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A J Baines

Publications and source records attributed to A J Baines.

At least 19 recordsLinked to original sources
PubMed2001

Protein 4.1 in forebrain postsynaptic density preparations: enrichment of 4.1 gene products and detection of 4.1R binding proteins.

4.1 Proteins are a family of multifunctional cytoskeletal components (4.1R, 4.1G, 4.1N and 4.1B) derived from four related genes, each of which is expressed in the nervous system. Using subcellular fractionation, we have investigated the possibility that 4.1 proteins are components of forebrain postsynaptic densities, cellular compartments enriched in spectrin and actin, whose interaction is regulated by 4.1R. Antibodies to each of 4.1R, 4.1G, 4.1N and 4.1B recognize polypeptides in postsynaptic density preparations. Of these, an 80-kDa 4.1R polypeptide is enriched 11-fold in postsynaptic density preparations relative to brain homogenate. Polypeptides of 150 and 125 kDa represent 4.1B; of these, only the 125 kDa species is enriched (threefold). Antibodies to 4.1N recognize polypeptides of approximately 115, 100, 90 and 65 kDa, each enriched in postsynaptic density preparations relative to brain homogenate. Minor 225 and 200 kDa polypeptides are recognized selectively by specific anti-4.1G antibodies; the 200 kDa species is enriched 2.5-fold. These data indicate that specific isoforms of all four 4.1 proteins are components of postsynaptic densities. Blot overlay analyses indicate that, in addition to spectrin and actin, postsynaptic density polypeptides of 140, 115, 72 and 66 kDa are likely to be 4.1R-interactive. Of these, 72 kDa and 66 kDa polypeptides were identified as neurofilament L and alpha-internexin, respectively. A complex containing 80 kDa 4.1R, alpha-internexin and neurofilament L was immunoprecipitated with anti-4.1R antibodies from brain extract. We conclude that 4.1R interacts with the characteristic intermediate filament proteins of postsynaptic densities, and that the 4.1 proteins have the potential to mediate the interactions of diverse components of postsynaptic densities.

Actins↗
PubMed2001

Properties of the C-terminal domain of 4.1 proteins.

At the C-terminus of all known 4.1 proteins is a sequence domain unique to these proteins, known as the C-terminal domain (CTD). Mammalian CTDs are associated with a growing number of protein-protein interactions, although such activities have yet to be associated with invertebrate CTDs. Mammalian CTDs are generally defined by sequence alignment as encoded by exons 18-21. Comparison of known vertebrate 4.1 proteins with invertebrate (Caenorhabditis elegans and Drosophila melanogaster) 4.1 proteins indicates that mammalian 4.1 exon 19 represents a vertebrate adaptation that extends the sequence of the CTD with a Ser/Thr-rich sequence. The CTD was first described as a 22/24-kDa domain by chymotryptic digestion of erythrocyte 4.1 (4.1R) [Leto, T.L. & Marchesi, V.T. (1984) J. Biol. Chem. 259, 4603-4608]. Here we show that in 4.1R the 22/24-kDa fragment is not stable but rapidly processed to a 15-kDa fragment by chymotrypsin. The 15-kDa fragment is extremely stable, being resistant to overnight digestion in chymotrypsin on ice. Analysis of this fragment indicates that it is derived from residues 709-858 (SwissProt accession no. P48193), and represents the CTD of 4.1R. The fragment behaves as a globular monomer in solution. Secondary-structure predictions indicate that this domain is composed of five or six beta strands with an alpha helix before the most C-terminal of these. Together these data indicate that the CTD probably represents an independent folding structure which has gained function since the divergence of vertebrates from invertebrates.

Amino Acid Sequence↗
PubMed2001

Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues.

The spectrin-based membrane skeleton of the humble mammalian erythrocyte has provided biologists with a set of interacting proteins with diverse roles in organization and survival of cells in metazoan organisms. This review deals with the molecular physiology of spectrin, ankyrin, which links spectrin to the anion exchanger, and two spectrin-associated proteins that promote spectrin interactions with actin: adducin and protein 4.1. The lack of essential functions for these proteins in generic cells grown in culture and the absence of their genes in the yeast genome have, until recently, limited advances in understanding their roles outside of erythrocytes. However, completion of the genomes of simple metazoans and application of homologous recombination in mice now are providing the first glimpses of the full scope of physiological roles for spectrin, ankyrin, and their associated proteins. These functions now include targeting of ion channels and cell adhesion molecules to specialized compartments within the plasma membrane and endoplasmic reticulum of striated muscle and the nervous system, mechanical stabilization at the tissue level based on transcellular protein assemblies, participation in epithelial morphogenesis, and orientation of mitotic spindles in asymmetric cell divisions. These studies, in addition to stretching the erythrocyte paradigm beyond recognition, also are revealing novel cellular pathways essential for metazoan life. Examples are ankyrin-dependent targeting of proteins to excitable membrane domains in the plasma membrane and the Ca(2+) homeostasis compartment of the endoplasmic reticulum. Exciting questions for the future relate to the molecular basis for these pathways and their roles in a clinical context, either as the basis for disease or more positively as therapeutic targets.

Amino Acid Sequence↗
PubMed2001

Synapsin I is expressed in epithelial cells: localization to a unique trans-Golgi compartment.

Synapsin I is abundant in neural tissues. Its phosphorylation is thought to regulate synaptic vesicle exocytosis in the pre-synaptic terminal by mediating vesicle tethering to the cytoskeleton. Using anti-synapsin antibodies, we detected an 85 kDa protein in liver cells and identified it as synapsin I. Like brain synapsin I, non-neuronal synapsin I is phosphorylated in vitro by protein kinase A and yields identical (32)P-peptide maps after limited proteolysis. We also detected synapsin I mRNA in liver by northern blot analysis. These results indicate that the expression of synapsin I is more widespread than previously thought. Immunofluorescence analysis of several non-neuronal cell lines localizes synapsin I to a vesicular compartment adjacent to trans-elements of the Golgi complex, which is also labeled with antibodies against myosin II; no sub-plasma membrane synapsin I is evident. We conclude that synapsin I is present in epithelial cells and is associated with a trans-Golgi network-derived compartment; this localization suggests that it plays a role in modulating post-TGN trafficking pathways.

Animals↗
PubMed2001

The postsynaptic spectrin/4.1 membrane protein "accumulation machine".

An important aspect of the function of the membrane-associated cytoskeleton has been suggested to be to trap and retain selected transmembrane proteins at points on the cell surface specified by cell adhesion molecules. In the process, cell adhesion molecules are cross-linked to each other, and so junctional complexes are strengthened. In this short review, we will discuss recent advances in understanding the role of this "accumulation machine" in postsynaptic structures. Function in the brain depends on correct ordering of synaptic intercellular junctions, and in particular the recruitment of receptors and other apparatus of the signalling system to postsynaptic membranes. Spectrin has long been known to be a component of postsynaptic densities, and recent advances in molecular cloning indicate that beta spectrins at PSDs are all "long" C-terminal isoforms characterised by pleckstrin homology domains. Isoforms of protein 4.1 are also present at synapses. All four 4.1 proteins are represented in PSD preparations, but it is 4.1R that is most enriched in PSDs. 4.1R binds to several proteins enriched in PSDs, including the characteristic PSD intermediate filament, alpha-internexin. Both 4.1 and spectrin interact with ionotropic glutamate receptors (AMPA and NMDA receptors, respectively): 4.1 stabilises AMPA receptors on the cell surface. By linking these receptors to the cytoskeletal and cell adhesion molecules that specify glutamatergic synapses, the membrane protein accumulation machine is suggested to direct the formation of postsynaptic signalling complexes.

Animals↗
PubMed2000

Identification of a novel C-terminal variant of beta II spectrin: two isoforms of beta II spectrin have distinct intracellular locations and activities.

It is established that variations in the structure and activities of betaI spectrin are mediated by differential mRNA splicing. The two betaI spectrin splice forms so far identified have either long or short C-terminal regions. Are analogous mechanisms likely to mediate regulation of betaII spectrins? Thus far, only a long form of betaII spectrin is reported in the literature. Five human expressed sequence tags indicated the existence of a short splice variant of betaII spectrin. The occurrence and DNA sequence of the short C-terminal variant was confirmed by analysis of human and rat cDNA. The novel variant lacks a pleckstrin homology domain, and has 28 C-terminal residues not present in the previously recognized longer form. Transcripts of the short C-terminal variant (7.5 and 7. 0 kb) were most abundant in tissues originating from muscle and nervous system. Antibodies raised to a unique sequence of short C-terminal variant recognized 240 kDa polypeptides in cardiac and skeletal muscle and in nervous tissue; in cerebellum and forebrain, additional 270 kDa polypeptides were detected. In rat heart and skeletal muscle, both long and short C-terminal forms of betaII spectrin localized in the region of the Z line. The central region of the sarcomere, coincident with the M line, was selectively labeled with antibodies to the short C-terminal form. In cerebellum, the short form was not detectable in parallel fibers, structures in which the long form was readily detected. In cultured cerebellar granule neurons, the long form was dominant in neurites, with the short form being most abundant in cell bodies. In vitro, the short form was found to lack the binding activity for the axonal protein fodaxin, which characterizes the C-terminal region of the long form. Subcellular fractionation of brain revealed that the short form was scarcely detectable in post-synaptic density preparations, in which the long form was readily detected. We conclude that variation in the structure of the C-terminal regions of betaII spectrin isoforms correlates with their differential intracellular targeting.

Alternative Splicing↗
PubMed1999

Constraints on the transport and glycosylation of recombinant IFN-gamma in Chinese hamster ovary and insect cells.

In this study we compare intracellular transport and processing of a recombinant glycoprotein in mammalian and insect cells. Detailed analysis of the N-glycosylation of recombinant human IFN-gamma by matrix-assisted laser-desorption mass spectrometry showed that the protein secreted by Chinese hamster ovary and baculovirus-infected insect Sf9 cells was associated with complex sialylated or truncated tri-mannosyl core glycans, respectively. However, the intracellular proteins were predominantly associated with high-mannose type oligosaccharides (Man-6 to Man-9) in both cases, indicating that endoplasmic reticulum to cis-Golgi transport is a predominant rate-limiting step in both expression systems. In CHO cells, although there was a minor intracellular subpopulation of sialylated IFN-gamma glycoforms identical to the secreted product (therefore associated with late-Golgi compartments or secretory vesicles), no other intermediates were evident. Therefore, anterograde transport processes in the Golgi stack do not limit secretion. In Sf9 insect cells, there was no direct evidence of post-ER glycan-processing events other than core fucosylation and de-mannosylation, both of which were glycosylation site-specific. To investigate the influence of nucleotide-sugar availability on cell-specific glycosylation, the cellular content of nucleotide-sugar substrates in both mammalian and insect cells was quantitatively determined by anion-exchange HPLC. In both host cell types, UDP-hexose and UDP-N-acetylhexosamine were in greater abundance relative to other substrates. However, unlike CHO cells, sialyltransferase activity and CMP-NeuAc substrate were not present in uninfected or baculovirus-infected Sf9 cells. Similar data were obtained for other insect cell hosts, Sf21 and Ea4. We conclude that although the limitations on intracellular transport and secretion of recombinant proteins in mammalian and insect cells are similar, N-glycan processing in Sf insect cells is limited, and that genetic modification of N-glycan processing in these insect cell lines will be constrained by substrate availability to terminal galactosylation.

Animals↗
PubMed1997

Ca2+-dependent interaction with calmodulin is conserved in the synapsin family: identification of a high-affinity site.

The synapsins are a family of proteins associated with small synaptic vesicles that are implicated in synaptic maintenance and in the supply of vesicles for exocytosis. They are well characterized as substrates for protein kinases, and one class of synapsin, synapsin I, has been shown to bind, and be regulated by, calmodulin. A representative of the synapsin II class is now shown to bind calmodulin. Optical biosensor assays of Ca2+-dependent calmodulin binding to recombinant rat synapsin IIb indicated an apparent KD for calmodulin of 31 +/- 5 nM. Phosphorylation at Ser 10 increased the rates of calmodulin association (by a factor of 10) and dissociation (by a factor of 20). Fragment analysis and predictions from the sequence indicated two potential calmodulin binding sequences in the conserved central (C) domain. Peptides representing these sequences (residues 122-143 and 313-334 in synapsin IIb) were synthesized. Peptide 122-143 was found to bind calmodulin (KD 32 +/- 10 nM) and inhibit interaction of synapsin IIb with calmodulin. The interaction of peptide 313-334 was much weaker. Sequences similar to residues 122-143 are present in all published synapsin sequences. Calmodulin binding by synapsins seems not to be confined to mammals: a recombinant Drosophila synapsin 1 fragment containing part of the C-domain showed Ca2+-dependent binding to mammalian calmodulin. We conclude that calmodulin binding to synapsins is likely to be a general aspect of regulation of synaptic function.

Amine Oxidase (Copper-Containing)↗
PubMed1997

Definition of a sequence unique in beta II spectrin required for its axon-specific interaction with fodaxin (A60).

Spectrin isotypes segregate in neurons and are differentially distributed between axons and somatodendritic compartments. Their functions in those compartments are likely to be mediated by proteins that interact selectively with one or other isotype. Fodaxin (an axon-specific protein previously termed A60) colocalizes in CNS neurons with axonal spectrin and in vitro binds brain spectrin (a mixture of alpha I, beta I, and beta II polypeptides) but not erythrocyte spectrin (alpha I and beta I). Because alpha II and beta II spectrin polypeptides are enriched in axons, we investigated a possible binding of fodaxin to the types of spectrin found in axons. Fodaxin did not bind to isolated brain alpha chains. Bacterially expressed C-terminal segments 18-19 of beta II spectrin bound to fodaxin and inhibited the binding of fodaxin to whole brain spectrin. By contrast, recombinant segments 18-19 of the somatodendritic beta I sigma 2 spectrin showed no interaction with fodaxin. Within beta II, fodaxin binding activity was localized to residues 2,087-2,198, which are unique to beta II and link between the end of segment 18 and the pleckstrin homology domain in segment 19. The divergent regions of sequence in segments 19 of beta II and beta I sigma 2 are candidates to mediate the isotype-specific functions of spectrin. Fodaxin is the first protein to be described that discriminates between the unique regions of beta spectrin isoforms.

Animals↗
PubMed1995

Coordinated regulation of synapsin I interaction with F-actin by Ca2+/calmodulin and phosphorylation: inhibition of actin binding and bundling.

The synapsins are a family of synaptic vesicle phosphoproteins whose role seems to be to limit the availability of small synaptic vesicles for exocytosis by linking them to the cytoskeleton. One member of the family, synapsin I, has been shown to bind calmodulin in a Ca(2+)-dependent manner. In this study, we have examined whether or not calmodulin can regulate one of the activities of synapsin I, namely, its interaction with F-actin. Synapsin I is an actin bundling protein: this activity is controlled by phosphorylation. Here we show that calmodulin in the presence of Ca2+ is a competitive inhibitor of both actin binding and bundling by synapsin I. Under the conditions of our assay (0.45 microM synapsin I, 4 microM F-actin), half-maximal inhibition of actin binding and bundling by unphosphorylated synapsin I was found with 4.3 and 3.7 microM calmodulin, respectively. The actin binding activity of synapsin I phosphorylated by cAMP-dependent protein kinase or by calmodulin-dependent protein kinase II showed similar sensitivity to calmodulin inhibition to unphosphorylated synapsin I. However, inhibition of bundling was potentiated. Half-maximal inhibition of bundling by synapsin I phosphorylated by cAMP-dependent kinase was achieved at approximately 0.5 microM calmodulin. Half-maximal inhibition of bundling by synapsin I phosphorylated by calmodulin-dependent protein kinase II was achieved at less than 0.2 microM calmodulin, although the maximum binding under the conditions of the assay was lower.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1995

A60, an axonal membrane-skeletal spectrin-binding protein.

A60 is a 60 kDa protein which is associated with the axonal cortical cytoskeleton in adult central nervous system neurons. It was originally defined by the use of a monoclonal antibody which showed that it was closely associated with the cytoplasic face of axonal plasma membranes. A60 appears to be neuron-specific and biochemical studies show that it is tightly bound to brain membranes. Affinity chromatography has revealed that A60 interacts with brain spectrin but not with erythrocyte spectrin. As erythrocyte spectrin is closely related to the isotype of spectrin that is localized in dendrites this raises the possibility that A60 is restricted to axons by interaction with the isotype of spectrin that is found in axons. During post-natal cerebellar development (days 1-13) A60 is initially located in the perikarya of precursor Purkinje cells and is then localized in the initial dendrites of these cells as well as in the white matter. In contrast, in the adult cerebellum the location of A60 is exclusively axonal. These data indicate that A60 has a spectrin-binding activity in the adult axonal membrane skeleton which is only required after initial axon growth has occurred. A60 is transiently expressed during embryonic and post-natal development of rat dorsal root ganglia (DRG). It is located in the large light DRG cells but is essentially absent from the small dark DRG cells.

Aging↗