Biomedical subjects

D R Storm

Publications and source records attributed to D R Storm.

At least 73 records · Page 4Linked to original sources

PubMed1994

Intracellular sorting of neuromodulin (GAP-43) mutants modified in the membrane targeting domain.

Neuromodulin (GAP-43) is a neurospecific calmodulin binding protein that is targeted to neuronal growth cones via fast axonal transport by an undefined mechanism. The protein is associated with membranes by palmitoylation of cys-3 and cys-4. The objective of this study was to examine the intracellular localization of neuromodulin and neuromodulin mutants modified in the membrane targeting domain of the protein in neurons and non-neuronal cells. The N-terminal palmitoylation domain of neuromodulin was found to be sufficient for membrane and Golgi targeting as well as neurite transport. A fusion protein consisting of the N-terminal 20 amino acids of neuromodulin and beta-galactosidase accumulated in neurite endings demonstrating that this sequence is sufficient for targeting to growth cone membranes. Mutations in the palmitoylation domain of neuromodulin that abolished its acylation and membrane association diminished its Golgi localization. Mutations that prevented Golgi accumulation of neuromodulin-beta-galactosidase fusion proteins also interfered with neurite transport of the fusion proteins. These data demonstrate a correlation between membrane targeting, Golgi localization, and neurite transport of neuromodulin.

Amino Acid Sequence↗

PubMed1993

Analysis of the palmitoylation and membrane targeting domain of neuromodulin (GAP-43) by site-specific mutagenesis.

Neuromodulin (GAP-43) is a neurospecific calmodulin binding protein which is implicated in neuronal growth and regeneration. It is concentrated in neuronal growth cones and associates with membranes through the palmitoylation of the N-terminal peptide MLCCMRRTK at Cys-3 and Cys-4. In the present study, we have identified critical amino acid residues required for palmitoylation and membrane association of neuromodulin in vivo. Several neuromodulin constructs with point mutations were tested for membrane association and palmitoylation. Wild-type neuromodulin expressed in COS-7 cells incorporated [3H]palmitic acid, whereas a mutant in which both Cys-3 and Cys-4 were substituted with glycine was not palmitoylated in vivo. Mutant proteins in which either Cys-3 or Cys-4 was substituted with leucine incorporated 75% and 25% of [3H]palmitic acid, respectively, compared to wild-type neuromodulin. The relative distribution of mutant neuromodulins expressed in COS-7 cells was quantitated by immunoblot analysis of the membrane and cytosolic fractions. There was a general correlation between membrane association of mutant neuromodulins and the extent to which they were palmitoylated in vivo. Additional point mutations in the acylation domain of neuromodulin indicated that a short hydrophobic amino acid sequence N-terminal to Cys-4 may be required for optimal palmitoylation and membrane association. We conclude that Cys-4 is critical for the palmitoylation and membrane association of neuromodulin.

Amino Acid Sequence↗

PubMed1993

Phorbol ester stimulation of the type I and type III adenylyl cyclases in whole cells.

Phorbol esters and activators of protein kinase C have been reported to either facilitate or inhibit increases in intracellular cAMP caused by activators of adenylyl cyclase. The variable responses to activators of protein kinase C may reflect, in part, the existence of distinct adenylyl cyclases present in animal cells. There are a family of adenylyl cyclases with different regulatory properties, and clones for six distinct types of adenylyl cyclase have been reported. Two of these enzymes, the type I and type III adenylyl cyclases, are stimulated by calcium and calmodulin whereas the others are not. In this study, we examined the effect of phorbol esters of the activity of the type I and type III adenylyl cyclases in whole cells. TPA markedly enhanced the forskolin responsiveness of the type I and type III adenylyl cyclases expressed in kidney 293 cells. The effect of TPA on the activity of the calmodulin-sensitive adenylyl cyclases was not mediated through increases in intracellular free calcium. These data suggest that activation of protein kinase C can elevate intracellular cAMP in animal cells that contain the type I or type III adenylyl cyclase.

1-Methyl-3-isobutylxanthine↗

PubMed1993

Cloning, chromosomal mapping, and expression of human fetal brain type I adenylyl cyclase.

The neural-specific calmodulin-sensitive adenylyl cyclase (type I), which was first cloned from bovine brain, has been implicated in learning and memory. The objective of this study was to clone and determine the chromosomal localization of human fetal brain type I adenylyl cyclase. A 3.8-kb cDNA clone was isolated that contained sequence coinciding with the 3' end 2553 nucleotides of the bovine open reading frame. This clone shows 87% nucleotide and 92% translated amino acid sequence identity to the bovine clone. The most significant sequence differences were in the carboxy-terminal 100 amino acid residues. This region contains one of several possible calmodulin binding domains and the only putative cAMP-dependent protein kinase A phosphorylation site. A chimera was constructed that contained the 5' half of the bovine type I adenylyl cyclase and the 3' half of the human type I adenylyl cyclase. The activity of the chimeric gene product and its sensitivity to calmodulin and calcium were indistinguishable from those of the bovine type I adenylyl cyclase. In situ hybridization was used to localize the human type I adenylyl cyclase gene to the proximal portion of the short arm of chromosome 7.

Adenylyl Cyclases↗

PubMed1993

Virulence of a Bordetella pertussis strain expressing a mutant adenylyl cyclase with decreased calmodulin affinity.

Bordetella pertussis, the pathogen responsible for whooping cough, produces a toxic calmodulin-sensitive adenylyl cyclase which enters animal cells and increases intracellular cAMP. A point mutant of B. pertussis with abolished adenylyl cyclase catalytic activity was over 1000-fold less pathogenic to newborn mice than wild-type bacteria, demonstrating the importance of the adenylyl cyclase for B. pertussis virulence (Gross et al.). The B. pertussis adenylyl cyclase is highly sensitive to calmodulin with an apparent Kd for calmodulin of approximately 1 nM. The importance of this high-affinity calmodulin binding for virulence in vivo was examined by the creation of a B. pertussis point mutant (Trp-242 to Glu-242) with 200-fold lower calmodulin affinity than the native enzyme. This mutant B. pertussis strain retained its virulence in a newborn mouse model of pertussis, but the time course for establishment of a lethal infection in vivo was significantly delayed for the mutant strain. These data illustrate that high-affinity calmodulin binding is not obligatory for the activity of this toxin but is important for the rate for establishment of a lethal infection.

Adenylyl Cyclases↗

PubMed1993

Identification of a domain in Bordetella pertussis adenylyl cyclase important for subunit interactions and cell invasion activity.

Bordetella pertussis produces a calmodulin-stimulated adenylyl cyclase that invades animal cells and raises intracellular cAMP levels. The enzyme does not enter animal cells by receptor-mediated endocytosis, but the mechanism for invasion of animal cells has not been defined. We have proposed that the 177 kDa adenylyl cyclase is proteolyzed to a 45 kDa catalytic subunit and one or more polypeptides (invasive factor) that facilitate entry of the catalytic subunit into animal cells. In this study, we report the identification of a sequence of amino acids within the adenylyl cyclase catalytic subunit that is important for entry of the enzyme into eukaryotic cells. A synthetic peptide corresponding to amino acids 313-339 within the catalytic subunit was shown to inhibit invasion of neuroblastoma cells by the adenylyl cyclase. In addition, this peptide inhibited the association of the catalytic subunit with invasive factor. We propose that this domain is a site for interaction between the catalytic subunit and invasive factor.

Adenylate Cyclase Toxin↗

PubMed1993

The regulatory diversity of the mammalian adenylyl cyclases.

Clones for six mammalian adenylyl cyclases have recently been isolated. One of these enzymes, the type I calmodulin-sensitive adenylyl cyclase, is neurospecific and is implicated in neuroplasticity. We propose that the type I adenylyl cyclase may be important for learning and memory because it allows Ca(2+)-amplified cAMP signals, synergism between Ca2+ and cAMP-activated kinases, and positive feedback regulation of Ca2+ channels by cAMP-dependent protein kinase.

Adenylyl Cyclases↗

PubMed1993

Type I calmodulin-sensitive adenylyl cyclase is neural specific.

The distribution of type I calmodulin-sensitive adenylyl cyclase in bovine and rat tissues was examined by northern blot analysis and in situ hybridization. Northern blot analysis using poly(A)(+)-selected RNA from various bovine tissues indicated that mRNA for type I adenylyl cyclase was found only in brain, retina, and adrenal medulla, suggesting that this enzyme is neural specific. In situ hybridization studies using bovine, rabbit, and rat retina indicated that mRNA for type I adenylyl cyclase is found in all three nuclear layers of the neural retina and is particularly abundant in the inner segment of the photoreceptor cells. The neural-specific distribution of type I adenylyl cyclase mRNA and its restricted expression in areas of brain implicated in neuroplasticity are consistent with the proposal that this enzyme plays an important role in various neuronal functions including learning and memory.

Adenylyl Cyclases↗

PubMed1992

Fluorescence energy transfer analysis of calmodulin-peptide complexes.

The interactions between calmodulin and the tryptophan residues of synthetic peptides corresponding to the calmodulin binding domains of skeletal muscle myosin light-chain kinase and the plasma membrane calcium pump were examined. The single tryptophan residue contained in each peptide became relatively immobilized and inaccessible to iodide ion upon binding to calmodulin, indicating that the indole side chain was inserted into a hydrophobic cleft in the surface of calmodulin. Fluorescence energy transfer from peptidyl tryptophan residues to an AEDANS moiety attached to cysteine-26 of spinach calmodulin was measured. Included in these analyses was a tryptophan-containing peptide analog of the calmodulin binding domain of neuromodulin. These data indicated that the indole ring of each peptide inserted 32-35 A away from cysteine-26 and may therefore interact with the carboxyl-terminal lobe of CaM in its "bent" conformation [Persechini & Kretsinger (1988a) J. Cardiovasc. Pharmacol. 12 (Suppl 5), S1-S12; Ikura et al. (1992) Science 256, 632-638; Vorherr et al. (1992) Eur. J. Biochem. 204, 931-937]. The interchange of tryptophan-3 and phenylalanine-21 of the calcium pump peptide increased the efficiency of energy transfer to the AEDANS-moiety approximately 12-fold, reducing the calculated distance to 20 A. These data suggest that phenylalanine-21 of the calcium pump peptide interacts with the hydrophobic cleft in the amino-terminal lobe of CaM.

Amino Acid Sequence↗

PubMed1992

Palmitylation of neuromodulin (GAP-43) is not required for phosphorylation by protein kinase C.

Neuromodulin (also designated GAP-43, B-50, and F-1) is a prominent protein kinase C substrate attached to the membranes of neuronal growth cones during development and to presynaptic membranes in discrete subsets of adult synapses. In this study, we have examined the relationship between the attachment of neuromodulin to membranes and its phosphorylation by protein kinase C. To address this issue, we have compared wild-type and mutant neuromodulins expressed in cells that normally lack the protein. Wild-type neuromodulin expressed in Chinese hamster ovary cells was associated with membranes, incorporated [3H]palmitic acid, and was phosphorylated in response to phorbol ester treatment. Substitution of serine 41, the in vitro protein kinase C site, abolished the phorbol ester response, indicating that serine 41 serves as the sole protein kinase C phosphorylation site in vivo. Substitution of the putative fatty acylation sites, cysteines 3 and 4, abolished membrane association as well as [3H]palmitic acid labeling of neuromodulin. Fatty acylation therefore appears to serve as the mechanism for anchoring neuromodulin to membranes. Surprisingly, the soluble cysteine substitution mutant was phosphorylated by protein kinase C at a rate indistinguishable from that of the wild-type protein. Therefore, membrane association may not be required for the phosphorylation of neuromodulin by protein kinase C.

Amino Acid Sequence↗

PubMed1992

High-affinity calmodulin binding is required for the rapid entry of Bordetella pertussis adenylyl cyclase into neuroblastoma cells.

Bordetella pertussis produces a calmodulin-stimulated adenylyl cyclase that invades animal cells and raises intracellular cAMP levels [Confer, D. L., & Eaton, J. W. (1982) Science 217, 948-950; Shattuck, R. L., & Storm, D. R. (1985) Biochemistry 24, 6323-6328]. The mechanism for invasion of animal cells by this enzyme has not been defined, but there is considerable evidence that it does not enter by receptor-mediated endocytosis [Gordon, V. M., Leppla, S. H., & Hewlett, E. L. (1988) Infect. Immun. 56, 1066-1069; Donovan, M. G., & Storm, D. R. (1990) J. Cell. Physiol. 145, 444-449]. In this study, the importance of high-affinity calmodulin (CaM) binding for entry of the enzyme into neuroblastoma cells was evaluated using a mutant enzyme that has significantly lower affinity for calmodulin than the wild-type enzyme. Oligonucleotide-directed site-specific mutagenesis was used to create a point mutant at a critical tryptophan residue (Trp-242) within the proposed CaM binding domain of the B. pertussis adenylyl cyclase. Substitution of Trp-242 with Glu lowered the apparent affinity of the enzyme for calmodulin by 250-fold; however, the maximal enzyme activity in the presence of saturating calmodulin was equivalent to the wild-type enzyme. The Glu-242 mutant adenylyl cyclase was returned to B. pertussis by homologous recombination, and the enzyme produced by this strain was examined for invasion of neuroblastoma cells. Although the mutant enzyme stimulated the production of intracellular cAMP in neuroblastoma cells, the rate of cAMP accumulation was at least 10-fold lower than that caused by the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗

PubMed1992

The type III calcium/calmodulin-sensitive adenylyl cyclase is not specific to olfactory sensory neurons.

A cDNA clone for type III adenylyl cyclase was originally isolated from a rat olfactory cDNA library and Northern analysis using total RNA suggested that the expression of the type III mRNA may be limited to the olfactory epithelium (Bakalyar and Reed, Science, 250 (1990) 1403-1406). In this study, the distribution of type III adenylyl cyclase mRNA in a number of bovine tissues and cultured cells was examined by Northern analysis using poly(A)+ RNA. Type III adenylyl cyclase mRNA was expressed in brain, spinal cord, adrenal medulla, adrenal cortex, heart atrium, aorta, lung, retina, 293 cells and PC-12 cells. Furthermore, the Ca2+ sensitivity of adenylyl cyclase activity in 293 cells indicated the presence of type III adenylyl cyclase. These data indicate that expression of the type III adenylyl cyclase is not limited to olfactory tissues, and that this enzyme probably has a number of physiological functions in addition to olfactory signal transduction.

Adenylyl Cyclases↗

PubMed1992

Stimulation of the type III olfactory adenylyl cyclase by calcium and calmodulin.

Characterization of adenylyl cyclases has been facilitated by the isolation of cDNA clones for distinct adenylyl cyclases including the type I and type III enzymes. Expression of type I adenylyl cyclase activity in animal cells has established that this enzyme is stimulated by calmodulin and Ca2+. Type III adenylyl cyclase is enriched in olfactory neurons and is regulated by stimulatory G proteins. The sensitivity of the type III adenylyl cyclase to Ca2+ and calmodulin has not been reported. In this study, type III adenylyl cyclase was expressed in human kidney 293 cells to determine if the enzyme is stimulated by Ca2+ and calmodulin. The type III enzyme was not stimulated by Ca2+ and calmodulin in the absence of other effectors. It was, however, stimulated by Ca2+ through calmodulin when the enzyme was concomitantly activated by either GppNHp or forskolin. The concentrations of free Ca2+ for half-maximal stimulation of type I and type III adenylyl cyclases were 0.05 and 5.0 microM Ca2+, respectively. These data suggest that the type III adenylyl cyclase is stimulated by Ca2+ when the enzyme is activated by G-protein-coupled receptors and that increases in free Ca2+ accompanying receptor activation may amplify the primary cyclic AMP signal.

Adenylyl Cyclases↗

PubMed1992

Calcium and muscarinic agonist stimulation of type I adenylylcyclase in whole cells.

The type I adenylylcyclase which was originally purified and cloned from bovine brain is stimulated by Ca2+ and calmodulin in vitro. Although it has been proposed that this enzyme may couple elevations in intracellular Ca2+ to increases in cAMP in whole cells, this has not been demonstrated in vivo. In this study, the type I adenylylcyclase was expressed in human 293 cells, and the influence of extracellular Ca2+ and Ca2+ ionophore on intracellular cAMP levels was examined. The cAMP levels of control cells were unaffected by Ca2+ and A23187. In contrast, intracellular cAMP in 293 cells expressing type I adenylylcyclase was markedly elevated by addition of A23187 and extracellular Ca2+. In the presence of forskolin, the muscarinic agonist carbachol also increased cAMP in 293 cells expressing the type I adenylylcyclase. These data indicate that the type I adenylylcyclase can be stimulated by Ca2+ in vivo, and that muscarinic agonists may indirectly stimulate the enzyme by increasing intracellular free Ca2+.

Adenylyl Cyclases↗

PubMed1992

Targeted mutations that ablate either the adenylate cyclase or hemolysin function of the bifunctional cyaA toxin of Bordetella pertussis abolish virulence.

Bordetella pertussis, the causative agent of whooping cough, secretes several toxins implicated in this disease. One of these putative virulence factors is the adenylate cyclase (AC) toxin that elevates intracellular cAMP in eukaryotic cells to cytotoxic levels. This toxin is a bifunctional protein comprising both AC and hemolysin (HLY) enzymatic domains. The gene encoding the AC toxin (cyaA) is expressed as part of an operon that includes genes required for secretion or activation of the toxin. Because of this genetic organization, it is difficult to create B. pertussis mutants of cyaA that are ablations of a single enzyme function by conventional means, such as transposon mutagenesis. Therefore, to clarify the role of individual toxin functions in the virulence of B. pertussis, we have used site-directed or deletion mutagenesis and genetic recombination to specifically target the cyaA gene of B. pertussis to produce mutants that lack only the AC or HLY activity of this toxin. A point mutant of B. pertussis with abolished AC catalytic activity was greater than 1000 times less pathogenic to newborn mice than wild-type bacteria, directly demonstrating the importance of the AC toxin in pertussis virulence. Similarly, an in-frame deletion mutant of B. pertussis that lacks HLY is equally avirulent, supporting observations that the HLY domain plays a critical role in AC toxin entry into cells. Furthermore, the genetically inactivated AC toxin produced by the point mutant is antigenically similar to the native toxin, suggesting that this strain may be useful in the development of pertussis component vaccines.

Adenylate Cyclase Toxin↗

PubMed1992

Functional domains of neuromodulin (GAP-43).

Although neuromodulin (GAP-43, B50, F1, pp46, protein 4) was first identified over a decade ago, the physiological function(s) of the protein and the molecular mechanism(s) for its biological activities are still an area of active investigation. Neuromodulin has been implicated in several biological processes in neurons, including growth and regeneration, synaptic plasticity and neurotransmitter release. The molecular mechanisms underlying these implied physiological roles have not been elucidated, but there are several molecular properties of neuromodulin that may be important for its function in neurons. In this review, we will discuss research which has defined several of the functional domains of neuromodulin, including its phosphorylation sites, calmodulin binding domain, membrane binding domain and growth cone targeting domain. We will also suggest possible molecular functions of neuromodulin based on its biochemical properties.

Amino Acid Sequence↗

PubMed1991

Phosphorylation of neuromodulin (GAP-43) by casein kinase II. Identification of phosphorylation sites and regulation by calmodulin.

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin-binding protein believed to play a role in regulation of neurite outgrowth and neuroplasticity. Neuromodulin is phosphorylated by protein kinase C, and this phosphorylation prevents calmodulin from binding to neuromodulin (Alexander, K. A., Cimler, B. M., Meier, K. E. & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). The only other protein kinase known to phosphorylate neuromodulin is casein kinase II (Pisano, M. R., Hegazy, M. G., Reimann, E. M. & Dokas, L. A. (1988) Biochem. Biophys. Res. Commun. 155, 1207-1212). Phosphoamino acid analyses revealed that casein kinase II modified serine and threonine residues in both native bovine and recombinant mouse neuromodulin. Two serines located in the C-terminal end of neuromodulin, Ser-192 and Ser-193, were identified as the major casein kinase II phosphorylation sites. Thr-88, Thr-89, or Thr-95 were identified as minor casein kinase II phosphorylation sites. Phosphorylation by casein kinase II did not affect the ability of neuromodulin to bind to calmodulin-Sepharose. However, calmodulin did inhibit the phosphorylation of neuromodulin by casein kinase II with a Ki of 1-2 microM. Calmodulin inhibition of casein kinase II phosphorylation was due to calmodulin binding to neuromodulin rather than to the protein kinase. These data suggest that the minimal secondary and tertiary structure exhibited by neuromodulin may be sufficient to juxtapose its calmodulin-binding domain, located at the N-terminal end, with the neuromodulin casein kinase II phosphorylation sites at the C-terminal end of the protein. We propose that calmodulin regulates casein kinase II phosphorylation of neuromodulin by binding to neuromodulin and sterically hindering the interaction of casein kinase II with its phosphorylation sites on neuromodulin.

Amino Acid Sequence↗

PubMed1991

Characterization of the calmodulin binding domain of neuromodulin. Functional significance of serine 41 and phenylalanine 42.

Neuromodulin (also designated P-57, GAP-43, B-50) is a major presynaptic substrate for protein kinase C. Phosphorylation of neuromodulin decreases its affinity for calmodulin, suggesting that neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons, releasing calmodulin locally in response to phosphorylation by protein kinase C (Alexander, K. A., Cimler, B. M., Meier, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). In the present study, we have constructed and characterized several mutant neuromodulins to demonstrate that the amino acid sequence 39-56 is required for calmodulin binding, and that this domain contains the sole in vitro protein kinase C phosphorylation site at serine 41. We also demonstrate that the adjacent phenylalanine 42, interacts hydrophobically with calmodulin. These hydrophobic interactions may be disrupted by the introduction of negative charge at serine 41, and thereby regulate the neuromodulin/calmodulin binding interactions. The sensitivity of the neuromodulin/calmodulin binding interaction to negative charge at serine 41 was determined by substitution of serine 41 with an aspartate or an asparagine residue. The asparagine mutant retained its affinity for calmodulin-Sepharose while the aspartate mutant did not adsorb to calmodulin-Sepharose. We conclude that protein kinase C phosphorylation of neuromodulin abolishes calmodulin binding by introducing negative charges within the calmodulin binding domain at a position adjacent to the phenylalanine.

Amino Acid Sequence↗