N-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons.

Ca2+ influx controls multiple neuronal functions including neurotransmitter release, protein phosphorylation, gene expression, and synaptic plasticity. Brain L-type Ca2+ channels, which contain either alpha 1C or alpha 1D as their pore-forming subunits, are an important source of calcium entry into neurons. Alpha 1C exists in long and short forms, which are differentially phosphorylated, and C-terminal truncation of alpha 1C increases its activity approximately 4-fold in heterologous expression systems. Although most L-type calcium channels in brain are localized in the cell body and proximal dendrites, alpha 1C subunits in the hippocampus are also present in clusters along the dendrites of neurons. Examination by electron microscopy shows that these clusters of alpha 1C are localized in the postsynaptic membrane of excitatory synapses, which are known to contain glutamate receptors. Activation of N-methyl-D-aspartate (NMDA)-specific glutamate receptors induced the conversion of the long form of alpha 1C into the short form by proteolytic removal of the C terminus. Other classes of Ca2+ channel alpha1 subunits were unaffected. This proteolytic processing reaction required extracellular calcium and was blocked by inhibitors of the calcium-activated protease calpain, indicating that calcium entry through NMDA receptors activated proteolysis of alpha1C by calpain. Purified calpain catalyzed conversion of the long form of immunopurified alpha 1C to the short form in vitro, consistent with the hypothesis that calpain is responsible for processing of alpha 1C in hippocampal neurons. Our results suggest that NMDA receptor-induced processing of the postsynaptic class C L-type Ca2+ channel may persistently increase Ca2+ influx following intense synaptic activity and may influence Ca2+-dependent processes such as protein phosphorylation, synaptic plasticity, and gene expression.

The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems.

The experiments reported here were designed to test the hypothesis that the two-electron quinone reductase DT-diaphorase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] functions to maintain membrane-bound coenzyme Q (CoQ) in its reduced antioxidant state, thereby providing protection from free radical damage. DT-diaphorase was isolated and purified from rat liver cytosol, and its ability to reduce several CoQ homologs incorporated into large unilamellar vesicles was demonstrated. Addition of NADH and DT-diaphorase to either large unilamellar or multilamellar vesicles containing homologs of CoQ, including CoQ9 and CoQ10, resulted in the essentially complete reduction of the CoQ. The ability of DT-diaphorase to maintain the reduced state of CoQ and protect membrane components from free radical damage as lipid peroxidation was tested by incorporating either reduced CoQ9 or CoQ10 and the lipophylic azoinitiator 2,2'-azobis(2,4-dimethylvaleronitrile) into multilamellar vesicles in the presence of NADH and DT-diaphorase. The presence of DT-diaphorase prevented the oxidation of reduced CoQ and inhibited lipid peroxidation. The interaction between DT-diaphorase and CoQ was also demonstrated in an isolated rat liver hepatocyte system. Incubation with adriamycin resulted in mitochondrial membrane damage as measured by membrane potential and the release of hydrogen peroxide. Incorporation of CoQ10 provided protection from adriamycin-induced mitochondrial membrane damage. The incorporation of dicoumarol, a potent inhibitor of DT-diaphorase, interfered with the protection provided by CoQ. The results of these experiments provide support for the hypothesis that DT-diaphorase functions as an antioxidant in both artificial membrane and natural membrane systems by acting as a two-electron CoQ reductase that forms and maintains the antioxidant form of CoQ. The suggestion is offered that DT-diaphorase was selected during evolution to perform this role and that its conversion of xenobiotics and other synthetic molecules is secondary and coincidental.

Chemical specificity and physical properties of the lipid bilayer in the regulation of protein kinase C by anionic phospholipids: evidence for the lack of a specific binding site for phosphatidylserine.

The association of protein kinase C (PKC) with membranes was found not to be specific for phosphatidyl-L-serine (PS). In particular, a synthetic phospholipid, dansyl-phosphatidylethanolamine, proved to be fully functional in the association of PKC with lipid bilayers and in mediating the interaction of this enzyme with diacylglycerol. Dansyl-phosphatidylethanolamine was also able to activate the enzyme in a Ca2+-dependent fashion. Differences in the ability to bind and activate PKC observed for an array of anionic lipids were not larger than alterations caused by changes in acyl chain composition. Thus, although different lipids interact to different extents with PKC, there are no specific binding sites for the PS headgroup on the enzyme. We found that lipids with a greater tendency to form inverted phases increased the binding of PKC to bilayers. However, these changes in lipid structure cannot be considered separately from the miscibility of lipid components in the membrane. For pairs of lipids with similar acyl chains, the dependence on PS concentration is sigmoidal, while for dissimilar acyl chains there is much less dependence of binding on PS concentration. The results can be explained in terms of differences in the lateral distribution of components in the membrane.

Two alternative processing pathways for a preprohormone: a bioactive form of secretin.

An N-terminally 9-residue elongated form of secretin, secretin-(-9 to 27) amide, was isolated from porcine intestinal tissue and characterized. Current knowledge about peptide processing sites does not allow unambiguous prediction of the signal peptide cleavage site in preprosecretin but suggests cleavage in the region of residues -10 to -14 counted upstream from the N terminus of the hormone. However, the structure of the isolated peptide suggests that the cleavage between the signal peptide and the N-terminal propeptide occurs at the C-terminal side of residue -10. Moreover, the isolated peptide demonstrates that secretin can be fully processed C-terminally prior to the final N-terminal cleavage. The results from this report, and those from earlier studies, where C-terminally elongated variants were isolated, show that the processing of the secretin precursor may proceed by one of two alternative pathways, in which either of the two ends is processed first. The bioactivity of the N-terminally extended peptide on exocrine pancreatic secretion was lower than that of secretin, indicating the importance of the finally processed free N terminus of the hormone for interaction with secretin receptors.

Cloning of a gamma-aminobutyric acid type C receptor subunit in rat retina with a methionine residue critical for picrotoxinin channel block.

Ionotropic receptors for gamma-aminobutyric acid (GABA) are important to inhibitory neurotransmission in the mammalian retina, mediating GABAA and GABAC responses. In many species, these responses are blocked by the convulsant picrotoxinin (PTX), although the mechanism of block is not fully understood. In contrast, GABAC responses in the rat retina are extremely resistant to PTX. We hypothesized that this difference could be explained by molecular characterization of the receptors underlying the GABAC response. Here we report the cloning of two rat GABA receptor subunits, designated r rho 1 and r rho 2 after their previously identified human homologues. When coexpressed in Xenopus oocytes, r rho 1/r rho 2 heteromeric receptors mimicked PTX-resistant GABAC responses of the rat retina. PTX resistance is apparently conferred in native heteromeric receptors by r rho 2 subunits since homomeric r rho 1 receptors were sensitive to PTX; r rho 2 subunits alone were unable to form functional homomeric receptors. Site-directed mutagenesis confirmed that a single amino acid residue in the second membrane-spanning region (a methionine in r rho 2 in place of a threonine in r rho 1) is the predominant determinant of PTX resistance in the rat receptor. This study reveals not only the molecular mechanism underlying PTX blockade of GABA receptors but also the heteromeric nature of native receptors in the rat retina that underlie the PTX-resistant GABAC response.

MyoD forms micelles which can dissociate to form heterodimers with E47: implications of micellization on function.

MyoD is a member of a family of DNA-binding transcription factors that contain a helix-loop-helix (HLH) region involved in protein-protein interactions. In addition to self-association and DNA binding, MyoD associates with a number of other HLH-containing proteins, thereby modulating the strength and specificity of its DNA binding. Here, we examine the interactions of full-length MyoD with itself and with an HLH-containing peptide portion of an E2A gene product, E47-96. Analytical ultracentrifugation reveals that MyoD forms micelles that contain more than 100 monomers and are asymmetric and stable up to 36 degrees C. The critical micelle concentration increases slightly with temperature, but micelle size is unaffected. The micelles are in reversible equilibrium with monomer. Addition of E47-96 results in the stoichiometric formation of stable MyoD-E47-96 heterodimers and the depletion of micelles. Micelle formation effectively holds the concentration of free MyoD constant and equal to the critical micelle concentration. In the presence of micelles, the extent of all interactions involving free MyoD is independent of the total MyoD concentration and independent of one another. For DNA binding, the apparent relative specificity for different sites can be affected. In general, heterodimer-associated activities will depend on the self-association behavior of the partner protein.

Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin.

Endoproteolytic processing of the human protein C (HPC) precursor to its mature form involves cleavage of the propeptide after amino acids Lys-2-Arg-1 and removal of a Lys156-Arg157 dipeptide connecting the light and heavy chains. This processing was inefficient in the mammary gland of transgenic mice and pigs. We hypothesized that the protein processing capacity of specific animal organs may be improved by the coexpression of selected processing enzymes. We tested this by targeting expression of the human proprotein processing enzyme, named paired basic amino acid cleaving enzyme (PACE)/furin, or an enzymatically inactive mutant, PACEM, to the mouse mammary gland. In contrast to mice expressing HPC alone, or to HPC/PACEM bigenic mice, coexpression of PACE with HPC resulted in efficient conversion of the precursor to mature protein, with cleavage at the appropriate sites. These results suggest the involvement of PACE in the processing of HPC in vivo and represent an example of the engineering of animal organs into bioreactors with enhanced protein processing capacity.

Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder.

Both stem cells and mast cells express c-kit and proliferate after exposure to c-kit ligand. Mutations in c-kit may enhance or interfere with the ability of c-kit receptor to initiate the intracellular pathways resulting in cell proliferation. These observations suggested to us that mastocytosis might in some patients result from mutations in c-kit. cDNA synthesized from peripheral blood mononuclear cells of patients with indolent mastocytosis, mastocytosis with an associated hematologic disorder, aggressive mastocytosis, solitary mastocytoma, and chronic myelomonocytic leukemia unassociated with mastocytosis was thus screened for a mutation of c-kit. This analysis revealed that four of four mastocytosis patients with an associated hematologic disorder with predominantly myelodysplastic features had an A-->T substitution at nt 2468 of c-kit mRNA that causes an Asp-816-->Val substitution. One of one patient examined who had mastocytosis with an associated hematologic disorder had the corresponding mutation in genomic DNA. Identical or similar amino acid substitutions in mast cell lines result in ligand-independent autophosphorylation of the c-kit receptor. This mutation was not identified in the patients within the other disease categories or in 67 of 67 controls. The identification of the point mutation Asp816Val in c-kit in patients with mastocytosis with an associated hematologic disorder provides insight not only into the pathogenesis of this form of mastocytosis but also into how hematopoiesis may become dysregulated and may serve to provide a means of confirming the diagnosis, assessing prognosis, and developing intervention strategies.

Relating aromatic hydrocarbon-induced DNA adducts and c-H-ras mutations in mouse skin papillomas: the role of apurinic sites.

Mouse skin tumors contain activated c-H-ras oncogenes, often caused by point mutations at codons 12 and 13 in exon 1 and codons 59 and 61 in exon 2. Mutagenesis by the noncoding apurinic sites can produce G-->T and A-->T transversions by DNA misreplication with more frequent insertion of deoxyadenosine opposite the apurinic site. Papillomas were induced in mouse skin by several aromatic hydrocarbons, and mutations in the c-H-ras gene were determined to elucidate the relationship among DNA adducts, apurinic sites, and ras oncogene mutations. Dibenzo[a,l]pyrene (DB[a,l]P), DB[a,l]P-11,12-dihydrodiol, anti-DB[a,l]P-11,12-diol-13,14-epoxide, DB[a,l]P-8,9-dihydrodiol, 7,12-dimethylbenz[a]anthracene (DMBA), and 1,2,3,4-tetrahydro-DMBA consistently induced a CAA-->CTA mutation in codon 61 of the c-H-ras oncogene. Benzo[a]pyrene induced a GGC-->GTC mutation in codon 13 in 54% of tumors and a CAA-->CTA mutation in codon 61 in 15%. The pattern of mutations induced by each hydrocarbon correlated with its profile of DNA adducts. For example, both DB[a,l]P and DMBA primarily form DNA adducts at the N-3 and/or N-7 of deoxyadenosine that are lost from the DNA by depurination, generating apurinic sites. Thus, these results support the hypothesis that misreplication of unrepaired apurinic sites generated by loss of hydrocarbon-DNA adducts is responsible for transforming mutations leading to papillomas in mouse skin.

Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo.

The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.

Direct observation of disordered regions in the major histocompatibility complex class II-associated invariant chain.

Invariant chain (Ii) is a trimeric membrane protein which binds and stabilizes major histocompatibility complex class II heterodimers in the endoplasmic reticulum and lysosomal compartments of antigen-presenting cells. In concert with an intracellular class II-like molecule, HLA-DM, Ii seems to facilitate loading of conventional class II molecules with peptides before transport of the class II-peptide complex to the cell surface for recognition by T cells. The interaction of Ii with class II molecules is thought to be mediated in large part through a region of 24 amino acids (the class II-associated Ii peptide, CLIP) which binds as a cleaved moiety in the antigenic peptide-binding groove of class II molecules in HLA-DM-deficient cell lines. Here we use nuclear magnetic resonance techniques to demonstrate that a soluble recombinant Ii ectodomain contains significant disordered regions which probably include CLIP.

Cooperative transcriptional activity of Jun and Stat3 beta, a short form of Stat3.

To identify proteins that regulate the transcriptional activity of c-Jun, we have used the yeast two-hybrid screen to detect mammalian polypeptides that might interact functionally with the N-terminal segment of c-Jun, a known regulatory region. Among the proteins identified is a short form of Stat3 (designated Stat3 beta). Stat3 beta is missing the 55 C-terminal amino acid residues of the long form (Stat3 alpha) and has 7 additional amino acid residues at its C terminus. In the absence of added cytokines, expression of Stat3 beta (but not Stat3 alpha) in transfected cells activated a promoter containing the interleukin 6 responsive element of the rat alpha 2-macroglobulin gene; coexpression of Stat3 beta and c-Jun led to enhanced cooperative activation of the promoter. Nuclear extracts of cells transfected with a Stat3 beta expression plasmid formed a complex with an oligonucleotide containing a Stat3 binding site, whereas extracts of cells transfected with a Stat3 alpha plasmid did not. We conclude that there is a short form of Stat3 (Stat3 beta), that Stat3 beta is transcriptionally active under conditions where Stat3 alpha is not, and that Stat3 beta and c-Jun are capable of cooperative activation of certain promoters.

A membrane-associated form of sucrose synthase and its potential role in synthesis of cellulose and callose in plants.

Sucrose synthase (SuSy; EC 2.4.1.13; sucrose + UDP reversible UDPglucose + fructose) has always been studied as a cytoplasmic enzyme in plant cells where it serves to degrade sucrose and provide carbon for respiration and synthesis of cell wall polysaccharides and starch. We report here that at least half of the total SuSy of developing cotton fibers (Gossypium hirsutum) is tightly associated with the plasma membrane. Therefore, this form of SuSy might serve to channel carbon directly from sucrose to cellulose and/or callose synthases in the plasma membrane. By using detached and permeabilized cotton fibers, we show that carbon from sucrose can be converted at high rates to both cellulose and callose. Synthesis of cellulose or callose is favored by addition of EGTA or calcium and cellobiose, respectively. These findings contrast with the traditional observation that when UDPglucose is used as substrate in vitro, callose is the major product synthesized. Immunolocalization studies show that SuSy can be localized at the fiber surface in patterns consistent with the deposition of cellulose or callose. Thus, these results support a model in which SuSy exists in a complex with the beta-glucan synthases and serves to channel carbon from sucrose to glucan.

Cytochrome c oxidase in Neurospora crassa contains myristic acid covalently linked to subunit 1.

Radiolabel from [3H]myristic acid was incorporated by Neurospora crassa into the core catalytic subunit 1 of cytochrome c oxidase (EC 1.9.3.1), as indicated by immunoprecipitation. This modification of the subunit, which was specific for myristic acid, represents an uncommon type of myristoylation through an amide linkage at an internal lysine, rather than an N-terminal glycine. The [3H]myristate, which was chemically recovered from the radiolabeled subunit peptide, modified an invariant Lys-324, based upon analyses of proteolysis products. This myristoylated lysine is found within one of the predicted transmembrane helices of subunit 1 and could contribute to the environment of the active site of the enzyme. The myristate was identified by mass spectrometry as a component of mature subunit 1 of a catalytically active, purified enzyme. To our knowledge, fatty acylation of a mitochondrially synthesized inner-membrane protein has not been reported previously.

Critical role of the interleukin 2 (IL-2) receptor gamma-chain-associated Jak3 in the IL-2-induced c-fos and c-myc, but not bcl-2, gene induction.

The interleukin 2 receptor (IL-2R) consists of three subunits, the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two Janus family protein tyrosine kinases (PTKs), Jak1 and Jak3, were shown to associate with IL-2R beta c and IL-2R gamma c, respectively, and their PTK activities are increased after IL-2 stimulation. A Jak3 mutant with truncation of the C-terminal PTK domain lacks its intrinsic kinase activity but can still associate with IL-2R gamma c. In a hematopoietic cell line, F7, that responds to either IL-2 or IL-3, overexpression of this Jak3 mutant results in selective inhibition of the IL-2-induced activation of Jak1/Jak3 PTKs and of cell proliferation. Of the three target nuclear protooncogenes of the IL-2 signaling, c-fos and c-myc genes, but not the bcl-2 gene, were found to be impaired. On the other hand, overexpression of the dominant negative form of the IL-2R gamma c chain, which lacks most of its cytoplasmic domain, in F7 cells resulted in the inhibition of all three protooncogenes. These results provide a further molecular basis for the critical role of Jak3 in IL-2 signaling and also suggest a Jak PTK-independent signaling pathway(s) for the bcl-2 gene induction by IL-2R.