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PubMed1993

A truncated human insulin receptor missing the COOH-terminal 365 amino acid residues does not undergo insulin-mediated receptor migration or aggregation.

A previous study of tyrosine kinase-defective insulin receptors demonstrated that receptor autophosphorylation or tyrosine kinase activity was required for concentrating insulin receptors in coated pits, but not for their migration or aggregation on the cell surface. Furthermore, receptor migration and aggregation on the cell surface were not sufficient to cause internalization of the occupied receptors in coated pits. In the present study, biochemical and ultrastructural techniques were used to compare insulin receptor mobility and internalization in Rat 1 fibroblasts expressing wild-type human insulin receptors (HIRc) with those in cells expressing receptors truncated at residues 978 (HIR delta 978) or 1301 of the carboxyl-terminus (HIR delta CT). There were no significant differences in the mobility or internalization of insulin receptors on HIR delta CT cells compared to those of insulin receptors on HIRc cells. Ultrastructural analysis revealed that truncated insulin receptors on HIR delta 978 cells failed to migrate from their initial location on the microvilli, move to the plasma membrane, and aggregate in coated pits. Receptor-mediated insulin internalization in HIR delta 978 cells was markedly decreased due entirely to a decrease in ATP-dependent, coated pit-mediated internalization. ATP-independent endocytosis in non-coated pinocytotic invaginations was not affected by receptor truncations. These results provide evidence of the roles that regions of the beta-subunit play in the processing of occupied insulin receptors. 1) The carboxyl-terminus of the insulin receptor is not involved in the events leading to receptor internalization, i.e. migration, aggregation, and concentration in coated pits. 2) Internalization of insulin receptors by the ATP-independent noncoated invagination pathway is not regulated by residues in the insulin receptor beta-subunit distal to 978. 3) Sequences in the beta-subunit between 978-1300, but not the autophosphorylation and kinase domains, are involved in insulin-induced receptor migration and aggregation.

Amino Acid Sequence↗

PubMed1999

[Thrombocyte receptors: current views and therapeutic options].

In the action of thrombocytes during stemming of a bleeding after damage to a blood vessel, receptors on the thrombocyte membrane play an important part. Adhesion of platelets takes place via specific binding of receptors; the main binding is that of glycoprotein (Gp) Ib to Von Willebrand factor which is synthetized by endothelial cells. Activation of thrombocytes is stimulated by adhesion and by agonists. Weak agonists, through production of thromboxane A2 and release of agonists from granules cause a self-fortifying process of thrombocyte stimulation; strong agonists (like thrombin) lead also to activation of Gp IIb/IIIa receptors. Aggregation of thrombocytes occurs after activation of Gp IIb/IIIa receptors. During stimulation, a change of shape occurs which enables binding to suitable plasma proteins of which the main one is fibrinogen. Knowledge of thrombocyte receptors enhances the insight into the prognosis and efficacy of certain treatments in diseases in which platelet aggregation is pivotal. Of the six categories of antiplatelet drugs, antagonists of Gp IIb/IIIa receptors are the most potent. In clinical trials good results have been obtained in patients with coronary disease of the intravenously administered form added to acetylsalicylic acid.

Blood Platelets↗

PubMed1986

The effects of N-ethylmaleimide on the phosphorylation and aggregation of insulin receptors in the isolated plasma membranes of 3T3-F442A adipocytes.

We have examined the insulin-dependent phosphorylation of the insulin receptor in the isolated plasma membranes of 3T3-F442A adipocytes. Phosphorylation of the insulin receptor is detected readily in the plasma membrane of these cells by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of sodium dodecyl sulfate and under nonreducing conditions, the predominant species of phosphorylated insulin receptor has an apparent molecular mass of 350,000 daltons with the beta-subunit (92,000 daltons) being phosphorylated. The phosphorylation of the insulin receptor in the isolated plasma membrane is absolutely dependent on insulin; tyrosine residues and, to a lesser extent, serine residues of the receptor are phosphorylated. Treatment of the plasma membrane with N-ethylmaleimide (NEM) has two effects; 1) NEM prevents the formation of a larger form (greater than 350,000 daltons) of the phosphorylated insulin receptor. The formation of this larger form of the receptor involves sulfhydryl oxidation which occurs at 37 degrees C under nondenaturing conditions, but does not occur at 0 degrees C or at room temperature even in the presence of sodium dodecyl sulfate. These results indicate that the larger form of the phosphorylated receptor can occur under physiological conditions and suggest that this process may be relevant to aggregation of the receptor-ligand complex. 2) Prior to treatment with insulin, NEM enhances the phosphorylation of the insulin receptor. Phosphopeptide analysis indicates that the site(s) of phosphorylation of the receptor is identical in the presence or absence of NEM.

Adipose Tissue↗

PubMed2006

The adhesion between a microvillus-bearing cell and a ligand-coated substrate: a Monte Carlo study.

In biology, specific cell adhesion is mediated by receptor-ligand interactions. Consequently, its strength correlates with the strength of single receptor-ligand bonds that can be measured with a variety of techniques. However, whether single receptor-ligand bonds are truly present in an experiment is often a concern. In this paper, we present a Monte Carlo simulation of the adhesion between a microvillus-bearing cell and a ligand-coated substrate. In the simulation, ligands were immobilized on the substrate either uniformly or in clusters of three and seven, while receptors were distributed uniformly on the microvillus tip and they moved randomly on the cellular surface. How ligand clustering affects the adhesion frequency and forward rate constant was studied. Other factors that were studied include receptor aggregation on the microvillus tip, ligand density, receptor density, contact time, and binding pocket size. In the case of uniformly distributed ligands, our simulation results agree well with those obtained from probabilistic analysis. We found that, even with clustered ligands on the substrate, most of the adhesion events were mediated by a single bond if the total adhesion frequency was less than 20%. Besides, ligand clustering decreased the total adhesion frequency and forward rate constant, but increased the single-bond adhesion frequency under comparable conditions. These findings should lend us some assistance in identifying single bonds in cell-substrate or cell-bead adhesion measurements and in illustrating some biological mechanisms that involve clustered ligands.

Cell Adhesion↗

PubMed1980

Progesterone-binding components of chick oviduct: analysis of receptor structure by limited proteolysis.

An endogenous calcium-activated sulfhydryl protease in chick oviduct cytosol has been utilized to study the structure of the chick oviduct progesterone receptor subunits, progestophilins A (79 000 g/mol) and B (117 000 g/mol). The protease is not a normal component of the native progesterone receptor aggregate (6 and 8 S) complexes. Both receptor protein subunits (A and B) can be cleaved to two hormone-binding fragments, form IV (43 000 g/mol) and meroreceptor (23 000 g/mol). The meroreceptors obtained from the A and B proteins are indistinguishable from each other on the basis of both size (gel filtration chromatography) and charge (isoelectric focusing, pI 8.3). These findings suggest a structural similarity between the A and B proteins. The discovery of a weak deoxyribonucleic acid (DNA) binding activity for the B protein suggests an even greater similarity between B and A subunits, since the A subunit has previously b:en shown to bind to DNA. The proteolytic fragments do not bind to DNA-cellulose, implying that the hormone- and DNA-binding regions of the A and B proteins exist in separate domains.

Animals↗

PubMed1981

Xenograft rejection in marine sponges. Isolation and purification of an inhibitory aggregation factor from Geodia cydonium.

In sponges there exists a graft rejection mechanism in which an inhibitory aggregation factor is involved. The inhibitory aggregation factor has been isolated from a culture medium containing dissociated cells of the sponge Geodia cydonium. Using ion-exchange and gel fractionation the factor was purified and shown to be electrophoretically pure. The factor has a molecular weight of 27000 and was characterized as a glycoprotein. The activity of the inhibitory aggregation factor was not affected by heat treatment, but treatment with trichloroacetic acid resulted in the irreversible loss of activity. The inhibitory aggregation factor affects the aggregation-factor-mediated reaggregation of dissociated sponge cells by extension of the lag phase preceding the aggregation process; the endpoint of the reaggregation process is not changed. The inhibitory aggregation factor competes with the aggregation receptor for the binding site on the aggregation factor. The inhibitory aggregation factor is not synthesized if homologous aggregation factor is added to the dissociated cells. The results indicate that the inhibitory aggregation factor is synthesized in xenografts of the system Geodia cydonium--Geodia rovinjensis after a grafting period of at least 3 days. Following the synthesis of the factor, the concentration of the extractable aggregation factor decreases.

Animals↗

PubMed2006

Physiological and pathophysiological aspects of ceramide.

Activation of cells by receptor- and nonreceptor-mediated stimuli not only requires a change in the activity of signaling proteins but also requires a reorganization of the topology of the signalosom in the cell. The cell membrane contains distinct domains, rafts that serve the spatial organization of signaling molecules in the cell. Many receptors or stress stimuli transform rafts by the generation of ceramide. These stimuli activate the acid sphingomyelinase and induce a translocation of this enzyme onto the extracellular leaflet of the cell membrane. Surface acid sphingomyelinase generates ceramide that serves to fuse small rafts and to form large ceramide-enriched membrane platforms. These platforms cluster receptor molecules, recruit intracellular signaling molecules to aggregated receptors, and seem to exclude inhibitory signaling factors. Thus ceramide-enriched membrane platforms do not seem to be part of a specific signaling pathway but may facilitate and amplify the specific signaling elicited by the cognate stimulus. This general function may enable these membrane domains to be critically involved in the induction of apoptosis by death receptors and stress stimuli, bacterial and viral infections of mammalian cells, and the regulation of cardiovascular functions.

Animals↗

PubMed1993

A structural and kinetic comparison of proto-oncogenic and oncogenic neu holo-receptors expressed in insect cells.

The proto-oncogenic and oncogenic forms of the rat neu receptors were expressed in the baculovirus system to characterize their structural and enzymatic differences. The epitopes of their extracellular domains, their molecular weights, and kinase activities were similar to rat neu receptors expressed in fibroblasts. The receptors were partially purified using a phospho-agarose column and were analyzed to compare kinetic parameters using ATP as a substrate. The oncogenic form of the receptor showed a significant increase in Vmax (56%) over the proto-oncogenic form. Structural analysis of these proteins using sucrose gradients showed the oncogenic receptors to have a 62.6% increase in aggregated receptors when compared to the proto-oncogenic receptors. These studies are the first to link enzymatic activation and the physical form of the receptor using isolated receptor species.

Animals↗

PubMed2002

Synaptically targeted narp plays an essential role in the aggregation of AMPA receptors at excitatory synapses in cultured spinal neurons.

Neuronal activity regulated pentraxin (Narp) has been implicated in the aggregation of AMPA-type glutamate receptors (GluR) at excitatory synapses. In the present paper, we examine the role of endogenous Narp in excitatory synapse formation by using novel, dominant-negative Narp mutants (dnNarp) that selectively bind endogenous Narp and prevent its accumulation at synapses. Axons from neurons transfected with wild-type Narp showed an increase in their ability to cluster AMPA receptors on spinal neurons, whereas axons from neurons transfected with dnNarp showed a marked decrease in their ability to induce GluR1 clusters on contacted dendrites. Despite their marked effect at excitatory synapses, dnNarp and wild-type Narp had no effect on the postsynaptic clustering of the inhibitory protein gephyrin or the percentage of contacts associated with staining for the presynaptic vesicle proteins GAD or synaptophysin. Use of the dnNarp mutants to suppress endogenous Narp expression by postsynaptic dendrites showed a complementary role for dendritic Narp in the clustering of synaptic AMPA receptors, as well as a reduction in the total number of excitatory synapses on transfected neurons. Together these experiments suggest an important role for Narp in the formation of excitatory synapses in cultured spinal neurons.

Animals↗

PubMed2005

Neuromuscular contacts induce nitric oxide signals in skeletal myotubes in vitro.

It has previously been shown that skeletal myotubes express nitric oxide synthase (NOS) and produce and release NO signals. NOS is also part of agrin-induced acetylcholine receptor aggregations on myotubes. As nerve-muscle interactions underlie reciprocal signaling mechanisms, we hypothesized that NO signals in target myotubes may be induced by neuromuscular contacts in development. Chimeric neuron-myotube co-cultures were prepared using p75-selected spinal cord neurons from embryonic chicken. Confocal imaging revealed robust 1,2-diaminoanthraquinone red fluorescence indicative of de novo formation of NO only in those myotubes which were contacted by neurites, also verified by pre- and postsynaptic marker costaining (anti-synaptotagmin and alpha-bungarotoxin). Neither soluble agrin nor sensory dorsal root ganglionic neurons showed comparable effects in this model. We concluded that in target skeletal muscle cells the NOS/NO system is controlled by motoneuron contacts by as yet incompletely understood signaling mechanisms. Endogenous NO signaling in myotubes may be essential during synapse formation and plasticity of the neuromuscular system.

Animals↗

PubMed1994

Making agonists of antagonists.

Cell-surface receptors can be divided into three classes, depending on whether they transmit information by allosteric conformational change, by receptor dimerization, or by receptor aggregation. So far, only the first class of receptors has proven readily accessible to chemical techniques; this is no accident, but times are changing.

Animals↗

PubMed2000

Intracellular trafficking of GABA(A) receptors.

Some of the mechanisms that control the intracellular trafficking of GABA(A) receptors have recently been described. Following the synthesis of alpha, beta, and gamma subunits in the endoplasmic reticulum, ternary receptor complexes assemble slowly and are inefficiently inserted into surface membranes of heterologous cells. While beta3, beta4, and gamma2S subunits appear to contain polypeptide sequences that alone are sufficient for surface targeting, these sequences are neither conserved nor essential for surface expression of heteromeric GABA(A) receptors formed from alpha1beta or alpha1betagamma subunits. At the neuronal surface, native GABA(A) receptor clustering and synaptic targeting require a gamma2 subunit and the participation of gephyrin, a clustering protein for glycine receptors. A linker protein, such as the GABA(A) receptor associated protein (GABARAP), may be necessary for the formation of GABA(A) receptor aggregates containing gephyrin. A substantial fraction of surface receptors are sequestered by endocytosis, another process which apparently requires a GABA(A) receptor gamma2 subunit. In heterologous cells, constitutive endocytosis seems to predominate while, in cortical neurons, internalization is evoked when receptors are occupied by GABA(A) agonists. After constitutive endocytosis, receptors are relatively stable and can be rapidly recycled to the cell surface, a process that may be regulated by protein kinase C. On the other hand, a portion of the intracellular GABA(A) receptors derived from ligand-dependent endocytosis is apparently degraded. The clustering of GABA(A) receptors at synapses and at coated pits are two mechanisms that may compete for a pool of diffusable receptors, providing a model for plasticity at inhibitory synapses.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Formation of mixed glycine and GABAergic synapses in cultured spinal cord neurons.

In the spinal cord, GABA and glycine mediate inhibition at separate or mixed synapses containing glycine and/or GABA(A) receptors (GlyR and GABA(A)R, respectively). We have analysed here the sequence of events leading to inhibitory synapse formation during synaptogenesis of embryonic spinal cord neurons between 1 and 11 days in vitro (DIV). We used immunocytochemical methods to detect simultaneously an antigen specific to inhibitory terminals, the vesicular inhibitory amino acid transporter (VIAAT), and one of the following postsynaptic elements: GlyR, GABA(A)R or gephyrin, the anchoring protein of GlyR, which is also associated with GABA(A)R. Quantitative analysis revealed that until 5 DIV most gephyrin clusters were not adjacent to VIAAT-positive profiles, but became associated with them at later stages. In contrast, GlyR and GABAAR clustered predominantly in front of VIAAT-containing terminals at all stages. However, about 10% of receptor aggregates were detected at nonsynaptic loci. The two receptors colocalized in 66.2+/-2.5% of the inhibitory postsynaptic domains after 11 DIV, while 30.3+/-2.6% and 3.4+/-0.8% of them contained only GlyR and GABA(A)R, respectively. Interestingly, at 3 DIV GABA(A)R clustered at a postsynaptic location prior to gephyrin and GlyR; GABA(A)R could thus be the initiating element in the construction of mixed glycine and GABAergic synapses. The late colocalization of gephyrin with GABA(A)R, and the demonstration by other groups that, in the absence of gephyrin, postsynaptic GABA(A)R is not detected, suggest that gephyrin is involved in the stabilization of GABA(A)R rather than in its initial accumulation at synaptic sites.

Amino Acid Transport Systems↗

PubMed1988

Metabolic stability and antigenic modulation of nicotinic acetylcholine receptors on bovine adrenal chromaffin cells.

Bovine adrenal chromaffin cells have nicotinic acetylcholine receptors (AChRs) that are activated by the splanchnic nerve, resulting in release of catecholamines from the cells. Examination of the AChRs can provide information about the regulation and turnover of synaptic components on neurons and endocrine cells. Previous studies have shown that mAb 35 recognizes the AChR on the cells. Here we show that mAb 35 can remove AChRs from the surface of the cells by antigenic modulation, and that the modulation can be used together with other methods to examine the stability and turnover of the receptors in the plasma membrane. Unexpectedly, the results indicate a disparity between the rate at which AChRs reappear on the cells and the rate at which the ACh response recovers after preexisting AChRs have been removed. Exposure of bovine adrenal chromaffin cultures to mAb 35 results in a parallel decrease in the magnitude of the nicotinic response and the number of AChRs on the cells. The decrease depends on the concentration and divalence of mAb 35, and on the time and temperature of the incubation. The antibody induces receptor aggregation in the plasma membrane under conditions where receptor loss subsequently occurs. After binding to receptor, mAb 35 appears to be internalized, degraded, and released from the cells through a temperature sensitive pathway that requires lysosomal function. These features are characteristic of antigenic modulation. Appearance of new AChRs on the cells either after antigenic modulation or after blockade of existing AChRs with monovalent antibody fragments occurs at a rate equivalent to 3% of the receptors present on control cells per hour. The rate of receptor loss from the cells was measured in the presence of either tunicamycin or puromycin to block appearance of new receptors. Both conditions indicated a receptor half-life of approximately 24 h and a rate of loss of approximately 3%/h. The finding that the rate of receptor loss equaled the rate of receptor appearance was consistent with the observation that the total number of AChRs on untreated cells did not increase with time. In the presence of tunicamycin, loss of receptor-mediated response to nicotine also occurred with a half-time of 24 h. Paradoxically, the rate of recovery of the nicotinic response, determined using two procedures, was more than twice as great as the rate at which new AChRs appeared on the cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Glands↗

PubMed1998

Distinct regulation of T-cell death by CD28 depending on both its aggregation and T-cell receptor triggering: a role for Fas-FasL.

CD28 is a major coreceptor that regulates cell proliferation, anergy, and viability of T cells. The negative selection by T-cell receptor (TCR)-induced cell death of immature thymocytes as well as of activated human antigen-specific T-cell clone, requires a costimulatory signal that can be provided by CD28. Conversely, CD28-mediated signals increase expression of Bcl-XL, a survival gene, and promote survival of naive T cells cultured in the absence of antigen or costimulation. Because CD28 appears to both protect from, or induce T-cell death, one important question is to define the activation and cellular parameters that dictate the differential role of CD28 in T-cell apoptosis. Here, we compared different CD28 ligands for their ability to regulate TCR-induced cell death of a murine T-cell hybridoma. In these cells, TCR triggering induced expression of Fas and FasL, and cell death was prevented by anti-Fas blocking monoclonal antibody (MoAb). When provided as a costimulus, both CD28 MoAb and the B7.1 and B7.2 counter receptors downregulated, yet did not completely abolish T-cell receptor-induced apoptosis. This CD28 cosignal resulted in both upregulation of Bcl-XL and prevention of FasL expression. In marked contrast, when given as a single signal, CD28 MoAb or B7.1 and B7.2 induced FasL expression and resulted in T-cell death by apoptosis, which was dependent on the level of CD28 ligation. Furthermore, triggering of CD28 upregulated FasL and induced a marked T-cell death of previously activated normal peripheral T cells. Our results identify Fas and FasL as crucial targets of CD28 in T-cell death regulation and show that within the same cell population, depending on its engagement as a single signal or as a costimulus together with the TCR, CD28 can either induce a dose-dependent death signal or protect from cell death, respectively. These data provide important insights into the role of CD28 in T-cell homeostasis and its possible implication in neoplastic disorders.

Animals↗

PubMed1979

The effects of alpha adrenergic agents on human platelet aggregation.

The effects of alpha adrenergic agonists and antagonists on human in vitro platelet aggregation were studied to characterize further the platelet alpha adrenergic receptor. Aggregation induced by ADP and U46619; a stable prostaglandin endoperoxide analog, was potentiated by alpha adrenergic agonists, an effect which was completely blocked by the alpha adrenergic antagonist phentolamine (1 X 10(-6) M) but not by prazosin (1 X 10(-6) M). The order of potency for the alpha adrenergic agonists in potentiating ADP-induced aggregation was clonidine greater than or equal to epinephrine greater than alpha-methylnorepinephrine greater than norepinephrine greater than phenylephrine greater than methoxamine. Epinephrine-induced platelet aggregation was blocked by phentolamine, yohimbine, dihydroergotamine, clonidine and lofexidine but not by phenoxybenzamine (1 X 10(-5) M). These findings suggest that: 1)clonidine and lofexidine are partial agonists and 2) that the alpha adrenergic receptor of the platelet is different from the classical postsynaptic alpha adrenergic receptor and more closely resembles presynaptic alpha adrenergic receptors.

Adenosine Diphosphate↗

PubMed1992

The mobile receptor hypothesis revisited: a mechanistic role for hormone receptor lateral mobility in signal transduction.

Recent application of the technique of fluorescence photobleaching recovery to direct measurement of the lateral mobility of plasma membrane-localized hormone receptors has shed new light on the role of receptor lateral mobility in signal transduction. Receptors for insulin and EGF have been known for some time to be largely immobile at physiological temperatures. This presumably relates to their signal transduction mechanism, which appears to require intermolecular autophosphorylation (receptor aggregation) for activation. In contrast, G-protein coupled receptors must interact with other membrane components to bring about signal transduction, and it is interesting in this regard that the adenylate cyclase (AC) activating vasopressin V2-receptor is highly laterally mobile at 37 degrees C. It has recently been possible to reversibly modulate the V2-receptor mobile fraction (f) to largely varying extents, and to demonstrate thereby a direct effect on the maximal rate of in vivo cAMP production at 37 degrees C in response to vasopressin. A direct correlation between f and maximal cAMP production indicates that f may be a key parameter in hormone signal transduction in vivo, especially at sub-KD (physiological) hormone concentrations, with mobile receptors being required to effect G-protein activation.

Adenylyl Cyclases↗

PubMed2005

Lipid rafts are involved in C95 (4,8) agrin fragment-induced acetylcholine receptor clustering.

During development of the neuromuscular junction, high densities of acetylcholine receptors accumulate beneath the overlying nerve terminal. A defining feature of mature synapses is the sharp demarcation of acetylcholine receptor density, which is approximately 1000-fold higher in the postsynaptic as compared with the contiguous extrasynaptic muscle membrane. These high densities of receptors accumulate by at least four mechanisms, re-distribution of existing surface receptors, local synthesis of new receptors, decreased turnover of synaptic receptors, and limitation of diffusion of sub-neural, aggregated receptors. The limitation of receptor diffusion within the membrane is likely in part due to the anchoring of acetylcholine receptor complexes to components of the cytoskeleton. Here we have tested the idea that lipid rafts--mobile, cholesterol enriched microdomains within the lipid bilayer--are another mechanism by which acetylcholine receptors are clustered in the postsynaptic apparatus. Using mouse C2C12 cells, a muscle cell line, we show that a carboxy terminal 95 amino acid fragment [C95 (4,8)] of the extracellular matrix molecule agrin that is essential for nerve-induced postsynaptic differentiation, promotes the redistribution of acetylcholine receptors into lipid rafts. Disruption of lipid rafts before agrin treatment largely inhibits de novo agrin-induced acetylcholine receptor clustering. Moreover, mature acetylcholine receptor clusters are destabilized if lipid rafts are disrupted. These results show that lipid rafts are important in both the initial clustering and later stabilization of agrin-induced acetylcholine receptor clusters and also suggest that lipid rafts may contribute to the postsynaptic localization of acetylcholine receptors in vivo.

Agrin↗