on acrylamide gels under reducing and denaturing conditions. Both methods gave identical results. In Fig. 10 the extent of degradation measured by Method a is shown after different concentrations of ‘““I-PNGF were incubated with a high concentration of cells for various times. Following a lag phase of 10 min the percentage of degraded ‘2’51-/3NGF increased lin- early and reached 15% after 60 min at the lowest concentration of ‘*“I+NGF tested. Degradation requires binding of “‘I- PNGF to the cells since no degradation occurred if ‘*“I+NGF was incubated with conditioned medium derived from a cell suspension. The incubation conditions used in this work to study the equilibrium binding of ‘251-PNGF generally involved lower cell densities (510” cells per ml) than those used for the experiments depicted in Fig. 10 and relatively short incubation times (545 min). When the concentration of “‘I-PNGF was 25 x lo-‘* M, the fraction of ‘““I-PNGF degraded under these conditions was negligible. The binding data have to be corrected, however, for the extent of degradation in experiments with very high cell concentrations and when long incubation times are necessary to achieve equilibrium binding at very low ‘““I-PNGF concentrations. If no correction for degradation under those conditions is made, the Scatchard plot at the lowest ‘““I-PNGF concentrations deviates from linearity and curves toward the ordinate.

For ligands like EGF (26) and low density lipoprotein (27), it has been shown that endocytosis is greatly suppressed at 4°C compared with 37°C. A comparison of the binding of ‘251- PNGF at low and high temperatures should, therefore, reveal if cell internalization of NGF contributes in a major way to the binding characteristics seen at 37°C. The finding that both the steady state binding (Fig. 11) and the kinetic data (Fig. 8) showed the same two binding sites at 2°C as well as at 37°C confirmed the validity of the conclusions drawn from the binding data at the higher temperature. It is of interest to note that, while the affinities for both binding sites at 2°C were similar to those at 37°C and still differed by 2 orders of magnitude (&(I) = 9.8 (+4) X lo-i2 M, &(II) = 1.4 (kO.4) X

lOmy M), the number of binding Sites I per cell was significantly lower at 2°C than 37°C. In three pairs of binding isotherms determined on the same cell suspension at both 2°C and 37”C, three times as many binding Sites I were seen at the higher as


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FIG. 9. Dissociation of ““1-PNGF at 37°C under dilution and dilution plus chase conditions. a, PNGF chase concentration > “‘I- PNGF pre-equilibration concentration. Preincubation conditions: cell concentration 5 X lO”/ml, “‘1-/3NGF concentration 1.5 x lo-” M,

preincubation time 90 min. Dissociation conditions: dilution 50-fold, /?NGF chase concentration 4 x 10eH M, dilution (0); dilution plus chase (A). b, PNGF chase concentration < ““1-PNGF pre-equilibra-

compared to the lower temperature. The fact that Site II numbers are not higher at 37°C than at 2°C might at least in part be attributable to the higher losses of lz51-PNGF bound to these sites during the gradient centrifugation step at 37°C compared with 2°C.

DISCUSSION

The data presented here confirm the earlier observations of Frazier et al. (5) that the binding of NGF to embryonic chick sensory ganglion cells is heterogeneous. At least for the viable cell dissociates used in this work the results support the idea that there are two distinct binding sites for ,8NGF on these cells. Key observations in this regard are the appearance of two separate linear segments in the Scatchard plot of the binding data (Figs. 3b and 8) and the agreement of steady state and kinetic binding data observed at 37°C and 2°C when the dissociation kinetics were studied under chase conditions (Figs. 6 and 11). There are many factors which can produce

nonlinear Scatchard plots; they depend on the inherent properties of ligand or receptor or on the interaction of receptor molecules with each other or with other components of the cell membrane. The following have been considered. (a) The native and radiolabeled ligand have different binding affni- ties. This will produce curvilinear Scatchard plots if a constant concentration of labeled ligand and increasing amounts of unlabeled ligand are used to generate the binding data for the Scatchard analysis. Under most circumstances different affln- ities of labeled and unlabeled ligand will produce linear Scat-

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0 20 40 60

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tion concentration. Preincubation conditions: cell concentration 4.5 x 10” cells/ml, ““I-PNGF concentration 3.8 x lo-” M, preincubation time 90 min. Dissociation conditions: dilution 150-fold, /lNGF chase concentration 7.7 x IO-” M, dilution (0); dilution plus chase (A). In both experiments the specific binding at to was measured in sextuplets and at all other times in quadruplicate.

chard plots if the ligand concentration is altered by varying the concentration of the labeled ligand alone (28), which is the procedure used in the present work. In any event it was shown directly that the /3NGF and the ‘251-/?NGF are practically identical in their binding properties (Fig. 2). The presence of several different labeled species of ligand with different binding affinities will also affect the binding data and give curvilinear Scatchard plots under certain conditions even with only one class of receptors. When low receptor concentrations are used in the binding studies such that no significant change in the ratio of the concentrations of the different species of free ligand occurs, linear Scatchard plots can be expected. If this ratio is affected by using high receptor concentrations, then the binding data will yield convex Scatchard plots, since the ratio of free ligand with high affinity to free ligand with low affinity will increase with increasing ligand concentrations. Since the Scatchard analysis of the binding of the ‘2”I-/3NGF preparation used in the present study gave concave plots, the heterogeneity of binding is not caused by different affinities of radioiodinated j?NGF in the preparation. That it is the heterogeneity of the binding sites which accounts for the observed binding characteristics rather than a heterogeneity of the 1251- PNGF preparation itself is also evident from studies on cultured cells. The cell bodies of cultured sensory neurons bind NGF only with the characteristics of Site II.” Since the binding assays were not carried out on the culture plate but on suspensions of cells without their neurites, it is possible that the cultured neurons do carry binding sites I but on their


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neurites rather than on their soma. (b) A monomer-oligomer equilibrium exists for the ligand with different binding affinities for the different molecular species. Again, this is not a factor to be considered because ,!?NGF is a stable dimer at all concentrations used in this work (29) and since it has already been shown that the binding characteristics of the cross-linked NGF dimers were the same as those of the native NGF (30). (c) Multiple ligand conformations exist. This hypothetical possibility has been considered for ligands containing highly flexible hydrocarbon chains (31) and probably does not include peptide hormones such as ,8NGF. (d) Ligand-induced changes in receptor affinities, that is, cooperativity. (e) There are two distinct ligand-binding sites. The last two possibilities are the ones which have been examined in detail in this work.

The Question of Cooperative Binding of NGF-A negatively cooperative model for hormone-receptor interaction was fist postulated for the binding of insulin to its receptor on lymphocytes (7). Further studies suggested that several peptide hormones (5, 9, 10, 19) as well as a number of neurotransmitters (11, 12) bind to their receptors in this manner. The presence of negative cooperativity was usually deduced from dissociation experiments in which the dissociation of labeled hormone from its receptors was measured under two conditions, namely (a) at dilutions of the hormone receptor complex where no measurable rebinding of dissociated hormone could occur (“dilution”); or (b) at the same dilutions but in the presence of an excess of unlabeled hormone (“chase”). The acceleration of the dissociation under chase conditions was interpreted as negatively cooperative hormone-receptor interaction. Frazier et al. (5) noted an acceleration of the dissociation of ““I-NGF under chase conditions from both sensory and sympathetic ganglia cells. This observation has been confirmed in the present study (Fig. 9a). However, the acceleration of the dissociation of ““I-PNGF is observed not only when excess unlabeled PNGF is added to increase receptor occupancy in the dissociation step but also when the amount of added PNGF results in decreasing receptor occupancy (Fig. 96). It is unlikely, therefore, that the phenomenon of negative cooperativity is sufficient to account for this behavior. The dissociation under dilution conditions follows first order kinetics until at least 50% of the label is released from the cells (Figs. 9a and b). This argues against negative cooperativity or a change in the binding state at receptor occupancies of 50 to 100% of that defined by the ““I-PNGF concentration of 3.8 X lo-” M used to pre-equilibrate the cells in the experiment depicted in Fig. 96. The dissociation of ‘““I-PNGF in both experiments shown in Fig. 9b was induced by dilution of the cells into a 150-fold or larger volume of buffer with or without 7.7 x lo-*’ M cold PNGF. The total /?NGF concentration during the dissociation in the dissociation plus chase experiment was 2.5 X lo-l3 M plus 7.7 X 10” M, or approximately 8 X 10-l” M. I f the system behaved in a negatively cooperative manner, the binding affinities at receptor occupancies defined by a PNGF concentration of 8 X 10-l’ M

should be the same (in the absence of negative cooperativity) or even larger (in a negative cooperative system) than that at the receptor occupancies which were analyzed in the dilution- only experiments (Fig. 96, upper curue). In spite of this a significant increase in the initial dissociation rates equivalent to an apparent lower affinity is observed in the dissociation experiment in the presence of 7.7 X lo-l2 M cold PNGF (Fig. 9b, lower curve). Moreover, as seen in Fig. 6, the dissociation of ‘““I-/3NGF is markedly dependent on the receptor occupancy which exists before initiation of dissociation by either dilution or addition of unlabeled PNGF. After pre-equilibration of cells with concentrations of ‘““I-PNGF less than 0.5 x

IO-” M, the dissociation is monophasic and follows fist order

kinetics in the presence or absence of excess unlabeled NGF. At higher pre-equilibrating concentrations of iZ51-PNGF, the dissociation pattern becomes biphasic, and the relative amounts of ‘251-PNGF which dissociate slowly or rapidly cor- relate well with the relative occupancies of the higher (I) and lower (II) affinity binding sites before dissociation begins. Such a pattern would not be expected for a cooperative interaction of NGF with its receptors, but rather is best explained by the existence of two non cooperative binding sites with different affinities for NGF.

The concentration of native PNGF used in the chase experiments described in Fig. 6 was more than IOO-fold greater than the & for Site II. At this PNGF concentration, Sites I and II are fully occupied with NGF within seconds or less, irrespec- tive of the ‘“‘I-PNGF concentration used for the pre-equilibration of the cells. Nonetheless, the dissociation behavior was still markedly dependent on the lZ51-PNGF concentration used in the pre-equilibration. According to the negative cooperativity model (and given the approximate loo-fold difference in the affinities of Sites I and II) the dissociation of lz51-j?NGF from the high affinity Site I should be accelerated approximately lOO-fold when all available binding sites are occupied. This was not observed (Fig. 6, upper curve). The rapid dissociation of ‘251-/3NGF was only observed following pre-equilibration of the cells at ‘251-/?NGF concentrations which, according to the data in Fig. 3, are high enough to achieve significant occupancy of Site II (Fig. 6, lower curves). The data in Fig. 6 are thus consistent with the two-site model with an independent dissociation of “‘I-PNGF from both sites. Even when allowance is made for the potential complications of ligand-receptor interactions on viable cells (see later), the kinetic experiments shown in Figs. 6 and 9 are incompatible with the model of negatively cooperative NGF binding. They do, however, support the two-site model.

An explanation is still required for the finding that unlabeled ,BNGF accelerates the dissociation of ‘““I-PNGF over a wide range of PNGF concentrations, including very low concentrations. “Retention effects” or “unstirred layers” might account for such phenomena (32, 33). Hydrophobic, polar, or steric interactions of a ligand with membrane components other than the specific receptor could create a diffusion barrier inhibiting the release of the ligand from the membrane surface. This would result, in the present instance, in the rebinding of a fraction of the ““I+NGF released from the receptors and, therefore, in a slower rate of dissociation than in the absence of a diffusion barrier. The addition of unlabeled PNGF even at low concentrations is apparently sufficient to dilute the specific radioactivity of the ‘““I-PNGF within the diffusion barrier and to prevent rebinding. Experiments in which sensory ganglion membranes were stirred in attempts to disturb the barrier indicate that the difference in dissociation rates in the presence and absence of unlabeled PNGF is significantly reduced, thus providing evidence for the presence of an “unstirred layer”.4

A Comparison of NGF Binding in Different Systems-* There are a number of reasons why the present results differ, in part, from those published previously. The use of different buffer systems is not, however, one of these, because identical steady state binding was obtained in both the buffer used by Frazier et al. (5, 6) and the one described here (data not shown). Following Herrup and Shooter (4), viable single cell suspensions were used in this investigation, whereas Frazier et al. (5) used a ganglion dissociate which, besides whole cells, contained an undefined amount of cell membranes. It has

’ H. J. Hiopelle, M. Klearman, and A. Sutter, manuscript in preparation.


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been noted that the rate of dissociation of insulin from membranes derived from cultured human lymphocytes, human blood peripheral monocytes, and rat and mouse liver is significantly slower than from whole cells (19)) and the same holds true for the dissociation of PNGF from membranes of sensory ganglia.4 It follows that there will not be complete agreement on the binding characteristics of PNGF in the two systems. Also, in contrast to the saturability of the specific binding of ‘?-/?NGF to intact cells, nonsaturability of the low affinity component of the binding of lz51-/3NGF on membranous material from sensory ganglia was observed. This result suggests that disruption of the cells exposes internal cellular binding sites for NGF which have different binding characteristics.

The different assay procedures used by the different inves- tigators may also have contributed to the observed differences. Because of the rapid dissociation of ‘““I-PNGF from binding site II on intact cells accurate binding characteristics of this site can only be obtained in assay systems where the cells are continuously exposed to the labeled hormone as they are separated from free ““I-PNGF. The centrifugation procedure b used in the present study meets this requirement. This procedure was used as a control for the centrifugation procedure a generally used in this work, because it rendered lower nonspecific binding backgrounds. Procedures for the separa- tion of bound from free hormone which include washing steps will remove all the NGF bound to binding site II on intact chick sensory ganglion cells. Thus the washing procedure in the assays reported by Banerjee et al. (3) on membranes from sympathetic ganglia may well have contributed to their finding a much smaller number of specific binding sites compared with the numbers of sites seen by Herr-up and Shooter (4), Frazier et al. (5), in this work, and on intact cells of mouse sensory ganglia.” It is not yet possible to decide if the use of two types of binding assays, each including washing steps, contributed to the fact that in the analysis of Frazier et al. only the high affinity component of the steady state data is seen in the kinetic data, whereas the low affinity component (KI, = lo-” to lo-” M) is not represented (5).

Besides the different assay procedures and methods of cell preparation used, it is possible that the use of various forms of the NGF protein and of different radioiodination protocols may have added to the variability of the data. Banerjee et al. (3) as well as Frazier et al. (5) used a 2.5 S NGF preparation where the proportion of NGF chains lacking the COOH-ter- minal arginine residues is significantly higher than in PNGF (34).

The most significant difference between the various 1251- NGF preparations is, however, the extent of nonspecific binding. The filtration through the Centriflo membranes produces lZ”I-/3NGF preparations which display less than 5% of their total binding as nonspecific binding at 1 to 3 nM and cell concentrations >2 X 10” cells per ml. In the 5 to 100 PM range practically all the binding is specific. In the earlier preparations (3-5) up to 50% of the total binding was nonspecific, and this precluded binding studies in the range of concentration needed to define the higher affinity receptor (Site I).

Secondary Events Following the Binding of NGF-In the binding studies discussed here the analysis of the interaction of NGF with its receptors was based on steady state binding data, on association kinetics, and on dissociation kinetics after ““I-PNGF binding reached steady state levels. Steady state binding data potentially reflect more than the reversible equilibrium binding of a ligand to its receptor. Cell surface receptors might, for example, undergo ligand-induced conforma- tional changes or cell internalization of the ligand or ligand. receptor complex, and subsequent degradation of one or both

of the components might occur. The latter process might in turn control the insertion of new receptors into the cell membrane. It has been shown for several ligands that the binding of the ligand to its receptor is followed by secondary processes of this kind. Indeed the compartmentalization of the ligands or their receptors (or both) has evolved as a major theme in the current research on the mode of action of growth factors and hormones (26,35-44). The binding of EGF to its receptor on fibroblasts or 3T3 cells leads to internalization of the EGF. receptor complex and to its degradation (26, 41). The low density lipoprotein is also rapidly taken up by fibroblasts after binding to its specific cell surface receptors (27,45). For insulin the occurrence of cell internalization and degradation of the hormone is well documented (see Ref. 42 for further references). Processing following the formation of the hormone. receptor complex is also indicated for glucagon, growth hormone, and gonadotropin. The dissociation characteristics for the first two hormones change with the time of incubation due to accumulation of slowly dissociating components (8, 14) while for the latter hormone endocytosis and degradation by lysosomal enzymes was shown (46).

The data presented here show that degradation and cell internalization of NGF do not interfere with the qualitative analysis of the NGF-binding data. A study on the time dependence of the dissociation kinetics of the PNGF label on the pre-equilibration time, however, indicates that secondary processes take place following the binding of NGF to its receptors. At a concentration of 4.3 X lo-‘” M ‘““I-PNGF the binding reaches a steady state in less than 15 min at 37°C. The dissociation kinetics of labeled NGF from cells pre-equilibrated with this concentration of ‘““I-/3NGF for 15 min is shown in the lower curve of Fig. 12. The relative proportions of the occupied binding sites I and II determined from this data are in reasonable agreement with the values predicted from the steady state binding data (Table I). However, the proportion of the occupied binding site I to binding site II changes with time after the apparent steady state has been reached, as shown by the upper dissociation curve in Fig. 10. The pre-equilibration time for this experiment was 60 min. Not only did the relative proportions of the site number change, but the dissociation curve for the slower component became markedly curved. Also in experiments in which the trypsin stability of the cell-bound label was studied a new component was seen which accumulated with time and which, unlike ‘2”I-PNGF bound to Sites I and II, was trypsin resistant. The accumulation of this component was not prevented by the starvation of the cells in glucose-free buffer nor by metabolic inhibitors which completely block the uptake of amino acids into the cells.” The mechanisms which bring about those changes in the characteristics of the bound ‘““I-PNGF are not yet known. However, these phenomena might explain why the ratios of Site I to Site II occupancy at any given ““I-PNGF concentration, determined from the dissociation data, are always consistently higher than those predicted from steady state data which were obtained after 45 min of binding. The shorter the pre-equilibration times, provided they are long enough to reach steady state, the closer is the agreement between the site ratios obtained by the two methods. Obser- vations on the kinetic binding behavior of human growth hormone have been used to indicate that site heterogeneity seen on Scatchard plots could be mimicked by ligand compartmentalization following the binding of the ligand to a homogeneous class of receptors (14). However, the steady state and kinetic data obtained at 2°C (Figs. 8 and ll), the observation of both sites in the presence of metabolic inhibi-

’ R. M. Harris-Warrick, unpublished data.


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tom and under conditions of glucose starvation, the detection of both sites on membrane preparations of sensory ganglia of &day-old chick embryos devoid of intact cells, and the characterization of Site I and Site II in competition experiments (Fig. 7) exclude this possibility in the case of NGF. The presence of two distinct cell surface binding sites for NGF on sensory ganglia cells was also confirmed in a study in which anti-NGF antibody, complement, and NGF-dependent cyto- toxicity assays where performed with live cells in order to differentiate between Sites I and II (47).

On the basis of the present data no conclusion can be drawn as to whether the two binding sites are two different receptor molecules or are the same receptor in two different confor- mational states. If the latter were true the different confor- mational states would not be due to cooperative hormone receptor interactions. Both sites appear as specific binding sites for NGF since none of the peptide hormones tested besides ,f3NGF competes with the binding of 12”I-PNGF to either site. The two NGF binding sites can be distinguished on the basis of their participation in the biological response of sensory neurons to NGF. The initiation of neurite outgrowth from sensory neurons requires only that NGF binds to the higher affinity Site I receptors6 It will be of interest to determine if mammalian sensory neurons, sympathetic neurons from both chick and mouse, and the clonal cell line PC 12 derived from a rat pheocbromocytoma (48) display the same two specific NGF-binding sites.

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A Sutter, R J Riopelle, R M Harris-Warrick and E M Shooterbinding sites on chick embryo sensory ganglia cells.

Nerve growth factor receptors. Characterization of two distinct classes of

1979, 254:5972-5982.J. Biol. Chem.

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