Nerve growth factor in the nucleus: interaction with receptors on the nuclear membrane.

Abstract

Cells of a continuous line of rat pheochromocytoma (PC12) were incubated with 125I-labeled P nerve growth factor (PNGF), and at given time intervals the cell nuclei were isolated by a procedure that used the detergent Triton X-100. NGF was detectable in the nucleus after 20 min and continued to accumulate in a linear fashion for several hours after the total binding to the cell had reached steady state. After 17 hr at 370C, about 60% of the NGF bound to the cell was in the nucleus. NGF was not translocated to the nucleus at 4VC. When nuclei were purified from PC12 cells and incubated with 125I-labeled j#NGF, specific binding sites were found. Binding was saturable and consistent with two sites: a high-affinity site with a Kd of 0.08 nM (+0.02 nM) and a lower-affinity site with a Kd of 9.0 nM (±2.0 nM). The receptors in the nucleus were shown to be localized to the nuclear membrane. Membrane-free chromatin did not bind NGF specifically. The translocation of NGF to the nucleus was accompanied by a commensurate decrease in the cell-surface binding capacity. In the nucleus, however, the receptor capacities of both sites were increased when PC12 cells were grown in the presence of NGF. Nerve growth factor (NGF) is a polypeptide hormone that is involved in the development and maintenance of several cell types of neural crest origin. The injection of NGF antiserum into neonatal mice results in the rapid degeneration of the sympathetic neurons of paravertebral and prevertebral ganglia. At the ultrastructural level, the first changes are observed in the nucleus; these include the disorganization of the nucleolus and the disruption of the nuclear membrane (1, 2). The most striking morphological effect of NGF is the stimulation of neurite extension. Pronounced metabolic changes are also observed; these include alterations in RNA and protein synthesis (3).The finding that NGF can be internalized in vivo by retrograde axonal transport into the cell body of the sympathetic neuron (4) prompts the consideration of an intracellular mechanism. Triton-resistant binding sites for NGF were recently demonstrated in a preparation of nuclei from the dorsal root ganglion. The sites were reported to be contained in chromatin (5).The clonal cell line of rat pheochromocytoma (PC12) responds to NGF by the extension of neuritic processes and the cessation of cell division (6, 7). In the present communication, we describe the uptake of NGF by the viable PC12 cell and the time course of its appearance in the nucleus. Receptors for NGF are found in the nucleus of PC12; they are localized exclusively to the nuclear membrane.MATERIALS AND METHODS Subfractionation of PC12. The PC12 cell line was kindly provided by Lloyd A. Greene. PC12 cells were plated on polystyrene tissue culture dishes and grown in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% fetal calf serum and 5% horse serum. Cultures were maintained in a water-saturated atmosphere of 88% air/12% CO2. Subconfluent cultures grown either in the presence or absence...

Microtubules and microfilaments in fixed and permeabilized cells are selectively decorated by nerve growth factor.

“…This finding suggests that, if the pool of NGF internalized in target cells (3,5,9,18), presumably via endocytosis, is subsequently released and diffuses before being degraded, as is known to occur for some internalized proteins (24,25), the chances ofan interaction with these cytoskeletal structures are high.…”

“…Our studies show that the specificity and affinity of NGF binding to MTs and MFs is in the range of that of antibodies against tubulin and actin. The possible relevance of these findings to the mechanism of action of NGF in target cells is discussed.After the protein nerve growth factor (NGF) (1) binds to receptors located on the cytoplasmic membrane or at the nerve terminals of target cells (2, 3), it is internalized into the cytoplasm (4,5). In pheochromocytoma cells (clone PC12), it is localized around the nuclear outline and within the nucleus in a paranucleolar position (6).…”

“…After the protein nerve growth factor (NGF) (1) binds to receptors located on the cytoplasmic membrane or at the nerve terminals of target cells (2, 3), it is internalized into the cytoplasm (4,5). In pheochromocytoma cells (clone PC12), it is localized around the nuclear outline and within the nucleus in a paranucleolar position (6).…”

Luteinizing hormone‐releasing hormone (LH‐RH) binding to purified rat pituitary nuclei

“…Since LH-RH stimulation of the anterior pituitary is known to increase the biosynthesis of gonadotrophic hormones [7] and the number of LH-RH receptors [8] it is possible that one of its sites of action is the nucleus. Internalisation and nuclear binding of peptide hormones has been demonstrated for insulin [9-l 11, EGF [12], NGF [13,14], LH [IS] and thyrotropin releasing hormone [ 161. Therefore, we have examined the binding of LH-RH and its analogues to purified anterior pituitary nuclei.…”

Normal liver chromatin contains a firmly bound and larger protein related to the principal cytosolic target polypeptide of a hepatic carcinogen.

“…Nuclei of normal rat livers were isolated at 40C by a procedure patterned after that of Yankner and Shooter (15) and Birnie (17). Livers were homogenized as above.…”