Nerve Growth Factor Receptors
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Proc. Nati. Acad. Sci. USA Vol. 74, No. 7, pp. 2785-2789, July 1977 Biochemistry Nerve growth factor receptors: Identification of distinct classes in plasma membranes and nuclei of embryonic dorsal root neurons (hormone binding/nonionic detergent/retrograde axonal transport) ROGER Y. ANDRES, INGMING JENG*, AND RALPH A. BRADSHAW Department of Biological Chemistry, Washington University School of Medicine, St. Louis, MO 63110 Communicated by P. Roy Vagelos, May 2, 1977 ABSTRACT Two classes of receptors for '25I-labeled nerve sulin receptors (15), rather than from receptor heterogene- growth factor in chick embryonic dorsal root neurons have been ity. observed. One type is associated with the plasma membrane (or Recently, it has also been demonstrated that NGF can be microsomal fraction) and can be completely solubilized by Triton X-100. These receptors display the nonsaturable binding taken up by the synaptic terminals of neurons of the superior isotherms and curvilinear Scatchard plots previously reported cervical ganglia after injection of 125I-NGF into the anterior for nerve growth factor receptors in whole cells. The second chamber of the eye of the adult rat (16, 17). This internalized class of binding sites is located in the nucleus, firmly bound to NGF is carried to the cell perikarya by retrograde axoplasmic chromatin. These receptors are not solubilized by detergent, transport where it can also effect the synthesis of tyrosine hy- show saturable binding, and yield linear Scatchard plots of the type associated with a single class of binding sites of high af- droxylase and dopamine-f3-hydroxylase (18). It seems unlikely finity. The presence of the two receptor types suggests a bimodal that these effects could be mediated by the plasma membrane mechanism of action for nerve growth factor. receptors that direct neurite proliferation, suggesting more than one mechanism of action for NGF. This study provides direct Nerve growth factor (NGF) is an insulin-like protein (1) that evidence for the existence of two classes of NGF receptors that induces the morphologic and metabolic differentiation of differ in their solubilization by Triton X-100, binding proper- sympathetic and sensory neurons in vertebrates (2). However, ties, and subcellular localization. These two discrete NGF re- the responsiveness of these two tissues is quite different. Sensory ceptors could provide the separate loci for the expression of the neurons bind and are stimulated by NGF only during a rela- different NGF responses. tively narrow period of embryonic development (2-4). In contrast, the sympathetic cells retain sensitivity, even in adult MATERIALS AND METHODS animals (5), although the nature of the response changes during NGF was prepared from mouse submaxillary glands according different stages of development. to Bocchini and Angeletti (19). Subsequent references to NGF The types of cellular processes effected by NGF are also refer to this preparation, commonly identified as 2.5S NGF or variable. With low levels of hormone (about 1-10 ng), both NGF(AB). Oxidized NGF derivatives were prepared as de- sensory and sympathetic neurons produce the characteristic scribed by Frazier et al. (20). NGF was labeled with 1251 as neurite outgrowth with accompanying general stimulation of described previously (12). The specific activity of these prep- anabolic metabolism and neurotubule polymerization (2, 6). arations varied from 200 to 2000 cpm/fmol. Cytochrome c was At higher concentrations (about 1-10,tg), the specific induction labeled with 125I according to the procedure of Greenwood and of enzymes involved in nonadrenergic neurotransmitter syn- Hunter (21), yielding preparations with specific activities of thesis is observed (7) as well as such effects as regeneration of 200-1000 cpm/fmol. Chick dorsal root ganglia (DRG) were adrenergic fibers in brain (8), inhibition of the biosynthesis of dissected from freshly decapitated chick embryos (8-day) and mucopolysaccharide in chrondrocytes (9), and stimulation of collected in an ice-cooled vessel by vacuum aspiration with a the temporal conversion of cell surface adhesive specificity in fine glass pipette. The inevitable crushing of the ganglia col- embryonic optic tectal cells (10). lected by this process did not alter their binding properties. The responses associated with the low concentrations of NGF Specific binding of 125I-NGF was measured according to appear to be mediated through receptors bound to cell surface Frazier et al. (12). Experiments were carried out in triplicate membranes. NGF covalently linked to Sepharose beads under and were corrected for the high-capacity, low-affinity binding conditions that prevent the release of soluble NGF produces a of NGF to the tube in the presence of incubation buffer stimulation of neurite outgrowth from embryonic dorsal root alone. sensory ganglia indistinguishable morphologically from that Triton X-100 extractions of particulate samples were per- of the unbound hormone (11). By using 125I-labeled NGF formed by incubating samples at room temperature for 10 min (125I-NGF), these receptors have been shown to possess an af- in Hanks' balanced salt solution (HBSS) (12) containing the finity of approximately 1010 liters/mol (12-14), corresponding appropriate concentration of detergent. The insoluble residue to the concentration range required for activity. However, the was collected by centrifugation (10 min, 3000 X g) and washed specific binding is a nonsaturable process with lower affinity twice with HBSS. DRG extracted with 1% (vol/vol) Triton are (about 2 X 107 liters/mol) of binding sites at higher concen- referred to as Triton-extracted DRG. trations of labeled NGF (12). The range of affinities observed Prior to the determination of specific binding to solubilized results from negatively cooperative interactions among a single components, 125I-NGF was purified by passage over a Sephadex class of receptor molecules, analogous to that observed for in- G-100 column previously equilibrated in 50 mM Tris, pH 7.4, Abbreviations: NGF, nerve growth factor; 125I-NGF, 125I-labeled NGF; containing 0.02% Triton and bovine serum albumin, 1 mg/ml. DRG, dorsal root ganglia; HBSS, Hanks' balanced salt solution. The peak eluting at a position corresponding to a molecular * Present address: Lipid Research Center, Department of Medicine, weight of 27,000 was used immediately in the binding assays. Washington University School of Medicine, St. Louis, MO 63110. Ganglia (usually 250 per experiment) were homogenized in 5 2785 Downloaded by guest on October 1, 2021 2786 Biochemistry: Andres et al. Proc. Nati. Acad. Sci. USA 74 (1977) 100 o 0 2, 'O 60 -005 A 80 40 Zo2~~~~~~~ 1 2 20 125I-NGF, nM FIG. 2. Specific binding of 125I-NGF to nuclei (0) and micro- 02 OA Os 08 1 somes (0) from chick embryonic (8-day) DRG as a function of hor- Triton, % mone concentration. Each assay tube contained the material isolated from three ganglia in a final volume of 300,ul. The data are corrected FIG. 1. Specific binding of 125I-NGF to various subcellular for nonspecific binding (about 30% of specific binding). fractions of chick embryonic (8-day) DRG after extraction with dif- ferent concentrations of Triton X-100. Ganglia, microsomes, or nuclei of the specific binding was solubilized. These results clearly were incubated with the indicated concentrations of Triton (room binding sites, not temperature, 10 min). The nonsolubilized material was collected by suggest that DRG contain additional specific centrifugation and washed twice with ice cold HBSS containing bo- present in plasma membranes, that are exposed in detergent- vine serum albumin at 1 mg/ml. Specific binding assays were per- extracted homogenates and are not solubilized by this treat- formed on tubes containing three ganglia (0) or the equivalent ment. amount of microsomes (0) or nuclei (-) in a final volume of 300 M1. Support for this conclusion was provided by examination of the insoluble pellet, collected by centrifugation after Triton ml of HBSS and centrifuged (10 min, 3000 X g). The pellet was extraction of the DRG homogenates, which showed significant resuspended in 1 ml of 50 mM Tris, pH 7.4, containing 0.1% specific binding. Fractionation of this material by sucrose Triton. After 60 min at room temperature, the nonsolubilized density centrifugation revealed that virtually all of this binding material was removed by centrifugation (60 min, 48,000 X g). was located in the highest density fraction, containing pre- 125I-NGF was added to the supernatant (final concentration, dominately nuclei. Purification of nuclei from DRG (22) es- 30 pM) and, after incubation at room temperature for 60 min, tablished that these organelles indeed contained the binding the samples were chilled to 40 and chromatographed (at the sites for NGF that are not solubilized by Triton X-100 (Fig. 1, same temperature) on a column of Sephadex G-100 (0.8 X 22 squares). It should be noted that, because of the differences in cm) previously equilibrated in 50 mM Tris, pH 7.4, containing the specific binding-versus-125I-NGF concentration relation- 0.02% Triton and bovine serum albumin, 1 mg/ml. The eluate ships of the microsomal and nuclear binding (see below), the was monitored for radioactivity. proportion of the binding to the two populations of binding sites Nuclei were prepared according to Biessmann and Rajewsky varies with the 1251-NGF concentration used in the binding (22). This procedure involves preparation of a crude nuclear assay. fraction by low-speed centrifugation, purification of nuclei by Properties of the Receptors Not Solubilized by Triton. The sedimentation in 2.3 M sucrose/i mM sodium cacodylate/1 specific binding of 125I-NGF, localized in the nuclear fraction mM MgCl2 at pH 6.4, and washing with 1% Triton. These nu- of homogenates of chick embryonic DRG, displayed distinctly clei were resuspended in HBSS by homogenization in a tightly different binding properties from those observed in either fitting glass-Teflon homogenizer. Aliquots were taken for the dissociated ganglia or the microsomal fraction.