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R G Anderson

Publications and source records attributed to R G Anderson.

At least 19 recordsLinked to original sources
PubMed1992

Annexin VI is required for budding of clathrin-coated pits.

Isolated plasma membranes attached to a solid substratum at 4 degrees C have numerous clathrin-coated pits. These pits initially are flat but become deeply invaginated after warming to 37 degrees C. The pits remain tethered to the membrane in this rounded condition unless supplied with ATP, Ca2+, and cytosol. We now show that when cytosol is treated to remove the Ca(2+)-dependent, phospholipid-binding protein annexin VI, coated pit budding no longer takes place. Addition of purified annexin VI back to the annexin VI-depleted cytosol restores budding activity to normal. Purified annexin VI alone shows only a modest budding activity, suggesting that the cytosol contains a factor(s) in addition to annexin VI that is required for full activity. Cytosol-dependent activation of annexin VI requires both ATP and Ca2+. Annexin VI appears to be not only an active component in the detachment of coated pits from the membrane but also a site for regulating the formation of coated vesicles.

Adenosine Triphosphate↗
PubMed1992

Folate receptor allows cells to grow in low concentrations of 5-methyltetrahydrofolate.

The folate receptor mediates the uptake of 5-methyltetrahydrofolate in certain cultured cells by a process called potocytosis. When these cells are grown in physiological concentrations of folate, the receptor increases the efficiency of vitamin uptake by 30-fold. We now show that PAM 212 cells, a mouse keratinocyte cell line, are unable to grow in 1 nM 5-methyltetrahydrofolate unless they express a functional folate receptor. These results suggest that under certain conditions, tissue cells in the body may depend on the folate receptor to obtain enough 5-methyltetrahydrofolate for growth.

Animals↗
PubMed1992

Caveolin, a protein component of caveolae membrane coats.

Caveolae have been implicated in the transcytosis of macromolecules across endothelial cells and in the receptor-mediated uptake of 5-methyltetrahydrofolate. Structural studies indicate that caveolae are decorated on their cytoplasmic surface by a unique array of filaments or strands that form striated coatings. To understand how these nonclathrin-coated pits function, we performed structural analysis of the striated coat and searched for the molecular component(s) of the coat material. The coat cannot be removed by washing with high salt; however, exposure of membranes to cholesterol-binding drugs caused invaginated caveolae to flatten and the striated coat to disassemble. Antibodies directed against a 22 kd substrate for v-src tyrosine kinase in virus-transformed chick embryo fibroblasts decorated the filaments, suggesting that this molecule is a component of the coat. We have named the molecule caveolin. Caveolae represent a third type of coated membrane specialization that is involved in molecular transport.

Antibodies↗
PubMed1992

The mouse peroxisome proliferator activated receptor recognizes a response element in the 5' flanking sequence of the rat acyl CoA oxidase gene.

Peroxisome proliferators are a diverse group of chemicals, including several hypolipidaemic drugs, that activate a nuclear hormone receptor termed the peroxisome proliferator activated receptor (PPAR). The peroxisomal enzyme acyl CoA oxidase (ACO) is the most widely used marker of peroxisome proliferator action. We have examined the 5' flanking region of the rat ACO gene for sequences that mediate the transcriptional effect of peroxisome proliferators and have identified an element located 570 bp upstream of the ACO gene that confers responsiveness to the hypolipidaemic peroxisome proliferator Wy-14,643. This peroxisome proliferator response element (PPRE) contains a direct repeat of the sequence motifs TGACCT and TGTCCT and binds PPAR. These data therefore indicate an important role of PPAR in mediating the action of peroxisome proliferators including the induction of ACO.

Acyl-CoA Oxidase↗
PubMed1992

Histidine-rich calcium binding protein, a sarcoplasmic reticulum protein of striated muscle, is also abundant in arteriolar smooth muscle cells.

Histidine-rich calcium binding protein (HRC) is a luminal sarcoplasmic reticulum protein abundant in skeletal and cardiac muscle. Using immunofluorescence to examine non-muscle tissues, we now show that HRC is also abundant in the smooth muscle cells lining the walls of small arteries and arterioles. Arterioles that contain only one or two layers of smooth muscle cells are very brightly stained while small muscular arteries demonstrate a lesser degree of immunoreactivity only in cells just adjacent to the lumen of the vessel. In contrast, visceral smooth muscle cells from the gastrointestinal and genitourinary tracts show no HRC immunofluorescence. We also examined the subcellular distribution of HRC in arteriolar smooth muscle by immunoelectron microscopy. HRC was found in electron-dense vesicles beneath the plasma membrane, in small electron-lucent vesicles and in the nuclear envelope, suggesting a location within a calcium-sequestering compartment. These findings suggest that HRC plays a role in sarcoplasmic reticulum function that is unique to striated and arteriolar smooth muscle.

Animals↗
PubMed1992

Cytoplasmic sequence required for basolateral targeting of LDL receptor in livers of transgenic mice.

When expressed in livers of transgenic mice, the human low density lipoprotein (LDL) receptor is specifically targeted to the basolateral (sinusoidal) surface of hepatocytes as determined by immunofluorescence and immunoelectron microscopy. The COOH-terminal cytoplasmic domain of the receptor (residues 790-839) contains a signal for this targeting. A mutant receptor truncated at residue 812 was localized exclusively to the apical (bile canalicular) surface. A mutant receptor terminating at residue 829 showed the normal basolateral distribution, as did a receptor in which alanine was substituted for serine 833, which was previously shown to be a site for phosphorylation in vitro. These data localize the basolateral targeting signal to the 17-residue segment between residues 812 and 828. A 10-amino acid stretch within this segment shows a 4/10 match with a sequence within a previously identified basolateral sorting motif for the receptor for polymeric IgA/IgM in MDCK cells. The four shared residues are spaced at intervals of three, raising the possibility that they all face the same side of an alpha-helix. We conclude that this 10-amino acid stretch may contain a signal that directs certain proteins, including the LDL receptor and the polymeric IgG/IgM receptor, to the basolateral surface of polarized epithelia.

Amino Acid Sequence↗
PubMed1992

Lowering the cholesterol content of MA104 cells inhibits receptor-mediated transport of folate.

The folate receptor is clustered on the surface of MA104 cells in association with caveolae. This relationship is thought to be essential for the proper internalization and recycling of the receptor during the delivery of 5-methyltetrahydrofolate to the cytoplasm of folate-depleted cells. Both the clustered organization of the receptor and the integrity of caveolae are disrupted when cells are deprived of cholesterol. We now show that cholesterol depletion of MA104 cells markedly reduces the rate of 5-methyltetrahydrofolate internalization and causes a 70% decline in the number of receptors present in the internal, recycling compartment. This effect is consistent with morphologic data showing that cholesterol-depleted MA104 cells have a reduced number of caveolae as well as fewer receptors per caveolae.

Animals↗
PubMed1992

Clathrin heavy chain is required for pinocytosis, the presence of large vacuoles, and development in Dictyostelium.

To investigate the intracellular role of the clathrin heavy chain in living cells, we have used "antisense" RNA to engineer mutant Dictyostelium discoideum cells that are severely deficient in clathrin heavy chain expression. Immunoblots stained with an anti-clathrin heavy chain antiserum revealed that mutant cells contained undetectable amounts of clathrin heavy chain protein. Similarly, Northern blots showed an absence of clathrin heavy chain mRNA. Clathrin heavy chain-deficient Dictyostelium cells were viable, but exhibited growth rates twofold slower than parental cells. Whereas many morphological features of the mutant cells were normal, mutant cells lacked coated pits and coated vesicles. Clathrin-deficient cells were also missing large translucent vacuoles that serve as endosomes and contractile vacuoles. In the absence of clathrin heavy chain, mutant cells displayed three distinct functional defects: (a) impairment in endocytosis of fluid phase markers, but competence in another endocytic pathway, the phagocytosis of solid particles; (b) defects in osmoregulation; and (c) inability to complete the starvation-induced development cycle.

Animals↗
PubMed1992

Characterization of the clathrin heavy chain from Dictyostelium discoideum.

We report the cloning and analysis of a clathrin heavy-chain cDNA from the eukaryotic microorganism, Dictyostelium discoideum. A single gene, designated chcA, for the clathrin heavy chain encoded a protein of 1,694 amino acids with a molecular mass of 193,618 daltons. Comparison of the amino acid sequence with the rat and with the yeast sequence showed that the highly conserved protein was more similar to the mammalian clathrin heavy chain (57% identity) than to the yeast heavy chain (45% identity). The mRNA for the clathrin heavy chain was regulated during development. mRNA levels were highest during vegetative growth and declined as the cells progressed through the 24-hr developmental cycle. The concentration of clathrin heavy-chain protein was the same in cells grown in liquid media (high rates of pinocytosis) as in cells grown with bacteria (low rates of pinocytosis), which suggests that regulation of pinocytosis in these cells is not achieved by altering the concentration of clathrin.

Amino Acid Sequence↗
PubMed1991

Short-term and long-term changes in renal function after donor nephrectomy.

We retrospectively examined the effect of nephrectomy on renal function in 55 living related donors. Renal function was measured with 131iodine-orthoiodohippurate scans. All patients were studied preoperatively, and 1 week and 1 year postoperatively. In 20 patients 10-year followup was available. Compensatory hypertrophy was complete 1 week postoperatively: effective renal plasma flow of the remaining kidney was 32.5% higher than preoperatively. The increase remained stable for at least a year. The degree of compensatory hypertrophy was significantly greater in male patients (46.9% after 1 week) than in female patients (26.7%). Compensatory hypertrophy occurred in all age groups studied and it was most pronounced in patients less than 30 years old. In the patients followed for 10 years effective renal plasma flow decreased from 387.7 ml. per minute 1 week after nephrectomy to 367.4 ml. per minute at 10 years. This result is similar to the decrease seen in the normal population. According to our results, renal donation by living related persons does not lead to long-term decrease in renal function.

Adaptation, Physiological↗
PubMed1991

Reconstitution of clathrin-coated pit budding from plasma membranes.

Receptor-mediated endocytosis begins with the binding of ligand to receptors in clathrin-coated pits followed by the budding of the pits away from the membrane. We have successfully reconstituted this sequence in vitro. Highly purified plasma membranes labeled with gold were obtained by incubating cells in the presence of anti-LDL receptor IgG-gold at 4 degrees C, attaching the labeled cells to a poly-L-lysine-coated substratum at 4 degrees C and then gently sonicating them to remove everything except the adherent membrane. Initially the gold label was clustered over flat, clathrin-coated pits. After these membranes were warmed to 37 degrees C for 5-10 min in the presence of buffer that contained cytosol extract, Ca2+, and ATP, the coated pits rounded up and budded from the membrane, leaving behind a membrane that was devoid of LDL gold. Simultaneous with the loss of the ligand, the clathrin triskelion and the AP-2 subunits of the coated pit were also lost. These results suggest that the budding of a coated pit to form a coated vesicle occurs in two steps: (a) the spontaneous rounding of the flat lattice into a highly invaginated coated pit at 37 degrees C; (b) the ATP, 150 microM Ca2+, and cytosolic factors(s) dependent fusion of the adjoining membrane segments at the neck of the invaginated pit.

Adenine Nucleotides↗
PubMed1991

The folate receptor works in tandem with a probenecid-sensitive carrier in MA104 cells in vitro.

Previous studies have defined a novel route of internalization for the essential vitamin 5-methyltetrahydrofolate in MA104 cells that begins with binding of the vitamin to the membrane receptor for folate. One of the critical steps in the pathway is the passage of 5-methyltetrahydrofolate through the membrane into the cytoplasm. Utilizing both probenecid and low temperature as selective inhibitors, we have successfully blocked transmembrane movement of the vitamin into the cytoplasm without affecting binding to the receptor or the internalization of the vitamin-receptor complex, which suggests that passage is through an anion carrier. This anion carrier, which mediates inward movement of 5-methyltetrahydrofolate after it dissociates from the receptor, also appears to mediate the efflux of folylmonoglutamate, but not folylpolyglutamate, when the concentration of the former in the cytoplasm is sufficiently high. Since we also found that the synthesis of folylpolyglutamates is regulated in these cells, most likely the intracellular concentration of the vitamin is controlled by regulating the flux of folylmonoglutamate through this carrier.

Animals↗
PubMed1991

Simultaneous visualization of LDL receptor distribution and clathrin lattices on membranes torn from the upper surface of cultured cells.

We have developed a method for simultaneous visualization by electron microscopy of both the distribution of cell surface receptors and architectural features of the inner membrane surface, such as clathrin-coated pits. Electron microscope grids were covered with formvar and coated with poly-L-lysine. These grids were then placed on a piece of buffer-impregnated cellulose acetate membrane filter maintained at 4 degrees C on an ice bath. Cells of interest were grown on glass coverslips and incubated with either a ligand-gold or an antibody-gold conjugate specific for the membrane determinant of interest. The coverslip with gold-labeled cells was then overlaid on the grids and pressure was applied. When the grid was removed, large areas of the upper cell surface, which had labeled determinants, remained adherent to the formvar support. With the proper staining, both the gold particles and internal membrane features could be seen at the same time in the electron microscope. This method is rapid, does not require extensive experience with electron microscopic technique, and permits viewing of membrane samples that are large enough to perform quantitative analysis of gold distribution in relation to membrane specializations.

Animals↗
PubMed1990

Clathrin-coated pits contain an integral membrane protein that binds the AP-2 subunit with high affinity.

Coated pits will assemble onto purified plasma membranes that are attached to a poly-L-lysine coated substratum (Moore, M. S., Mahaffey, D. T., Brodsky, F. M., and Anderson, R. G. W. (1987) Science 236, 558-563; Mahaffey, D. T., Moore, M. S., Brodsky, F. M., and Anderson, R. G. W. (1989) J. Cell Biol. 108, 1615-1624). To better understand the assembly reaction, we have purified both clathrin triskelion and AP-2 subunits from bovine brain and assayed for their ability to bind to the cytoplasmic surface of attached membranes. Two types of membranes were analyzed: those washed with a high pH buffer that selectively removes triskelions and those washed with a high salt buffer that removes both the AP-2 and the triskelion subunits. We found that purified AP-2 subunits bind with high affinity (Kd approximately 3 x 10(-8) M) to salt stripped membranes. Binding is saturable and abolished by treating membranes with less than 20 micrograms/ml of elastase. When membranes were treated with elastase before the salt wash and then salt washed and assayed for AP-2 binding, normal binding was seen, which indicates that the presence of clathrin-coated pits protects the binding site from the protease. Membranes that had rebound AP-2 did not bind purified triskelions, even though high pH buffer-washed membranes that bear endogenous AP-2 bound triskelions with high affinity (Kd approximately 3 x 10(-9) M) and supported lattice assembly. We conclude that coated pit assembly is initiated by the binding of AP-2 to an integral membrane protein but that the AP-2 complex must be activated by an unknown process before the coated pit lattice will assemble.

Animals↗
PubMed1990

The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway.

The folate receptor, also known as the membrane folate-binding protein, is maximally expressed on the surface of folate-depleted tissue culture cells and mediates the high affinity accumulation of 5-methyltetrahydrofolic acid in the cytoplasm of these cells. Recent evidence suggests that this receptor recycles during folate internalization and that it is anchored in the membrane by a glycosyl-phosphatidylinositol linkage. Using quantitative immunocytochemistry, we now show that (a) this receptor is highly clustered on the cell surface; (b) these clusters are preferentially associated with uncoated membrane invaginations rather than clathrin-coated pits; and (c) the receptor is not present in endosomes or lysosomes. This receptor appears to physically move in and out of the cell using a novel uncoated pit pathway that does not merge with the clathrin-coated pit endocytic machinery.

Animals↗