Quantitation of an alpha subunit splicing intermediate: evidence for transcriptional activation in the control of acetylcholine receptor expression in denervated chick skeletal muscle.

We have investigated the mechanisms responsible for the increase in acetylcholine receptor subunit mRNAs during the induction of denervation supersensitivity in skeletal muscle. Using a cRNA probe specific for exon 7 (224 nucleotides; with flanking intron sequences of 105 nucleotides on the 3' end, and of 70 nucleotides on the 5' end) of the alpha subunit of the chicken muscle acetylcholine receptor gene, we were able to quantitate the concentration of mature alpha subunit mRNA and its precursor. In 3-wk-old chicks, the concentration of alpha subunit message in leg muscle was found to be 4.0 attomoles per microgram total RNA, and to increase 40-fold within 1 wk after section of the sciatic nerve. The molar ratio of precursor/mature mRNA, which was approximately 0.023 in innervated as well as denervated muscle, transiently rose to 0.047 at the beginning of the second postoperative day when mature message content increased 20-fold; the rise in precursor level preceded the increase in mature message content. These findings suggest that an accelerated rate of transcription of the message coding for the alpha subunit causes increased message content and the stimulation of receptor synthesis characteristic of denervated muscle.

Alpha-2-antiplasmin: a serpin with two separate but overlapping reactive sites.

Although the proteinase inhibitor alpha-2-antiplasmin (alpha 2AP) is known to control the activity of plasmin through rapid formation of stable complexes, it also efficiently inactivates chymotrypsin. These interactions are shown to occur at adjacent, overlapping sites so that plasmin attacks the inhibitor at an Arg364-Met365 peptide bond, while chymotrypsin interacts at a Met365-Ser366 sequence one residue downstream. Thus, a naturally occurring plasma serine proteinase inhibitor can have multiple specificities through interactions at adjacent sites. It also illustrates the potential flexibility of the reactive site loop in this class of inhibitors.

Identification of a naturally occurring peroxidase-lipoxygenase fusion protein.

A distant relative of catalase that is specialized for metabolism of a fatty acid hydroperoxide was identified. This heme peroxidase occurs in coral as part of a fusion protein, the other component of which is a lipoxygenase that forms the hydroperoxide substrate. The end product is an unstable epoxide (an allene oxide) that is a potential precursor of prostaglandin-like molecules. These results extend the known chemistry of catalase-like proteins and reveal a distinct type of enzymatic construct involved in the metabolism of polyunsaturated fatty acids.

A novel protein encoded by the InaD gene regulates recovery of visual transduction in Drosophila.

InaDp215 is a point mutation that affects photoreceptor function in Drosophila. To understand the molecular basis of the defect, we isolated the InaD gene and found it encodes a photoreceptor-specific polypeptide of 674 residues. Within its sequence are two repeats that share remarkable homology with a family of cytoskeleton-associated proteins that are involved in signal transduction. Patch-clamp recordings from isolated photoreceptor cells of InaDp215 show a slow deactivation of the light-induced current. This defective deactivation of InaD appears dependent on calcium influx; removal of extracellular calcium masks its abnormal phenotype. Moreover, InaD photoreceptors show increases sensitivity to dim light. We propose that InaD is involved in the negative feedback regulation of the light-activated signaling cascade in Drosophila photoreceptors.

Regulation of the TRP Ca2+ channel by INAD in Drosophila photoreceptors.

Drosophila vision involves a G protein-coupled phospholipase C-mediated signaling pathway that leads to membrane depolarization through activation of Na+ and Ca2+ channels. InaD mutant flies have a M442K point mutation and display a slow recovery of the Ca2+ dependent current. We report that anti-INAD antibodies coimmunoprecipitate TRP, identified by its electrophoretic mobility, cross reactivity with anti-TRP antibody, and absence in a null allele trp mutant. This interaction is abolished by the InaD point mutation in vitro and in vivo. Interaction was localized to the 19 amino acid C-terminus of TRP by overlay assays, and to the PDZ domain of INAD, encompassing the point mutation. Given the impaired electrophysiology of the InaD mutant, this novel interaction suggests that INAD functions as a regulatory subunit of the TRP Ca2+ channel.

A unique isoform of phospholipase Cbeta4 highly expressed in the cerebellum and eye.

We report a unique isoform of PLCbeta4 in rat, PLCbeta4c, that has an additional 37-nucleotide exon inserted between nucleotides 3459-3460 of the previously published PLCbeta4a coding sequence. This insertion results in replacement of 22 amino acid residues at the carboxyl terminal tail of PLCbeta4a with 41 unique residues. A human EST for PLCbeta4 also contains this exon and this exon was mapped to within a 5.5 kb intron of the human PLCbeta4 gene. PLCbeta4c is the third PLCbeta4 isoform to be identified which has a unique carboxyl-terminal tail. PLCbeta4b differs from PLCbeta4a by truncation 162 amino acid residues from the carboxyl terminus which are replaced with 10 distinct amino acid residues. Reverse transcription-polymerase chain reaction experiments show that both PLCbeta4a and PLCbeta4c mRNA are expressed throughout the rat brain and that PLCbeta4c mRNA is highly expressed in the eye and cerebellum. RNase protection assays demonstrate that both PLCbeta4a and PLCbeta4c transcripts are abundant in the cerebellum. The different carboxyl terminal tails of PLCbeta4 isoforms may allow for differential targeting and subcellular localization, contributing to regulation of PLC beta4-mediated signal transduction.

Acetylcholine receptor synthesis rate and levels of receptor subunit messenger RNAs in chick muscle.

Levels of mRNAs specific for the alpha-, gamma- and delta-subunit of the nicotinic acetylcholine receptor were measured in chick skeletal muscle by solution hybridization, using a genomic DNA probe containing the intramembrane segments M2 and M3 of the alpha-subunit and probes comprising exons 2-6 and exons 4-8, respectively, of the gamma- and delta-subunit. In the innervated calf musculature of adult chickens, receptor-specific messages were detected in approx. 100-fold excess over the amount required to account for the observed synthesis rate. Within 1 week after section of the sciatic nerve, alpha-, gamma- and delta-subunit message levels rose 112-, 42- and 24-fold, respectively, while receptor expression rate increased about 150-fold. The rise in message levels preceded the denervation-induced increase in receptor concentration. In differentiating myogenic cells all three messages were found in excess over the amounts required for the observed rate of receptor synthesis. Treatment of differentiated myotubes with drugs that change receptor synthesis rate selectively affects alpha-subunit mRNA. In all situations in vitro and in vivo the alpha-subunit mRNA was found to reach final levels faster, and to be from 3 to over 30 times more abundant, than the other messages. These observations corroborate earlier evidence for a regulatory mechanism in which the supply of mRNA determines acetylcholine receptor synthesis rate. They also suggest that receptor expression is not simply proportional to acetylcholine receptor subunit mRNA concentrations, but rather is controlled, to a considerable extent, by the efficiency with which the receptor-specific mRNAs and/or the subunits they code for are subsequently utilized.

Extracellular potassium and the regulation of acetylcholine receptor synthesis in embryonic chick muscle cells.

The effect of elevated extracellular potassium on acetylcholine receptor synthesis was studied in chick embryonic muscle cultures. At physiological ionic strength, potassium chloride, in the 3.3 to 50 mM range, gave rise to a complex dose-response curve whose prominent features are a considerable reduction of receptor appearance rate at 20 mM and a more than 2-fold increase at higher concentrations. The effect of potassium chloride on receptor synthesis appears to be fairly specific: neither was there a duplication of its effect by other electrolytes or solutes, nor did it alter total protein synthesis or receptor stability by more than 30% at any concentration tested; cellular acetylcholinesterase levels actually declined with increasing KCl concentrations. In order to explore the mechanism of the potassium effect, tetrodotoxin (10(-6) M), veratridine (3 X 10(-6) M), D-600 (1.6 X 10(-5) M), and ryanodine (3 X 10(-7) M) were tested in the presence of various concentrations of potassium. Sodium channel toxins as well as calcium effectors modified the potassium response. Based on these findings we propose that the effects of potassium are due to: (a) cessation of spontaneous muscle activity upon raising KCl from 3 to 10 mM; (b) depolarization of the muscle membrane and persistent activation of a calcium channel as concentration is raised from 10 to 20 mM; (c) finally, inactivation or desensitization of the calcium channel, or some other signaling element proximal to the sarcoplasmic reticulum, upon further depolarization.

A comparison of the antagonisms by neostigmine and diaminopyridine against the neuromuscular block caused by cobrotoxin and (+)-tubocurarine.

Cobrotoxin was about 11-fold more potent than (+)-tubocurarine on a weight basis in blocking neuromuscular transmission in mouse isolated phrenic nerve-diaphragm preparations. Neostigmine and diaminopyridine increased the concentrations of cobrotoxin for 70% inhibition of indirect contraction by 290 and 320%, and increased those of (+)-tubocurarine by 180 and 230%, respectively. More than additive increases were obtained when neostigmine and diaminopyridine were used simultaneously. Cobrotoxin, however, was only 6-fold more toxic than (+)-tubocurarine after intraperitoneal injection in mice. The lethal dose of (+)-tubocurarine was increased by 80% when both antidotes were used together, but only by 15-20% when used alone. In contrast, the lethality of cobrotoxin was not decreased by these drugs. Unexpectedly, the time to death after treatment with cobrotoxin was shortened when mice were pretreated with these antidotes.

The functional role of acute phase plasma proteinase inhibitors.

Human plasma contains an array of proteinase inhibitors which are utilized in the regulation of a host of biological activities, including coagulation, fibrinolysis, connective tissue turnover, and complement activation. The concentration of several of these inhibitors increase at varying rates in the acute phase state while others remain constant or actually decrease. Increases are presumably an attempt to retain rigid control over certain critical reactions, while decreases are probably due to inhibitor turnover either through consumption during complex formation or inactivation by other endogenous proteinases. Virtually all of these latter reactions take place in a reactive site loop which is an exposed region present in at least eight related serine proteinase inhibitors (Serpins) in plasma. Complex formation and inhibitor inactivation presumably act as signals for inhibitor production and turnover in the acute phase state. However, exactly how this initial stimulus for increased protein synthesis is manifested at the protein level remains to be established.

The reactive site of human alpha 2-antiplasmin.

Human alpha 2-antiplasmin rapidly forms a stable, equimolar complex with either its target enzyme, plasmin, or with trypsin. Perturbation of the inhibitor-trypsin complex results in peptide bond cleavage at the reactive site of the inhibitor with the concomitant release of a small peptide fragment which apparently represents the carboxyl-terminal segment of the inhibitor. Sequence analysis of this fragment, together with that of an overlapping peptide obtained by treatment of native inhibitor with either Staphylococcus aureus V8 proteinase or human neutrophil elastase, yields data which indicate that the reactive site of alpha 2-antiplasmin encompasses a P1-P'1 Arg-Met sequence. However, unlike alpha 1-1-proteinase inhibitor which has a Met residue in the P1-position, oxidation of alpha 2-antiplasmin has no effect on its inhibitory activity toward either plasmin, trypsin, or chymotrypsin, indicating the lesser mechanistic importance of the P'1-residue during enzyme inactivation by this inhibitor.

The second PDZ domain of INAD is a type I domain involved in binding to eye protein kinase C. Mutational analysis and naturally occurring variants.

INAD is a scaffolding protein containing five PSD95/dlg/zonular occludens-1 (PDZ) domains that tether NORPA (phospholipase Cbeta(4)), the TRP calcium channel, and eye-PKC in Drosophila photoreceptors. We previously showed that eye-PKC interacted with the second PDZ domain (PDZ2) of INAD. Sequence comparison with a prototypical type I PDZ domain predicts that PDZ2 is the best candidate among the five PDZ domains to recognize eye-PKC that contains a type I PDZ ligand, Ile-Thr-Ile-Ile, at its carboxyl terminus. Replacement of Ile(-3) in eye-PKC with charged residues resulted in a drastic reduction of the PDZ2 interaction. Substitution of a conserved His with Arg at the second alpha-helix of PDZ2 led to a reduced binding; however, a Leu replacement resulted in an enhanced eye-PKC association. We isolated and sequenced the InaD gene. The coding sequence of InaD contains nine exons spanning 3 kilobases. Translation of coding sequences from three wild-type alleles revealed three SNPs affecting residues, 282, 319, and 333 of INAD. These polymorphisms are localized in PDZ2. Interestingly, we found two of three PDZ2 variants displayed a greater affinity for eye-PKC. In summary, we evaluated the molecular basis of the eye-PKC and PDZ2 association by mutational analysis and concluded that PDZ2 of INAD is a type I domain important for the eye-PKC interaction.

The use of alpha 2-antiplasmin as a model for the demonstration of complex reversibility in serpins.

Human alpha 2-antiplasmin readily forms 1:1 complexes with either trypsin or chymotrypsin at independent but overlapping reactive sites. In the absence of alpha 2-macroglobulin, complex dissociation and enzyme release can be demonstrated without regeneration of inhibitory activity. However, in the presence of this inhibitor the dissociation of alpha 2-antiplasmin-chymotrypsin complexes or alpha 2-antiplasmin-trypsin complexs yields functionally active inhibitors which can now inactivate trypsin and chymotrypsin, respectively. These results clearly indicate that Serpin-proteinase complexes can dissociate to give both active inhibitor and enzyme. If the enzyme is trapped by alpha 2-macroglobulin, in vivo, it is possible that the inhibitor may be recycled for further use.

Isolation and structure of an arrestin gene from Drosophila.

A Drosophila gene encoding a homologue of vertebrate arrestin was isolated by subtractive hybridization and identified as a member of a set of genes that are preferentially expressed in the visual system. This gene encodes a 364-amino acid protein that displays greater than 40% amino acid sequence identity with human and bovine arrestin. Interestingly, the Drosophila homologue lacks the C-terminal sequences that were postulated to interact with rhodopsin during the quenching of the phototransduction cascade in the vertebrate visual response. These findings are discussed in terms of invertebrate phototransduction. The Drosophila gene was mapped cytogenetically to chromosomal position 36D1-2, near the ninaD locus. However, the arrestin gene does not appear to be the ninaD locus, as sequence analysis of three ethylmethane sulfate-induced ninaD mutant alleles reveals no alteration in amino acid sequence.

Trifluoperazine stimulates acetylcholine receptor synthesis in cultured chick myotubes.

Acetylcholine receptor appearance rate in the presence of the phenothiazines trifluoperazine and chlorpromazine was measured in cultured embryonic chick myotubes by means of 125I-alpha-bungarotoxin. At drug concentrations of 5 to 10 X 10(-6) M, receptor appearance rate was significantly enhanced while receptor half-life, cellular protein, net protein synthesis rate, and acetylcholinesterase levels were not similarly affected. The sulfoxide derivatives were without effect. At concentrations of 3 X 10(-5) M and above, both trifluoperazine and chlorpromazine caused myotube contracture and cell loss. Drug combination experiments revealed that receptor stimulation caused by phenothiazines is overcome by low concentrations of veratridine and ryanodine, but not by membrane depolarization with 20 mM KCl. These results lend support to the role of calcium as an intracellular messenger in acetylcholine receptor synthesis regulation, but are difficult to reconcile with the notion that cytosolic calmodulin serves as the calcium receptor in this signaling pathway. Since the trifluoperazine effect resembles that caused by the calcium antagonist D-600, phenothiazines may stimulate receptor synthesis by blocking a voltage-gated calcium channel.

The cyclophilin homolog ninaA is a tissue-specific integral membrane protein required for the proper synthesis of a subset of Drosophila rhodopsins.

Mutations in the Drosophila ninaA gene cause dramatic reductions in rhodopsin levels, leading to impaired visual function. The ninaA protein is a homolog of peptidyl-prolyl cis-trans isomerases. We find that ninaA is unique among this family of proteins in that it is an integral membrane protein, and it is expressed in a cell type-specific manner. We have used transgenic animals misexpressing different rhodopsins in the major class of photoreceptor cells to demonstrate that ninaA is required for normal function by two homologous rhodopsins, but not by a less conserved member of the Drosophila rhodopsin gene family. This demonstrates in vivo substrate specificity in a cyclophilin-like molecule. We also show that vertebrate retina contains a ninaA-related protein and that ninaA is a member of a gene family in Drosophila. These data offer insights into the in vivo role of this important family of proteins.

The ninaA gene required for visual transduction in Drosophila encodes a homologue of cyclosporin A-binding protein.

Mutations of the Drosophila melanogaster ninaA gene affect phototransduction: ninaA mutant flies have a 10-fold reduction in the levels of rhodopsin in the R1-R6 photoreceptor cells. The ninaA gene was isolated and found to encode a 237-amino-acid protein that has over 40% amino-acid sequence identity with the vertebrate cyclosporin A-binding protein, cyclophilin, a protein that seems to be involved in T-lymphocyte activation. The remarkable evolutionary conservation of cyclophilin in two phylogenetically distant organisms and its involvement in diverse transduction processes suggests that this protein plays an important role in cellular metabolism. Indeed, cyclophilin has recently been shown to be a prolyl cis-trans isomerase that catalyses, in vitro, rate-limiting steps in the folding of a number of proteins. Here, we present evidence for the involvement of cyclophilin-like molecules in a defined cellular process. The availability of mutations in a cyclophilin gene provides a new model system for the study of cyclophilin and cyclosporin action.

Association of INAD with NORPA is essential for controlled activation and deactivation of Drosophila phototransduction in vivo.

Visual transduction in Drosophila is a G protein-coupled phospholipase C-mediated process that leads to depolarization via activation of the transient receptor potential (TRP) calcium channel. Inactivation-no-afterpotential D (INAD) is an adaptor protein containing PDZ domains known to interact with TRP. Immunoprecipitation studies indicate that INAD also binds to eye-specific protein kinase C and the phospholipase C, no-receptor-potential A (NORPA). By overlay assay and site-directed mutagenesis we have defined the essential elements of the NORPA-INAD association and identified three critical residues in the C-terminal tail of NORPA that are required for the interaction. These residues, Phe-Cys-Ala, constitute a novel binding motif distinct from the sequences recognized by the PDZ domain in INAD. To evaluate the functional significance of the INAD-NORPA association in vivo, we generated transgenic flies expressing a modified NORPA, NORPAC1094S, that lacks the INAD interaction. The transgenic animals display a unique electroretinogram phenotype characterized by slow activation and prolonged deactivation. Double mutant analysis suggests a possible inaccessibility of eye-specific protein kinase C to NORPAC1094S, undermining the observed defective deactivation, and that delayed activation may similarly result from NORPAC1094S being unable to localize in close proximity to the TRP channel. We conclude that INAD acts as a scaffold protein that facilitates NORPA-TRP interactions required for gating of the TRP channel in photoreceptor cells.

Reversible phosphorylation of the signal transduction complex in Drosophila photoreceptors.

In the Drosophila visual cascade, the transient receptor potential (TRP) calcium channel, phospholipase Cbeta (no-receptor-potential A), and an eye-specific isoform of protein kinase C (eye-PKC) comprise a multimolecular signaling complex via their interaction with the scaffold protein INAD. Previously, we showed that the interaction between INAD and eye-PKC is a prerequisite for deactivation of a light response, suggesting eye-PKC phosphorylates proteins in the complex. To identify substrates of eye-PKC, we immunoprecipitated the complex from head lysates using anti-INAD antibodies and performed in vitro kinase assays. Wild-type immunocomplexes incubated with [(32)P]ATP revealed phosphorylation of TRP and INAD. In contrast, immunocomplexes from inaC mutants missing eye-PKC, displayed no phosphorylation of TRP or INAD. We also investigated protein phosphatases that may be involved in the dephosphorylation of proteins in the complex. Dephosphorylation of TRP and INAD was partially suppressed by the protein phosphatase inhibitors okadaic acid, microcystin, and protein phosphatase inhibitor-2. These phosphatase activities were enriched in the cytosol of wild-type heads, but drastically reduced in extracts prepared from glass mutants, which lack photoreceptors. Our findings indicate that INAD functions as RACK (receptor for activated PKC), allowing eye-PKC to phosphorylate INAD and TRP. Furthermore, dephosphorylation of INAD and TRP is catalyzed by PP1/PP2A-like enzymes preferentially expressed in photoreceptor cells.

Purification and molecular cloning of an 8R-lipoxygenase from the coral Plexaura homomalla reveal the related primary structures of R- and S-lipoxygenases.

Lipoxygenases that form S configuration fatty acid hydroperoxides have been purified or cloned from plant and mammalian sources. Our objectives were to characterize one of the lipoxygenases with R stereospecificity, many of which are described in marine and freshwater invertebrates. Characterization of the primary structure of an R-specific enzyme should help provide a new perspective to consider the enzyme-substrate interactions that are the basis of the specificity of all lipoxygenases. We purified an 8R-lipoxygenase of the prostaglandin-containing coral Plexaura homomalla by cation and anion exchange chromatography. This yielded a colorless enzyme preparation, a band of approximately 100 kDa on SDS-polyacrylamide gel electrophoresis, and turnover numbers of 4000 min-1 of 8R-lipoxygenase activity in peak chromatographic fractions. The full-length cDNA was cloned by PCR using peptide sequence from the purified protein and by 5'- and 3'-rapid amplification of cDNA ends. The cDNA encodes a polypeptide of 715 amino acids, including over 70 amino acids identified by peptide microsequencing. A peptide presequence of 52 amino acids is cleaved to give the mature protein of 76 kDa; the difference from the estimated size by SDS-PAGE implies a post-translational modification of the P. homomalla enzyme. All of the iron-binding histidines of S-lipoxygenases are conserved in the 8R-lipoxygenase. However, the C-terminal amino acid is a threonine, as opposed to the isoleucine that provides the carboxylate ligand to the iron in all known S-lipoxygenases. These results establish that the 8R-lipoxygenase is related in primary structure to the S-lipoxygenases. A model of the basis of R and S stereospecificity is described.