Multiple forms of the nerve growth factor protein and its subunits

Smith, Andrew P.; Varon, Silvio; Shooter, Eric M.

doi:10.1021/bi00849a032

Cited by 176 publications

(68 citation statements)

References 10 publications

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“…The p subunit of NGF was purified from mouse submaxillary glands by the 7 s oligomeric NGF procedure (Smith et al, 1968;Stach et al, 1977;Woodruff & Neet, 1986) and ran as a single band of 13 kDa on SDS polyacrylamide gel electrophoresis. NGF was radioiodinated to a specific activity of 53 cpm/pg using lactoperoxidase (Calbiochem) and Na"'1 (Amersham) with modifications of established procedures (Sutter et al, 1979).…”

Section: Methodsmentioning

confidence: 99%

Circular dichroism and crosslinking studies of the interaction between four neurotrophins and the extracellular domain of the low‐affinity neurotrophin receptor

Timm

Ross²,

Neet

1994

Protein Science

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Interactions between the purified recombinant receptor extracellular domain (RED) of the human low-affinity neurotrophin receptor (LANR) and recombinant human brain-derived neurotrophic factor, neurotrophin-3 (NT-3) and neurotrophin-4/5 have been studied by chemical crosslinking and circular dichroism. Conformational changes subsequent to binding have been shown by these procedures. First, relative affinities of the neurotrophins for RED were determined by binding competition assays in which radioiodinated nerve growth factor (NGF) from mouse submaxillary gland was crosslinked to RED in the presence of varying amounts of unlabeled neurotrophin competitors. RED bound each of the 3 recombinant human neurotrophins with affinities that were indistinguishable from authentic mouse NGF. These results are the first measurement of binding of the neurotrophin family to their common receptor using purified components. In order to study the effect of binding on the conformation of the proteins, CD measurements were made before and after mixing neurotrophins and RED, as had previously been done with NGF and RED (Timm DE, Vissavajjhala P, Ross AH, Neet KE, 1992, Protein Sci 1:1023-1031). Similar changes in CD spectra occurred upon combination of each of the neurotrophins and RED, with negative changes near 220-225 nm and positive changes near 190-200 nm; however, significant differences existed among the various neurotrophin-RED difference spectra. The NT-3/RED complex showed the largest spectral change and NGF the smallest. Thus, specific conformational changes in secondary structure of neurotrophin, RED, or both accompany the binding of each neurotrophin to the extracellular domain of the LANR.Keywords: circular dichroism; conformational change; low-affinity NGF receptor; nerve growth factor; neurotrophin; receptorThe neurotrophins are a family of target-derived trophic factors required for the development and survival of specific neuronal populations (Levi-Montalcini, 1987). This family of homologous proteins includes nerve growth factor, brain-derived neuro-

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Section: Methodsmentioning

confidence: 99%

Circular dichroism and crosslinking studies of the interaction between four neurotrophins and the extracellular domain of the low‐affinity neurotrophin receptor

Timm

Ross²,

Neet

1994

Protein Science

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“…Renin Tram/&ion NGF is synthesized in the male mouse submaxillary gland, It is found as a complex of 3 proteins with a stoichiametry CY~ :fl: y2 [ 1,2]. The p-subunit contains the neurotrophic activity.…”

Section: Nerve Growth Factormentioning

confidence: 99%

“…The cy-and yNGF were purified as in [2]. Although the protein samples which served for inoculation of rabbits appeared pure after coloration of polyacrylamide gels with silver stain, antisera showed cross-reactivity in reconstruction experiments performed with the purified radioiodinated proteins.…”

Section: Preparation Of the Purified Cy- And And Y-subunits And Of Thmentioning

confidence: 99%

Synthesis and partial maturation of the α‐ and γ‐subunits of the mouse submaxillary gland nerve growth factor in Xenopus laevis oocytes

1984

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Sera raised against the cy-, @-and y-subunits of the mouse 7 S NGF were used to characterize translation products coded by submaxillary gland mRNAs m~~ro~nje~ed into Xenctpus oocytes. Anti-@NGF sera did not cross-react with any material. In contrast, the precursors of the cy-and y-subunits, as well as that of renin were identified. Use of tunicamycin, and a comparison of the translation products obtained in oocytes or in the reticulocyte lysate indicated that oocytes achieved the cleavage of signal sequences, the glycosylation of the (Y-and y-precursors, and the subsequent secretion of the 3 proteins. In the submaxillary gland, however, the mature forms of CCNGF, yNGF and renin are composed of peptides of smaller size than those produced by the oocytes. These latter appear to lack specific proteases invoked in the terminal processing of the submaxillary gland proteins.

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“…These cells respond reversibly to NGF by cessation of cell division and the development of a number of properties characteristic of mature sympathetic neurons, including neurite outgrowth (9,10 Binding Assays. The method of Smith et al (14) was used to prepare ,BNGF (referred to subsequently as NGF). NGF was radioiodinated as described (4) to give a product with a final specific activity of 1300-3600 cpm/fmol.…”

mentioning

confidence: 99%

Nerve growth factor receptors on PC12 cells: ligand-induced conversion from low- to high-affinity states.

Landreth¹,

Shooter²

1980

Proc. Natl. Acad. Sci. U.S.A.

217

125

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The pheochromocytoma PC12 cell possesses specific cell surface receptors that bind nerve growth factor (NGF) with two different affinities. The rate of dissociation of NGF from the higher affinity receptor is slower than from the lower affinity receptor; this rate is reduced to essentially zero at low temperature, allowing the extent of high-affinity binding to be determined. When NGF is added to PC12 cells, only lowaffinity binding is observed. After a short lag period, high-affinity binding also appears and increases slowly. If NGF is removed from the medium after binding is initiated, high-affinity receptors continue to be formed at the expense of low-affinity receptors. The increase in receptor affinity is accompanied by a transfer of the NGF-receptor complex from a trypsin-sensitive to a trypsin-resistant state. This transfer does not involve internalization of the NGF. The data show that NGF binds first to receptors of low affinity and that the binding induces a conversion of a proportion of the receptors to a higher affinity state. It is also consistent with a model in which the change in affinity is due either to conformational changes in the receptor or to interaction of the occupied receptor with other receptors or with effector proteins in cell plasma membrane.Peptide hormones act on their target cells by initially binding to cell surface receptors. The hormone-receptor interaction has been extensively studied in a number of systems, and the data that have been obtained have been variously interpreted as evidence for multiple independent receptor sites (1-3) or, alternatively, for negatively cooperative site-site interactions within a homogeneous receptor population (4). A third model has been suggested in which hormone-receptor interactions are described by two or more sequential steps. In it the hormone first binds to its receptor and the hormone-receptor complex then associates with other membrane components, thus altering the apparent affinity with which the hormone is bound (5, 6). These models, although predicting similar binding characteristics at steady state, can be distinguished by analysis of the kinetic behavior of the hormone-receptor interaction. A useful system for exploring the validity of these models is that which involves the binding of nerve growth factor (NGF) to its specific NGF receptor. Evidence from earlier work (7) has shown that sensory neurons display two saturable and specific binding components for NGF. The two components differ -100-fold in affinity for NGF. it is clear from both steady-state and kinetic analyses that the two components cannot be ascribed to cooperative ligand-receptor interactions (7).NGF is required for the maintenance and development of sympathetic and certain sensory neurons (8). Greene and Tischler (9) have established an NGF-responsive clonal line of pheochromocytoma, PC12, which resembles the pluripotent neural crest cell. These cells respond reversibly to NGF by cessation of cell division and the development of a number of properties cha...

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Multiple forms of the nerve growth factor protein and its subunits

Cited by 176 publications

References 10 publications

Circular dichroism and crosslinking studies of the interaction between four neurotrophins and the extracellular domain of the low‐affinity neurotrophin receptor

Circular dichroism and crosslinking studies of the interaction between four neurotrophins and the extracellular domain of the low‐affinity neurotrophin receptor

Synthesis and partial maturation of the α‐ and γ‐subunits of the mouse submaxillary gland nerve growth factor in Xenopus laevis oocytes

Nerve growth factor receptors on PC12 cells: ligand-induced conversion from low- to high-affinity states.

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