7S Nerve growth factor alpha and gamma subunits are closely related proteins

The polypeptide composition and partial amino acid sequence of the 7S nerve growth factor (NGF) alpha subunit have been determined. Residues in 76 unique positions corresponding to 35% of the molecule were identified. The sequence shows that the NGF alpha subunit is closely related to the NGF gamma subunit and thus a member of the same protein family as the serine proteases. This finding is unexpected since the NGF alpha subunit is devoid of detectable protease activity. However, the NGF alpha subunit differs in one important respect from the NGF gamma subunit and related serine proteases. The highly conserved amino-terminal activation cleavage structure, common to most serine proteases, has been deleted, and an uncleaved activation peptide remains attached to the amino terminus of the mature NGF alpha subunit. It is suggested that this feature is causally related to the apparent lack of proteolytic activity.     

Subunit structure of high molecular weight mouse nerve growth factor

Studies from several laboratories have shown that mouse submandibular glands and mouse saliva contain nerve growth factor (NGF) as part of a high molecular weight oligomeric macromolecule composed of three different subunits, termed alpha, beta, and gamma. The beta-subunit is the nerve growth-promoting protein. The gamma-subunit is a serine protease class enzyme of highly restricted substrate specificity. The alpha-subunit has no known function. This high molecular weight form of nerve growth factor is also a Zn(II)-containing metalloprotein. In the present study, measurements of multiple physicochemical parameters have been used to deduce the subunit structure of high molecular weight NGF. Results demonstrate that it contains two alpha-, one beta- and one gamma-subunit together with one tightly bound Zn(II) ion per molecule.     

Nerve growth factor: biosynthetic products of the mouse salivary glands. Characterization of stable high molecular weight and 32,000-dalton nerve growth factors

Nerve growth factor (NGF) is a protein required for the growth and development of sensory and sympathetic neurons. The NGF is present in high concentrations in male mouse salivary glands, bovine seminal plasma, and snake venom. The physiological significance of NGF in these sources is not known: it might be a part of a high molecular weight (HMW) protein with possibly different biological function and be cleaved to the functional size by proteases. In an attempt to isolate a HMW protein containing as part of its structure the low molecular weight (LMW) NGF (2.5S), mouse salivary glands were homogenized in the presence of either 8 M urea or 6 M guanidine hydrochloride (Gdn X HCl) in order to denature proteases. This procedure revealed that the LMW NGF is a part of two HMW proteins that are biologically and immunologically homologous to the mouse 2.5S NGF. One of these HMW proteins (Mr 32,000 NGF) was purified and shown to be biologically active in the NGF bioassay. Furthermore, this Mr 32,000 NGF was cleaved by the gamma subunit of mouse HMW NGF to the 2.5S NGF. Evidence is also presented that there may be a HMW protein(s) with apparent molecular weights ranging from 94,000 to 200,000 and immunologically homologous to the three subunits (alpha, beta, gamma) of 7S NGF. This HMW NGF is biologically active in the NGF bioassay, and its activity is inhibited by antibody to the beta subunit. Furthermore, in contrast to mouse 7S NGF, this HMW NGF does not dissociate in either 8 M urea or 6 M Gdn X HCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

The sequence of a cDNA clone coding for a novel kallikrein from mouse submaxillary gland.

Mouse submaxillary gland contains many proteolytic enzymes, the most widely studied of which are the kallikreins. This gland also contains high levels of nerve growth factor (NGF), which is isolated as a complex of three subunits, alpha, beta, and gamma. We report here the cloning and sequence analysis of a novel kallikrein from mouse submaxillary gland. Antibodies directed against the alpha subunit precipitate the product of this clone, but do not precipitate the homologous gamma subunit. This new kallikrein is therefore closely related to alpha NGF, yet in contrast to the alpha subunit, its sequence suggests it has proteolytic activity.     

Comparison of 7S nerve growth factor and nerve growth factor I from mouse submandibular glands

7S nerve growth factor (7S NGF) and nerve growth factor I (NGFI) are NGF-containing protein complexes isolated from mouse submandibular glands by different protocols, and reports suggest that the molecules differ chemically. In this study, we compared the molecular properties and subunit compositions of the two proteins. Purified 7S NGF and NGFI electrophoresed to identical positions on polyacrylamide gels in nondissociating buffers, with electrophoretic mobilities indistinguishable from that of unpurified NGF in salivary gland extracts. Ultraviolet absorption curves were identical, and sedimentation coefficients were similar (7.3 +/- 0.25 S for 7S NGF; 7.2 +/- 0.2 S for NGFI) as determined by sedimentation velocity analysis. By sedimentation equilibrium analysis, molecular weights of 135 000-140 000 were obtained for both complexes at protein concentrations in the centrifuge cell greater than 85 micrograms/mL; when protein concentrations within the centrifuge cell ranged from approximately 30 to 100 micrograms/mL at equilibrium, both complexes dissociated. Molecular weight values determined by gel filtration on Bio-Gel P300 and Sephadex G200 resins were similar for both proteins, and the values determined on Sephadex agreed with those obtained by ultracentrifugation. The subunit compositions of the complexes were also similar as determined by nonequilibrium isoelectric focusing, NGFI being composed of proteins that migrated to positions identical with those of the alpha, beta, and gamma subunits of 7S NGF. Furthermore, the stoichiometry of the subunits was similar in the two complexes as determined by radioimmunoassays to each of the subunits and by densitometric analysis of electrophoretic gels. Both methods showed that the complexes contain approximately 2 mol of the alpha and gamma subunits per mole of beta-NGF.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the relationship of zinc ion to the structure and function of the 7S nerve growth factor protein.

The 7S nerve growth factor (7S NGF) is an oligomeric protein consisting of three distinct classes of subunits, alpha,beta, and gamma (A. P. Smith, S. Varon, and E. M. Shooter (1968), Biochemistry 7, 3259). The beta subunit contains the growth promoting activity while gamma is a potent esteropeptidase. The proteolytic activity of gamma is virtually completely inhibited in the 7S NGF aggregate (L. A. Greene, E. M. Shooter, and S. Varon (1969), Biochemistry 8, 3735). In this paper, we report that divalent metal ion chelating agents effect a seven- to tenfold increase in the esteropeptidase activity of 7S NGF at pH 7.40. Plots of esteropeptidase activity vs. chelator concentration give saturation curves which are either sigmoidal (EDTA) or hyperbolic (o-phenanthroline) depending on the chemical structure of the chelator. A survey of common divalent metal ions shows that only zinc ion (Ki = 8 times 10(7) M) and, to a lesser extent, cadmium ion are effective, reversible inhibitors of both 7S NGF and the gamma subunit esteropeptidase activities. We have found that during isolation of 7S NGF, Zn2+ is selectively associated with the oligomer in a ratio of approximately 1-2 g-atoms of zinc/mol of 7S NGF with an apparent affinity which is orders of magnitude tighter than is indicated by the Ki value for the gamma subunit. Dialysis to pH 4.0 where 7S NGF is known to undergo a reversible dissociation (A. P. Smith, S. Varon, and E. M. Shooter (1968), Biochemistry 7, 3259) brings about a tenfold reduction in the zinc ion content of the protein. This reduction is reversed on dialysis back to pH 7.4. In contrast, the isolated subunits contain only trace amounts of zinc ion at pH 7.4. Preliminary metal ion exchange experiments indicate that, of the common metal ions known to substitute for zinc in other zinc-metalloproteins, only cadmium ion is effective in substituting for zinc ion in 7S NGF. The fact that zinc ion is specifically bound to native 7S NGF, and that the zinc ion content of the system is critically dependent on the subunit aggregation state strongly suggests that zinc ion is an integral structural component of native 7S NGF.     

Absence of the alpha and gamma subunits of 7S nerve growth factor in denervated rodent iris: immunocytochemical studies

Immunocytochemical studies were performed to determine if denervated rodent iris produces nerve growth factor (NGF) in a form chemically similar to that of the 7S NGF complex in mouse submandibular glands. Antisera to the alpha, beta, and gamma subunits of 7S NGF were raised in rabbits and characterized on immunoblots of SDS-containing polyacrylamide gels. Antisera were applied to stretch preparations of rat and mouse irides that were cultured for periods of 2 to 6 days or sympathetically denervated by superior cervical ganglionectomy and left in situ 4 days. Antibody binding was visualized by indirect immunofluorescence. In control studies done on plastic sections of mouse submandibular glands, antisera co-localized the three subunits of 7S NGF within secretory granules of granular tubule cells. In denervated rat iris, beta NGF immunoreactivity was evident in a cellular plexus that resembled in distribution and morphology nerve fibers in the normal iris, in agreement with a previous study (R.A. Rush (1984). Nature (London) 312, 364-367). Identical staining patterns were observed in mouse iris. In neither rat or mouse, however, did the nerve-like processes stain with antibodies suggests that the NGF-like protein in denervated rodent iris is not synthesized as part of the 7S NGF complex. Iris also did not react with antibodies to epidermal growth factor, a protein co-localized with NGF in mouse submandibular glands and in guinea pig prostate.     

Characterization of the gamma subunits of the 7S nerve growth factor complex.

The gamma subunits of the 7S nerve growth factor complex (7S NGF) display arginine esteropeptidase activity. By varying the conditions of electrophoresis in acrylamide gel, it has been demonstrated that the gamma-subunit fraction of 7S NGF contains five different proteins, in contrast to the three (gamma1, gamma2, and gamma3) originally described (Smith, A.P., Varon, S. and Shooter, E.M. (1968), Biochemistry 7, 3259-3268); the gamma1 and gamma2 subunits, previously thought to be single species, can each be resolved into two components. The two components of the gamma1 subunit have the same isoelectric point, as do the two components of the gamma2 subunit. The distribution of protein among the two components of each of the gamma1 and gamma2 subunits varied from preparation to preparation. Moreover, a shift in the distribution for the gamma1 subunit was accompanied by a parallel shift for the gamma2 subunit. All of the different gamma proteins have the same molecular weight. On the basis of the molecular weights of the peptide chains of the gamma subunits and of the species which are formed by cross-linking with dimethyl suberimidate, it was concluded, that both the gamma1 and gamma2 subunits contain one species with two peptide chains and another with three peptide chains, while the gamma3 subunit is a single species with three peptide chains. The results also suggest that two of the chains in the three-chain species are derived, by proteolytic cleavage, from the larger chain in the two-chain species.     

Subunit interactions inhibit the binding of beta nerve growth factor to receptors on embryonic chick sensory neurons

The binding of the beta nerve growth factor subunit in the 7 S nerve growth factor complex to the two nerve growth factor receptors on chick embryo dorsal root ganglion cells was investigated. Under conditions where the 7 S nerve growth factor complex is maximally stable (in the presence of excess alpha and gamma subunits and of 20 to 30 microM zinc ion), no binding to either receptor was detectable. The time course of the decrease in the binding of beta nerve growth factor to the receptors upon addition of alpha and gamma subunits and zinc ion paralleled the formation of the 7 S complex. Addition of alpha and gamma subunits and zinc ion to the bisdes-arginine118-nerve growth factor, which does not re-form the 7 S complex, failed to inhibit the binding of the derivative to either receptor. While the alpha subunit alone had no effect on beta nerve growth factor binding, the gamma subunit decreased its binding in proportion to the amount of complex formed between these two subunits, suggesting that the beta . gamma complex, like the 7 S complex, does not bind to nerve growth factor receptors.     

Synthetic chimeras of mouse growth factor-associated glandular kallikreins. II. Growth factor binding properties.

Six chimeric constructs of the sequentially similar growth factor-associated kallikreins-epidermal growth factor binding protein (EGF-BP) and the gamma-subunit of nerve growth factor (gamma-NGF)--have been expressed, and their ability to generate complexes with epidermal growth factor (EGF) and beta-NGF, analogous to the high molecular weight forms (7S NGF and HMW-EGF) found in the mouse submaxillary gland, evaluated. The chimeras are distinguished by the interchange of three regions composing the amino, middle, and carboxyl terminal regions that encompass four surface loops possibly involved in specific growth factor interactions. Native beta-NGF (along with native alpha-NGF) formed complexes indistinguishable from naturally occurring 7S NGF, characterized by an alpha 2 beta gamma 2 structure (where beta-NGF is itself a dimer), with recombinant (r) gamma-NGF and with a chimera in which the amino terminal region from EGF-BP was substituted. Two other chimeras containing either the middle or carboxyl terminal regions of gamma-NGF showed weaker ability to form 7S complexes. Thus, all chimeras containing two segments from gamma-NGF retained at least some ability to form the 7S complex. rEGF-BP reacted weakly with EGF, but the chimera composed of the amino and middle segments of EGF-BP and the carboxyl terminal segment of gamma-NGF formed a nativelike HMW-EGF complex. None of the other chimeras appeared to bind EGF. These results identify amino acid positions within each kallikrein that participate in strong growth factor interactions and demonstrate that, outside of active site contacts, different regions of the kallikreins are involved in the binding of EGF and beta-NGF, respectively.     

Molecular weight and structure of 7 S nerve growth factor protein.

The molecular weight of 7 S nerve growth factor has been studied in the analytical ultracentrifuge between pH values 6.8 and 7.8. At pH 6.8, where no dissociation is observed, the molecular weight was found to be 137,000 plus and minus 7,000. Between pH values 7.4 and 7.8 there is some dissociation. Using the data from this study and results in the literature, a model of 7 S nerve growth factor, (alpha beta gamma)2, in reversible equilibrium with a subunit complex, (alpha beta gamma), is proposed.     

Dietary regulation of levels of active mRNA coding for amylase and serine protease zymogens in the rat pancreas

Translation products of a reticulocyte lysate reaction, programmed with poly(A)-rich RNAs from the male mouse submaxillary gland, were subjected to affinity chromatography on a tubulin-Sepharose column. Analysis of the bound proteins in sodium dodecylsulfate/polyacrylamide gels revealed two polypeptides of Mr 27 000 and 45 000, that were shown to bind to tubulin in a specific manner. These polypeptides were absent from the translation products coded by poly(A)-rich RNAs from the female mouse. They were eluted from the tubulin-Sepharose resin under conditions similar to those employed for the dissociation of immune complexes. The Mr-27 000 and Mr-45 000 proteins were identified by immunoprecipitation with specific antisera as the precursors of the gamma subunit of the nerve growth factor (NGF) and renin respectively. These two precursors as well as a third, unidentified polypeptide of Mr 38 000, probably unrelated to the beta subunit of NGF, bound also to microtubules. The mature form of renin, purified from the submaximillary gland, also displayed an affinity for the microtubules. In contrast, the mature form of the gamma subunit of NGF did not bind to the microtubules. The possible involvement of the microtubules (tubulin) in the biosynthesis of these two secretory proteins is discussed.     

Complete structure of the mouse mast cell receptor for IgE (Fc epsilon RI) and surface expression of chimeric receptors (rat-mouse-human) on transfected cells

The high affinity receptor for IgE (Fc epsilon RI) found on mast cells and basophils is a tetrameric complex of a single alpha subunit, a single beta subunit, and two identical gamma subunits. The genes for the three subunits of mouse Fc epsilon RI have now been cloned from the mast cell line, PT18. When compared at the DNA level, the rat and mouse subunits are similarly conserved. However, at the protein level the homology between mouse and rat alpha is surprisingly low (71% identities) especially in the cytoplasmic regions (57% identities) which are of different length (25 and 20 residues, respectively). By contrast the beta and gamma are homogeneously conserved between mouse and rat (83 and 93% identities, respectively). The consensus amino acid sequence of the alpha subunit derived from three species (rat, mouse, and human) shows that the cytoplasmic tail diverges to the same extent as the leader peptide. Conversely, the transmembrane domain of the alpha is highly conserved and contains 10 consecutive residues that are identical. Comparisons between mouse Fc epsilon RI and other mouse proteins reveal regions of high homology between the alpha subunit and Fc gamma RIIa and between the gamma subunit and the zeta chain of the T cell receptor. Cells transfected with the alpha gene express the alpha subunit on their surface very inefficiently. Efficient expression is only achieved after co-transfection of the three rodent genes or of the human alpha gene together with the rodent gamma without apparent need for beta. The subunits are completely interchangeable upon transfection so that various chimeric mouse-rat-human receptors can be expressed.     

G protein-effector coupling: binding of rod phosphodiesterase inhibitory subunit to transducin

The cyclic GMP phosphodiesterase of retinal rods is composed of three distinct polypeptides: alpha (90 kDa), beta (86 kDa), and gamma (10 kDa). In this multimeric form, the enzyme is inhibited. Its activity is stimulated by the interaction with the GTP-bound form of the T alpha subunit of transducin and reversed upon the recombination of the inhibitory gamma subunit with the catalytic alpha beta subunit. We show here by a novel coimmunoprecipitation technique that the gamma subunit, but not the alpha beta subunit, forms a 1:1 complex with T alpha. The binding of gamma to T alpha is nucleotide-dependent and is facilitated by GTP gamma S or Gpp(NH)p. This study provides convincing evidence that the T alpha-GTP subunit of transducin stimulates phosphodiesterase activity by binding to gamma and physically carrying it away from alpha beta.     

Serine 171, a conserved residue in the gamma-aminobutyric acid type A (GABAA) receptor gamma2 subunit, mediates subunit interaction and cell surface localization

Serine 171 in the GABA(A) receptor gamma2 subunit is highly conserved in the ligand-gated ion channel superfamily. In this paper, we report that mutating serine 171 within gamma2 to glycine or cysteine prevents the interaction of gamma2 with alpha2 and beta1 when these subunits are co-expressed in human embryo kidney 293 cells, resulting in intracellular retention of gamma2. Structure analysis based on a three-dimensional homology model of gamma2 (Ernst, M., Brauchart, D., Boresch, S., and Sieghart, W. (2003) Neuroscience 119, 933-943) reveals that serine 171 may play a critical role in the formation and stabilization of an exposed turn structure that is part of the subunit interaction site. Mutation of serine 171 in the gamma2 subunit could therefore result in alteration of the structure of the subunit interaction site, preventing correct subunit assembly.     

Role of the GABA(A)beta2, GABA(A)alpha6, GABA(A)alpha1 and GABA(A)gamma2 receptor subunit genes cluster in drug responses and the development of alcohol dependence

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter of the central nervous system and it acts at the GABA(A) and GABA(B) receptors. A possible role for the GABA(A) receptors in alcohol action has been derived from in vitro cell models, animal studies and human research. GABA(A) subunit mRNA expression in cell models has suggested that the long form of the gamma2 subunit is essential for ethanol enhanced potentiation of GABA(A) receptors, by phosphorylation of a serine contained within the extra eight amino acids. Several animal studies have demonstrated that alterations in drug and alcohol responses may be caused by amino-acid differences at the GABA(A)alpha6 and GABA(A)gamma2 subunits. An Arg(100)/Glu(100) change at the GABA(A)alpha6 subunit conferring altered binding efficacy of the benzodiazepine inverse agonist Ro 15-4513, was found between the AT (alcohol tolerance) and ANT (alcohol non-tolerance) rats. Several loci related to alcohol withdrawal on mouse chromosome 11 which corresponds to the region containing four GABA(A) subunit (beta2, alpha6, alpha1 and gamma2) genes on human chromosome 5q33-34, were also identified. Gene knockout studies of the role of GABA(A)alpha6 and GABA(A)gamma2 subunit genes in mice have demonstrated an essential role in the modulation of other GABA(A) subunit expression and the efficacy of benzodiazepine binding. Absence of the GABA(A)gamma2 subunit gene has more severe effects with many of the mice dying shortly after birth. Disappointingly few studies have examined the effects of response to alcohol in these gene knockout mice. Human genetic association studies have suggested that the GABA(A)beta2, alpha6, alpha1 and gamma2 subunit genes have a role in the development of alcohol dependence, although their contributions may vary between ethnic group and phenotype. In summary, in vitro cell, animal and human genetic association studies have suggested that the GABA(A)beta2, alpha6, alpha1 and gamma2 subunit genes have an important role in alcohol related phenotypes (300 words).     

Biological and biochemical properties of nerve growth factor synthesized by mouse S-180 cells in culture

Nerve Growth Factor (NGF), a protein involved in the maintenance and differentiation of sensory and sympathetic neuronal cells [1], is synthesized by several different types of cells in culture [2-7]. In this paper, the biochemical and biological properties of NGF synthesized by a mouse S-180 sarcoma cell line were examined. These cells do not appear to produce the 7S-NGF molecule, a form of NGF found in high concentrations in the mouse submandibular gland [8]. The 7S-NGF is comprised of three distinct protein subunits named beta-NGF, alpha and gamma [9]. Although the S-180 cells do not produce 7S-NGF, the cells do synthesize one of the component subunits of 7S-NGF, the beta-NGF subunit. Biological, electrophoretic, immunological and molecular weight criteria were used to establish that the beta-NGF synthesized by the S-180 cells is very similar to the submandibular gland beta-NGF. The S-180 beta-NGF was bound to an unidentified binding component(s) which is not immunologically similar to either the alpha- or gamma-subunit. The functional significance of this interaction is not known.     

A RECOMBINATION ASSAY OF HIGH MOLECULAR WEIGHT NERVE GROWTH FACTOR PROTEIN COMPLEXES

Abstract— The 7S Nerve Growth Factor (NGF) found in mouse is a protein composed of three dissimilar subunits designated α, γ, and β. Mouse NGF reversibly dissociates into its component subunits at pH values lower than 5 or greater than 8. The 7S protein complex is in fast equilibrium wiih the free subunits. A quantitative determination of 7S NGF based on the reversible dissociation of the molecule into its subunits is reported here. The basis for this procedure is the addition of [¹²⁵I]α subunit which competes with the native alpha in the 7S complex. The level of NGF present in a sample can be measured in terms of the displacement of [¹²⁵I]α from the α to the NGF position in a linear sucrose gradient. Measurements are sensitive to 3.5ng of NGF, linear, and specific for the NGF molecule. This exchange is unaffccted by competing unrelated growth factors and inhibiting enzymes. Its specificity was checked by the standard bioassay and by radioimmunoassay. In addition, the assay technique overrides masking by biological inhibitors and/or by the presence of antibodies directed against the IgG contaminant in murine 75 NGF.