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A H Sharp

Publications and source records attributed to A H Sharp.

At least 37 records · Page 2Linked to original sources
PubMed1997

Huntingtin-associated protein 1 (HAP1) interacts with the p150Glued subunit of dynactin.

Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine repeat in the HD protein huntingtin. Huntingtin's localization within the cell includes an association with cytoskeletal elements and vesicles. We previously identified a protein (HAP1) which binds to huntingtin in a glutamine repeat length-dependent manner. We now report that HAP1 interacts with cytoskeletal proteins, namely the p150 Glued subunit of dynactin and the pericentriolar protein PCM-1. Structural predictions indicate that both HAP1 and the interacting proteins have a high probability of forming coiled coils. We examined the interaction of HAP1 with p150 Glued . Binding of HAP1 to p150 Glued (amino acids 879-1150) was confirmed in vitro by binding of p150 Glued to a HAP1-GST fusion protein immobilized on glutathione-Sepharose beads. Also, HAP1 co-immunoprecipitated with p150 Glued from brain extracts, indicating that the interaction occurs in vivo . Like HAP1, p150 Glued is highly expressed in neurons in brain and both proteins are enriched in a nerve terminal vesicle-rich fraction. Double label immunofluorescence experiments in NGF-treated PC12 cells using confocal microscopy revealed that HAP1 and p150 Glued partially co-localize. These results suggest that HAP1 might function as an adaptor protein using coiled coils to mediate interactions among cytoskeletal, vesicular and motor proteins. Thus, HAP1 and huntingtin may play a role in vesicle trafficking within the cell and disruption of this function could contribute to the neuronal dysfunction and death seen in HD.

Animals↗
PubMed1997

Huntingtin-associated protein 1 (HAP1) binds to a Trio-like polypeptide, with a rac1 guanine nucleotide exchange factor domain.

Huntington's disease (HD) occurs when the widely expressed protein huntingtin contains an expanded glutamine repeat. The selective degeneration and neuronal morphologic abnormalities of HD may involve interactions with proteins that bind to huntingtin, such as HAP1. The biological significance of this interaction is unclear because neither HAP1 nor huntingtin have significant homology to known proteins. Therefore, we sought to identify HAP1-binding proteins. Using the yeast two-hybrid system, we isolated a rat cDNA encoding part of a protein that interacts with HAP1, and we confirmed the specificity of this interaction using an in vitro protein-binding assay. We called the protein Duo because it is closely related to the human protein Trio but is shorter. Northern blot analysis indicates brain-specific expression of Duo. Human Duo contains a guanine nucleotide exchange factor (GEF) domain that is likely to be rac1-specific, a pleckstrin homology (PH) domain and spectrin-like repeat units. These data support the hypothesis that huntingtin is involved in vesicle trafficking and cytoskeletal functions, and raise the possibility of a role for huntingtin in the regulation of a ras-related signaling pathway.

Amino Acid Sequence↗
PubMed1997

Huntington's disease and dentatorubral-pallidoluysian atrophy: proteins, pathogenesis and pathology.

Each of the glutamine repeat neurodegenerative diseases has a particular pattern of pathology largely restricted to the CNS. However, there is considerable overlap among the regions affected, suggesting that the diseases share pathogenic mechanisms, presumably involving the glutamine repeats. We focus on Huntington's disease (HD) and Dentatorubral-pallidoluysian atrophy (DRPLA) as models for this family of diseases, since they have striking similarities and also notable differences in their clinical features and pathology. We review the pattern of pathology in adult and juvenile onset cases. Despite selective pathology, the disease genes and their protein products (huntingtin and atrophin-1) are widely expressed. This presents a central problem for all the glutamine repeat diseases-how do widely expressed gene products give rise to restricted pathology? The pathogenic effects are believed to occur via a "gain of function" mechanism at the protein level. Mechanisms of cell death may include excitotoxicity, metabolic toxicity, apoptosis, and free radical stress. Emerging data indicate that huntingtin and atrophin-1 may have distinct protein interactions. The specific interaction partners may help explain the selective pathology of these diseases.

Adult↗
PubMed1996

Lymphocyte apoptosis: mediation by increased type 3 inositol 1,4,5-trisphosphate receptor.

B and T lymphocytes undergoing apoptosis in response to anti-immunoglobulin M antibodies and dexamethasone, respectively, were found to have increased amounts of messenger RNA for the inositol 1,4,5-trisphosphate receptor (IP3R) and increased amounts of IP3R protein. Immunohistochemical analysis revealed that the augmented receptor population was localized to the plasma membrane. Type 3 IP3R (IP3R3) was selectively increased during apoptosis, with no enhancement of type 1 IP3R (IP3R1). Expression of IP3R3 antisense constructs in S49 T cells blocked dexamethasone-induced apoptosis, whereas IP3R3 sense, IP3R1 sense, or IP3R1 antisense control constructs did not block cell death. Thus, the increases in IP3R3 may be causally related to apoptosis.

Amino Acid Sequence↗
PubMed1996

Huntingtin-associated protein (HAP1): discrete neuronal localizations in the brain resemble those of neuronal nitric oxide synthase.

Huntington disease stems from a mutation of the protein huntingtin and is characterized by selective loss of discrete neuronal populations in the brain. Despite a massive loss of neurons in the corpus striatum, NO-generating neurons are intact. We recently identified a brain-specific protein that associates with huntingtin and is designated huntingtin-associated protein (HAP1). We now describe selective neuronal localizations of HAP1. In situ hybridization studies reveal a resemblance of HAP1 and neuronal nitric oxide synthase (nNOS) mRNA localizations with dramatic enrichment of both in the pedunculopontine nuclei, the accessory olfactory bulb, and the supraoptic nucleus of the hypothalamus. Both nNOS and HAP1 are enriched in subcellular fractions containing synaptic vesicles. Immunocytochemical studies indicate colocalizations of HAP1 and nNOS in some neurons. The possible relationship of HAP1 and nNOS in the brain is reminiscent of the relationship of dystrophin and nNOS in skeletal muscle and suggests a role of NO in Huntington disease, analogous to its postulated role in Duchenne muscular dystrophy.

Animals↗
PubMed1996

Expansion of polyglutamine repeat in huntingtin leads to abnormal protein interactions involving calmodulin.

Huntington's disease (HD) is an inherited neurodegenerative disorder associated with expansion of a CAG repeat in the IT15 gene. The IT15 gene is translated to a protein product termed huntingtin that contains a polyglutamine (polyGln) tract. Recent investigations indicate that the cause of HD is expansion of the polyGln tract. However, the function of huntingtin and how the expanded polyGln tract causes HD is not known. We investigate potential protein-protein interactions of huntingtin using affinity resins. Huntingtin from brain extracts is retained on calmodulin(CAM)-Sepharose in a calcium-dependent fashion. We purify rat huntingtin to apparent homogeneity using a combination of DEAE-cellulose column chromatography, ammonium sulfate precipitation, and preparative SDS/PAGE. Purified rat huntingtin does not interact with CAM directly as revealed by 125I-CAM overlay. Huntingtin forms a large CAM-containing complex of over 1,000 kDa in the presence of calcium, which partially disassociates in the absence of calcium. Furthermore, an increased amount of mutant huntingtin from HD patient brains is retained on CAM-Sepharose compared to normal huntingtin from control patient brains, and the mutant allele is preferentially retained on CAM-Sepharose in the absence of calcium. These results suggest that huntingtin interacts with other proteins including CAM and that the expansion of polyGln alters this interaction.

Animals↗
PubMed1996

cDNA cloning and characterization of an atrophin-1 (DRPLA disease gene)-related protein.

Dentatorubral and pallidoluylsian atrophy (DRPLA) is a progressive neurological disorder characterized by neuronal degeneration, especially in the cerebellar dentate nucleus. DRPLA is caused by an unstable expansion of a CAG trinucleotide repeat coding for glutamine in a gene of unknown function, termed atrophin-1, located on chromosome 12. To gain additional understanding of atrophin-1, we have isolated a second member of the atrophin-1 gene family by screening rat cDNA libraries. The 1006-amino-acid product of this gene, which we have termed rat atrophin related protein(rARP), does not contain a glutamine repeat, but it does contain two regions of alternating acidic and basic amino residues similar to those found in atrophin-1. rARP is widely expressed as both a 7.4- and a 9.4-kb message, with enrichment in cerebellum and testis. Like atrophin-1, the rARP in vitro translation product migrates more slowly on SDS-polyacrylamide gel electrophoresis than predicted by molecular weight. We conclude that, at least in the rat, polyglutamine is not an essential feature of the atrophin family of genes.

Amino Acid Sequence↗
PubMed1996

Inositol 1,4,5-trisphosphate receptors in endocrine cells: localization and association in hetero- and homotetramers.

The inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular calcium channel involved in coupling cell membrane receptors to calcium signal transduction pathways within cells including endocrine cells. Several isoforms (I, II, and III) of IP3Rs have been identified, which are encoded by separate genes, and are expressed in many tissues with differing patterns of cellular expression. We have generated specific affinity-purified polyclonal anti-peptide antibodies to each of the three isoforms. Western blot analysis of RINm5F and ATt20 cells shows high levels of endogenously expressed type I and type III IP3R, but undetectable levels of type II. Immunofluorescence studies revealed an endoplasmic reticulum-like pattern similar to BiP, an ER marker. In contrast with previous claims, both type I and type III IP3Rs were absent from the secretory granules of ATt20 cells. Western blots of sucrose gradients and gel filtration probed with antibodies to either type I or type III showed a molecular weight of greater than 1,000 kDa consistent with a tetrameric structure. Co-immunoprecipitation experiments indicated that most of the receptors were present as heterotetramers. Homotetramers were identified for the type III IP3R; however, type I homotetramers were undetectable. These data suggest that molecular association of IP3Rs into heterotetrameric forms can contribute to the complexity of the regulation of Ca2+ release from ER by IP3Rs within cells.

Amino Acid Sequence↗
PubMed1996

Absence of disease phenotype and intergenerational stability of the CAG repeat in transgenic mice expressing the human Huntington disease transcript.

The mutation underlying Huntington disease (HD) is CAG expansion in the first exon of the HD gene. In order to investigate the role of CAG expansion in the pathogenesis of HD, we have produced transgenic mice containing the full length human HD cDNA with 44 CAG repeats. By 1 year, these mice have no behavioral abnormalities and morphometric analysis at 6 (one animal) and 9 (two animals) months age revealed no changes. Despite high levels of mRNA expression, there was no evidence of the HD gene product in any of these transgenic mice. In vitro transfection studies indicated that the inclusion of 120 bp of the 5' UTR in the cDNA construct and the presence of a frameshift mutation at nucleotide 2349 prevented expression of the HD cDNA. These findings suggest that the pathogenesis of HD is not mediated through DNA-protein interaction and that presence of the RNA transcript with an expanded CAG repeat is insufficient to cause the disease. Rather, translation of the CAG is crucial for the pathogenesis of HD. In contrast to that seen in humans, the CAG repeat in these mice was remarkably stable in 97 meioses. This suggests that genomic sequences may play a critical role in influencing repeat instability.

Animals↗
PubMed1995

A huntingtin-associated protein enriched in brain with implications for pathology.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanding polyglutamine repeat in the IT15 or huntingtin gene. Although this gene is widely expressed and is required for normal development, the pathology of HD is restricted to the brain, for reasons that remain poorly understood. The huntingtin gene product is expressed at similar levels in patients and controls, and the genetics of the disorder suggest that the expansion of the polyglutamine repeat induces a toxic gain of function, perhaps through interactions with other cellular proteins. Here we report the identification of a protein (huntingtin-associated protein (HAP)-1) that binds to huntingtin. This binding is enhanced by an expanded polyglutamine repeat, the length of which is also known to correlate with the age of disease onset. The HAP-1 protein is enriched in the brain, suggesting a possible basis for the selective brain pathology of HD.

Amino Acid Sequence↗
PubMed1995

Inositol 1,4,5-trisphosphate receptors: immunocytochemical localization in the dorsal cochlear nucleus.

In the cochlear nucleus of mammals, the relatively homogeneous responses of auditory nerve fibers are transformed into a variety of different response patterns by the different classes of resident neurons. The spectrum of these responses is hypothesized to depend on the types and distribution of receptors, ion channels, G proteins, and second messengers that form the signaling capabilities in each cell class. In the present study, we examined the immunocytochemical distribution of the inositol 1,4,5-triphosphate (IP3) receptor in the dorsal cochlear nucleus to better understand how this second messenger might be involved in shaping the neural signals evoked by sound. Affinity-purified polyclonal antibodies directed against the IP3 receptor labeled a homogeneous population of neurons in the dorsal cochlear nucleus of rats, guinea pigs, mustache bats, cats, New World owl monkeys, rhesus monkeys, and humans. These cells were all darkly immunostained except in the human where the labeling was less intense. Immunoblots of dorsal cochlear nucleus tissue from the rat revealed a single band of protein of molecular weight approximately 260 kD, which is the same size as the purified receptor, indicating that our antibodies reacted specifically with the IP3 receptor. These immunolabeled neurons were identified as cartwheel cells on the basis of shared characteristics across species, including cell body size and distribution, the presence of a highly invaginated nucleus, and a well-developed system of cisternae. Reaction product was localized along the membranes of rough and smooth endoplasmic reticulum, subsurface cisternae, and the nuclear envelope. This label was distributed throughout the cartwheel cell body and dendritic shafts but not within dendritic spines, axons, or axons terminals. The regular pattern of immunolabeling across mammals suggests that IP3 and cartwheel cells are conserved in evolution and that both play an important but as yet unknown role in hearing.

Aged↗
PubMed1995

Widespread expression of Huntington's disease gene (IT15) protein product.

Huntington's Disease (HD) is caused by expansion of a CAG repeat within a putative open reading frame of a recently identified gene, IT15. We have examined the expression of the gene's protein product using antibodies developed against the N-terminus and an internal epitope. Both antisera recognize a 350 kDa protein, the predicted size, indicating that the CAG repeat is translated into polyglutamine. The HD protein product is widely expressed, most highly in neurons in the brain. There is no enrichment in the striatum, the site of greatest pathology in HD. Within neurons, the protein is diminished in nuclei and mitochondria and is present in the soluble cytoplasmic compartment, as well as loosely associated with membranes or cytoskeleton, in cell bodies, dendrites, and axons. It is concentrated in nerve terminals, including terminals within the caudate and putamen. Thus, the normal HD gene product may be involved in common intracellular functions, and possibly in regulation of nerve terminal function. The product of the expanded allele is expressed, consistent with a gain of function mechanism for HD at the protein level.

Amino Acid Sequence↗
PubMed1995

Expression of the Huntington's disease (IT15) protein product in HD patients.

Huntington's disease (HD) is an inherited, neurodegenerative disorder caused by expansion of a CAG repeat in the IT15 gene, leading to an expanded glutamine repeat in the HD protein. The mechanism by which the expanded repeat causes expression of the disease is not known, though there do not appear to be changes in the mRNA levels. We have conducted quantitative Western blot analyses of HD patients and controls. Expression of the IT15 protein is essentially equal in control and HD frontal cortex. In caudate from HD patients, IT15 protein is decreased in parallel with the decrease in a neuronal marker, suggesting that loss of IT15 protein is secondary to neuronal loss. In order to determine expression of the two alleles of the IT15 protein we used Western blots of 4% polyacrylamide gels. Both alleles of the IT15 protein were expressed at similar levels in HD lymphoblastoid cell lines and HD post-mortem hippocampus and cerebellum (regions relatively spared in HD), indicating that even very long CAG repeats can be translated into polyglutamine. In contrast, in cerebral cortex and caudate (regions severely affected in HD), in the longer expanded repeat cases the expanded allele of the IT15 protein was present at a significantly lower level (compared with the normal length allele), often with a smear of more slowly migrating reactivity above it. These data suggest the possibility of altered structure, abnormal processing or abnormality of protein-protein interactions involving the IT15 protein with the expanded glutamine repeat.

Aged↗
PubMed1994

Epithelial inositol 1,4,5-trisphosphate receptors. Multiplicity of localization, solubility, and isoforms.

In an earlier subcellular fractionation study of epithelial tissue (liver and pancreas), we demonstrated that the inositol 1,4,5-trisphosphate receptor (IP3R) is found in association with biochemically distinct cellular membranes, including the endoplasmic reticulum (ER) and plasma membrane (Sharp, A. H., Snyder, S. H., and Nigam, S. K. (1992) J. Biol. Chem. 267, 7444-7449). To further characterize epithelial IP3Rs, we have now employed cultured Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell type. Indirect immunofluorescence with an antiserum which specifically recognizes IP3R in MDCK cells by immunoblotting and immunoprecipitation gave an ER-like staining pattern as well as a basolateral plasma membrane-like staining pattern, the latter being particularly evident in highly confluent monolayers. In sections of adult rat kidney tubules a similar staining pattern was observed. Interestingly, whereas known basolateral proteins (Na+,K(+)-ATPase and the facilitated glucose transporter) gave a continuous basolateral staining pattern, that seen for IP3R was discontinuous (punctate). A highly similar staining pattern was observed for the caveolar protein, caveolin, suggesting that the punctate basolateral plasma membrane-like staining pattern observed for IP3R reflects its localization to basolateral caveolae. Biotinylation of non-permeabilized and permeabilized MDCK cells, followed by immunoprecipitation of IP3R and detection with streptavidin, indicated that while most IP3R is localized to biotin-inaccessible compartments (i.e. ER), a fraction (10-20%) of IP3R was accessible to externally added biotin primarily from the basolateral side. This result is compatible with the dual ER and basolateral caveolar localization suggested by immunocytochemistry, although it does not exclude the presence of some IP3R in the basolateral plasma membrane as well. Solubility studies revealed IP3R to be considerably more insoluble than the basolateral proteins, Na+,K(+)-ATPase and the liver cell adhesion molecule, as well as the cytoskeletal proteins, ankyrin and fodrin. In the most insoluble fraction, IP3R was found along with caveolin, further supporting the notion that part of the cellular IP3R pool associates with caveolae. Since multiple localizations of IP3R within a cell might reflect the existence of multiple isoforms, polymerase chain reaction amplification of first strand cDNA with primers specific for the three isotypes of IP3R was performed. All three isoforms of IP3R were expressed in the homogeneous population of MDCK cells. The existence of distinct membrane localizations and multiple isoforms of IP3R within the same cell type suggests an explanation for the complex spatiotemporal patterns of Ca2+ release from inositol 1,4,5-trisphosphate-sensitive Ca2+ pools in epithelial and other cells.

Animals↗
PubMed1994

Localization of inositol trisphosphate receptor subtype 3 to insulin and somatostatin secretory granules and regulation of expression in islets and insulinoma cells.

Calcium ions play a central role in stimulus-secretion coupling in pancreatic beta cells, and an elevation of cytosolic Ca2+ levels is necessary for insulin secretion. Inositol 1,4,5-trisphosphate mobilizes intracellular Ca2+ stores in the beta cell by binding to specific receptors that are ligand-activated Ca2+ channels. The inositol trisphosphate receptors comprise a family of structurally related proteins with distinct but overlapping tissue distributions. Previous studies indicated that the predominant inositol trisphosphate receptor subtype expressed in rat pancreatic islets was the protein designated IP3R-3. We have confirmed the expression of IP3R-3 in pancreatic islets by immunohistocytochemistry and localized this protein to the secretory granules of insulin-secreting beta cells and somatostatin-secreting delta cells by immunogold electron microscopy. Secretory granules contain high levels of Ca2+, and the presence of IP3R-3 in the granule provides a mechanism for mobilizing granule Ca2+ stores in response to glucose and/or hormones. The release of Ca2+ from granule stores would increase the Ca2+ concentration in the surrounding cytoplasm and promote rapid exocytosis of granules, especially those granules in close proximity to the plasma membrane. The levels of IP3R-3 were increased in pancreatic islets of diabetic rats and rats that had been refed after a period of fasting. They were also increased in rat insulinoma RINm5F cells cultured in 25 mM glucose compared with cells cultured in 5 mM glucose. The localization of IP3R-3 to secretory granules of insulin-secreting beta cells and somatostatin-secreting delta cells suggests that granule Ca2+ stores actively participate in the secretory process and that their release is regulated by inositol 1,4,5-trisphosphate. The regulation of IP3R-3 levels by glucose, diabetes, and refeeding may allow the beta cell to adjust the insulin secretory response to changing physiological conditions.

Amino Acid Sequence↗
PubMed1993

Neuronal inositol 1,4,5-trisphosphate receptor localized to the plasma membrane of olfactory cilia.

Both the cyclic adenosine 3',5'-monophosphate and the phosphoinositide second messenger systems are involved in olfactory signal transduction. The inositol 1,4,5-trisphosphate receptor is one of the principal intracellular calcium channels responsible for mobilizing stored calcium. The precise location of the 1,4,5-trisphosphate receptor (endoplasmic reticulum vs surface) and its role in the events of olfactory signal transduction need to be defined. By light microscopic and confocal immunohistochemistry we show expression of the olfactory-enriched G-protein, G(olf), associated with cyclic AMP responses, and of the inositol 1,4,5-trisphosphate receptor in the dendritic projections and cilia of essentially all olfactory receptor neurons, indicating that individual neurons express components of both second messenger systems. By immunoelectron microscopy, we demonstrate that the inositol 1,4,5-trisphosphate receptor is localized to the surface membrane of rat olfactory cilia. This is the first morphological demonstration of the surface membrane localization of inositol 1,4,5-trisphosphate receptor in olfactory cilia. Our findings, taken in conjunction with electrophysiological data from other workers, are supportive of the inositol 1,4,5-trisphosphate receptor playing a novel role in regulating calcium flux at the ciliary surface membrane.

Animals↗
PubMed1993

Inositol 1,4,5-trisphosphate receptors: immunohistochemical localization to discrete areas of rat central nervous system.

The second messenger inositol 1,4,5-trisphosphate triggers the release of intracellular Ca2+ stores upon binding to the inositol 1,4,5-trisphosphate receptor protein, a calcium channel that has been purified and molecularly cloned. To clarify the roles of inositol 1,4,5-trisphosphate receptor in the central nervous system, we have examined in detail the distribution of inositol 1,4,5-trisphosphate receptors in the rat brain and spinal cord using immunohistochemical methods. Inositol 1,4,5-trisphosphate receptors are present in neuronal cells, fibers and terminals in a wide distribution of areas throughout the central nervous system. These include a number of areas not previously reported, such as the olfactory bulb, thalamic nuclei and dorsal horn of the spinal cord. In addition, we have noted a strikingly high density of inositol 1,4,5-trisphosphate receptors in circumventricular organs and neuroendocrine structures such as the area postrema, choroid plexus, subcommisural organ, pineal gland and pituitary. The distribution of inositol 1,4,5-trisphosphate receptors in discrete structures throughout the central nervous system, including interconnected neuronal systems and neuroendocrine and circumventricular organ structures, presumably reflects the importance of Ca2+ release mediated by the phosphoinositide second messenger system in control of diverse physiological processes.

Animals↗