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C D Strader

Publications and source records attributed to C D Strader.

126 records · Page 7Linked to original sources
PubMed1986

Potentiation of specific association of insulin with HepG2 cells by phorbol esters.

The effects of tumour-promoting phorbol esters on the receptor-mediated endocytosis of insulin were investigated in the human hepatoma cell line HepG2. Treatment of these cells with the biologically active phorbol 12-O-tetradecanoylphorbol 13-acetate (TPA), but not with the non-tumour-promoting analogue 4 alpha-phorbol 12,13-didecanoate, resulted in dramatic morphological changes, which were accompanied by a 1.5-2.5-fold increase in specific 125I-insulin association with the cells at 37 degrees C. This increase in insulin binding was not observed when the binding reaction was performed at 4 degrees C. The potentiation of 125I-insulin association with TPA-treated cells at 37 degrees C could be completely accounted for by an increase in the intracellular pool of internalized insulin; there was no concomitant increase in cell-surface insulin binding. Dissociation studies showed that the enhanced internalization of insulin by cells after treatment with TPA resulted from a decrease in the rate of intracellular processing of the insulin after receptor-mediated endocytosis. The phorbol-ester-induced enhancement of internalized insulin in HepG2 cells was additive with the potentiation of endocytosed insulin induced by both the lysosomotropic reagent chloroquine and the ionophore monensin; this indicates that TPA affects the intracellular processing of the insulin receptor at a point other than those disrupted by either of these two reagents. The potentiation of insulin receptor internalization by tumour-promoting phorbol esters could be completely mimicked by treatment with phospholipase C, but not with phospholipase A, and partially mimicked by treatment with the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol. By these criteria, the effects of phorbol esters on the insulin receptor in HepG2 cells appear to be mediated through protein kinase C. These results support the concept that the activation of protein kinase C by treatment with phorbol esters causes a perturbation of the insulin-receptor-mediated endocytotic pathway in HepG2 cells, reflected in a long-term decreased rate of dissociation of internalized insulin by the phorbol-ester-treated cells.

Animals↗
PubMed1984

Association of sequestered beta-adrenergic receptors with the plasma membrane: a novel mechanism for receptor down regulation.

Chronic exposure of frog erythrocytes to beta-adrenergic agonists leads to desensitization of the responsiveness of adenylate cyclase to isoproterenol and is accompanied by "down-regulation", a decrease in the number of beta-adrenergic receptors on the cell surface. When frog erythrocyte plasma membranes are prepared by osmotic lysis of cells, the receptors lost from the cell surface during desensitization can be recovered in a "light membrane fraction", obtained by centrifuging the cell cytosol at 158,000 X g for 1 hr. These receptors are sequestered away from the plasma membrane fraction which contains the adenylate cyclase and the guanine nucleotide regulatory protein. If desensitized frog erythrocytes are disrupted by gentler freeze/thaw procedures, however, the sequestered beta-adrenergic receptors can be demonstrated to be physically associated with the plasma membrane. Typically, plasma membranes prepared in this fashion do not demonstrate a significant down regulation despite attenuation of isoproterenol-stimulated adenylate cyclase activity. Under these conditions, beta-adrenergic receptors from control and desensitized preparations co-migrate on sucrose density gradients in exactly the same place as the plasma membrane marker, adenylate cyclase. In contrast, when membranes from osmotically lysed desensitized cells are fractionated on sucrose gradients the down regulated receptors are sequestered in a light membrane fraction which barely enters the gradient and which is physically separated from adenylate cyclase activity. The data are consistent with a novel mechanism of receptor down-regulation which appears to involve the sequestration of the beta-adrenergic receptors away from the cell surface into a membrane compartment which remains physically associated with the plasma membrane.

Adenylyl Cyclases↗
PubMed1984

Morphologic demonstration of clathrin-coated pits in frog and turkey erythrocytes.

We have examined nucleated erythrocytes of frog and turkey for the presence of clathrin-coated structures using electron microscopy and immunocytochemistry. By electron microscopy, coated pits were found on the plasma membrane of peripheral blood erythrocytes of both species. These structures had an appearance similar to coated pits seen in non-erythroid mammalian cells. Using immunofluorescence with anti-(bovine) clathrin antibody, erythrocytes of both species showed punctate membrane fluorescence similar to the pattern of coated pits seen in other cells. By both methods, frog erythrocytes showed considerable heterogeneity, such that only about 50% of the cells showed significant numbers of coated pits, usually fewer than 20-50 per cell. In contrast, the vast majority of turkey erythrocytes showed no detectable coated pits, but occasional cells (less than 10%) showed large numbers of coated structures. These results suggest that a functional endocytic system may be present in a subpopulation of these nucleated erythrocytes. These findings may be of significance in understanding the ligand-induced loss of some receptors from the surface of these cells, and may serve as an indication of morphologic differentiation.

Animals↗
PubMed1983

Reconstitution of beta-adrenergic receptors in lipid vesicles: affinity chromatography-purified receptors confer catecholamine responsiveness on a heterologous adenylate cyclase system.

The binding function of purified receptors can be assessed with radioligands, but the interaction of receptors with their biochemical effectors has not been amenable to direct study. Toward this end, procedures have been developed for directly demonstrating functionality of purified beta-adrenergic receptor preparations. Digitonin-solubilized beta-adrenergic receptors from frog erythrocytes or rat lung were purified approximately equal to 100- to 5,000-fold by affinity chromatography and inserted into a mixture of frog erythrocyte lipids and dimyristoyl phosphatidylcholine in the presence of octyl glucoside. Reconstitution of beta-adrenergic receptor binding was typically 25-50% and could also be effected with soybean phosphatidylcholine in the presence of octyl glucoside. The reconstituted beta-adrenergic receptors were then fused with Xenopus laevis erythrocytes, which contain prostaglandin E1-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] but few beta-adrenergic receptors and little or no catecholamine-sensitive adenylate cyclase. Fusion of reconstituted receptor with Xenopus laevis erythrocytes establishes a substantial (2- to 10-fold) stimulation of the hybrid adenylate cyclase by the beta-agonist isoproterenol. The extent of stimulation depends on the amount of reconstituted beta-adrenergic receptor added, is blocked by propranolol, and is eliminated by boiling the beta-adrenergic receptor prior to reconstitution. The successful coupling of a purified receptor to a heterologous adenylate cyclase opens the way to the study of receptor structure---function relationships.

Adenylyl Cyclases↗
PubMed1983

Antibodies to the beta-adrenergic receptor: attenuation of catecholamine-sensitive adenylate cyclase and demonstration of postsynaptic receptor localization in brain.

Antibodies to the beta 2-adrenergic receptor of frog erythrocytes have been raised in rabbits by immunization with purified receptor preparations. Binding of the antibodies to the receptors was demonstrated by immunoprecipitation and by the altered mobility of the antibody-bound receptors on steric-exclusion HPLC columns. As assessed by a radioimmunoassay developed with the antibody, beta 2-adrenergic receptors from several sources showed various degrees of immunological crossreactivity whereas several beta 1-adrenergic receptors did not crossreact. The antibody appeared to not bind at the ligand binding site of the receptor and did not perturb antagonist radioligand binding to the receptor. Nonetheless, the antibodies selectively attenuated catecholamine-stimulated adenylate cyclase. This suggests that the antibodies recognize and bind to domains of the receptor other than the binding site and that may be involved in coupling to other components of the adenylate cyclase system. Immunocytochemical techniques were used with the antibodies to delineate a postsynaptic localization of beta-adrenergic receptors in rat and frog brain. Thus, these anti-beta-adrenergic receptor antibodies provide a useful reagent for probing beta-adrenergic receptor structure, function, and localization.

Adenylyl Cyclases↗
PubMed1980

Acetylcholine receptor: complex of homologous subunits.

The acetylcholine receptor from the electric ray Torpedo californica is composed of five subunits; two are identical and the other three are structurally related to them. Microsequence analysis of the four polypeptides demonstrates amino acid homology among the subunits. Further sequence analysis of both membrane-bound and Triton-solubilized, chromatographically purified receptor gave the stoichiometry of the four subunits (40,000:50,000:60,000:65,000 daltons) as 2:1:1:1, indicating that this protein is a pentameric complex with a molecular weight of 255,000 daltons. Genealogical analysis suggests that divergence from a common ancestral gene occurred early in the evolution of the receptor. This shared ancestry argues that each of the four subunits plays a functional role in the receptor's physiological action.

Acetylcholine↗
PubMed1980

Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins.

A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures.

Alkalies↗
PubMed1980

Topographic studies of Torpedo acetylcholine receptor subunits as a transmembrane complex.

The exposure of the four subunits of the acetylcholine receptor from Torpedo californica on both the extracellular and cytoplasmic faces of the postsynaptic membranes of the electroplaque cells has been investigated. Sealed membrane vesicles containing no protein components other than the receptor were isolated and were shown to have 95% of their synaptic surfaces facing the medium. The susceptibility of the four receptor subunits in these preparations to hydrolysis by trypsin both from the external and from the internal medium was used to investigate the exposure of the subunits on the synaptic and cytoplasmic surfaces of the membrane. It was shown by sodium dodecyl sulfate gel electrophoresis of the tryptic products that all four subunits are exposed on the extracellular surface to a similar degree. All four subunits are also exposed on the internal surface of the membrane, but the apparent degree of exposure varies with the subunit size, the larger subunits being more exposed. The results are discussed in terms of a possible topographic model of the receptor as a transmembrane protein complex.

Animals↗
PubMed1980

Hemocyanin-protein A, an immunochemical reagent for scanning and transmission electron microscopy.

A method is described for preparing hemocyanin-protein A (Hcy/A), a versatile labeling reagent for immunochemical electron microscopy. Because protein A has a specific affinity for the Fc region of immunoglobulins, Hcy/A can be used in indirect labeling with antisera prepared in several different animal species. Because of the distinctive shape and size of hemocyanin, Hcy/A labeling is adaptable to a number of preparative techniques for scanning and transmission electron microscopy. Hcy/A is a stable, highly monomeric conjugate (greater than 90% monomeric, i.e., units of approximately 35 X 50 nm) with a molar ratio of protein A to hemocyanin near unity. Hcy/A labeling is demonstrated using antisera from rabbits, mice and humans to localize cell surface and subcellular antigens. Included are the localization of developmentally expressed erythrocyte antigens, blood group antigens, acetylcholine receptors on Torpedo electroplax membrane fragments, and actin within a subcellular matrix.

Actins↗
PubMed1977

Interaction of the unique competitive inhibitor imidazole with human carbonic anhydrase B.

Imidazole was previously found to be unique among the inhibitors of human carbonic anhydrase B (HCAB) in that it binds competitively with the CO2 substrate (Khalifah, R. G. (1971), J. Biol. Chem. 246, 2561). We report here an aromatic ultraviolet difference spectral study of its interaction with HCAB and compare it with a variety of other inhibitors. Imidazole is found to be unique in that: (1) it generates a different spectrum upon binding that is also much supressed in intensity; (2) its affinity for HCAB is maximal at high pH, being abolished upon its protonation and being independent of active-site ionizations. Imidazole differs from CO2 in that it binds competitively with the anionic inhibitor iodide. The unique properties of imidazole binding are consistent with the recently determined crystal structure of its complex with HCAB showing it to bind as a weak and distant fifth ligand of the essential zinc atom, rather than displacing the solvent molecule in the fourth ligand position (Kannan, K.K., Petef, M., Fridborg, K., Cid-Dresdner, H., and Lövgren, S. (1977), FEBS Lett 73, 115).

Binding, Competitive↗
PubMedDate not supplied

Cloning of the gene and cDNA for mammalian beta-adrenergic receptor and homology with rhodopsin.

The adenylate cyclase system, which consists of a catalytic moiety and regulatory guanine nucleotide-binding proteins, provides the effector mechanism for the intracellular actions of many hormones and drugs. The tissue specificity of the system is determined by the particular receptors that a cell expresses. Of the many receptors known to modulate adenylate cyclase activity, the best characterized and one of the most pharmacologically important is the beta-adrenergic receptor (beta AR). The pharmacologically distinguishable subtypes of the beta-adrenergic receptor, beta 1 and beta 2 receptors, stimulate adenylate cyclase on binding specific catecholamines. Recently, the avian erythrocyte beta 1, the amphibian erythrocyte beta 2 and the mammalian lung beta 2 receptors have been purified to homogeneity and demonstrated to retain binding activity in detergent-solubilized form. Moreover, the beta-adrenergic receptor has been reconstituted with the other components of the adenylate cyclase system in vitro, thus making this hormone receptor particularly attractive for studies of the mechanism of receptor action. This situation is in contrast to that for the receptors for growth factors and insulin, where the primary biochemical effectors of receptor action are unknown. Here, we report the cloning of the gene and cDNA for the mammalian beta 2AR. Analysis of the amino-acid sequence predicted for the beta AR indicates significant amino-acid homology with bovine rhodopsin and suggests that, like rhodopsin, beta AR possesses multiple membrane-spanning regions.

Amino Acid Sequence↗
PubMedDate not supplied

Ligand binding to the beta-adrenergic receptor involves its rhodopsin-like core.

Recently the genes for several hormone receptors that interact with guanine nucleotide binding proteins (G proteins) have been cloned, including the hamster beta 2-adrenergic receptor (beta 2AR), a human beta AR, the turkey erythrocyte beta AR and the porcine muscarinic acetylcholine receptor (MAR). All these receptors share some amino-acid homology with rhodopsin, particularly in 7 hydrophobic stretches of residues that are believed to represent transmembrane helices. To determine whether differences in ligand specificity result from the divergence in the sequences of the hydrophilic regions of these receptors, we have expressed in mammalian cells genes for the wild-type hamster and human beta AR proteins, and a series of deletion mutant genes of the hamster beta 2AR. The pharmacology of the expressed receptors indicates that most of the hydrophilic residues are not directly involved in the binding of agonists or antagonists to the receptor. In addition, we have identified a mutant receptor that has high agonist affinity but does not couple to adenylate cyclase.

Adenylyl Cyclases↗
PubMedDate not supplied

Molecular biology of adrenergic receptors in the rat and frog central nervous system.

Recent developments in the characterization of the adrenergic receptors have led to the identification and purification of the binding subunits of the various catecholamine receptors. beta-Adrenergic receptors have been identified in a wide variety of tissues by photoaffinity labeling with the antagonist [125I]p-azidobenzylcrazolol and have been purified to apparent homogeneity from several of these tissues. Thus, beta 1- and beta 2-adrenergic receptor binding sites appear to reside on peptides with molecular weights of 60,000 to 65,000. The alpha 1-adrenergic receptor binding subunit has been identified in several peripheral tissues by photoaffinity labeling with a newly developed probe (4-amino-6,7-dimethoxy-2[4(5(3-[125I]-iodo-4-azidophenyl) pentanoyl)-1-piperazinyl]-quinazoline, or [125I]APDQ). This binding site resides on a peptide with a molecular weight of 80,000. These techniques have been applied to the elucidation of the binding subunit structure of these receptors in the rat central nervous system with the result that beta 1-, beta 2-, and alpha 1-adrenergic binding sites appear to reside on peptides of similar molecular weight to those identified in peripheral tissues (i.e., 60,000-65,000 and 80,000). Using immunocytochemical techniques with antibodies raised to the frog erythrocyte, beta 2-adrenergic receptor, beta-adrenergic receptors were identified at the light microscopic level in regions of the rat and frog brain previously found by ligand binding and autoradiography to be richest in beta-adrenergic receptors. At the electron microscopic level, beta-receptor immunoreactivity was found throughout dendritic processes with local accumulations at certain postsynaptic sites. This finding is consistent with the idea that the density of the receptors might be significantly increased at postsynaptic junctions of adrenergic neurons.

Affinity Labels↗
PubMedDate not supplied

Purification and reconstitution of a recombinant human neurokinin-1 receptor.

Recombinant human neurokinin-1 receptors expressed in insect cells have been purified to near homogeneity by sequential metal-chelating chromatography and gel filtration chromatography. The purified receptor consists of a single polypeptide with an apparent molecular weight of 50 kD as revealed by SDS gel electrophoresis, and exhibits a specific activity of 19 nmol of L-703,606 bound per mg of protein. Immunoblot experiments further confirm the identity of the stained protein band. The purified receptor binds the antagonist L-703,606 with an affinity similar to that of native human neurokinin-1 receptor, and binds the agonist substance P with an affinity similar to that of the low affinity state of uncoupled native receptor. The purified receptor can be reconstituted with membranes from uninfected insect cells, and the reconstitution results in an increased affinity for substance P, consistent with the reappearance of the high affinity state of the receptor for agonist in the presence of endogenous G proteins. These data indicate that the purified neurokinin-1 receptor is functional with respect to agonist and antagonist binding and G protein coupling.

Amino Acid Sequence↗