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C G Davis

Publications and source records attributed to C G Davis.

At least 37 records · Page 2Linked to original sources
PubMed1987

Self-association of the low density lipoprotein receptor mediated by the cytoplasmic domain.

When the low density lipoprotein (LDL) receptor was solubilized from bovine adrenal cortex membranes and subjected to electrophoresis in the absence of reducing agents, a disulfide-bonded dimeric species was demonstrated. Formation of these covalent bonds was blocked when the tissue was homogenized in the presence of sulfhydryl alkylating agents, indicating that the native receptor was self-associated noncovalently and that the disulfide bond formation occurred only after homogenization. The disulfide-linked dimers were disrupted and the receptor was restored to a monomeric form when inside-out adrenal vesicles were treated with trypsin, suggesting that the disulfide bond formation involved the 50-amino acid cytoplasmic domain of the receptor. When the receptor was solubilized from bovine adrenal cortex membranes and then purified by ion exchange and affinity chromatography, it could be covalently coupled into dimers and trimers in the presence of bivalent cross-linking agents. Receptor dimers could also be demonstrated by chemical cross-linking of intact cells that were transfected with an expressible cDNA encoding the normal human LDL receptor. Dimer formation was markedly reduced in transfected cells expressing mutated cDNAs that had premature termination codons at positions 792, 807, and 812, which produced shortened receptors that retained 2, 17, and 22 of the original 50 amino acids of the cytoplasmic domain, respectively. The first two mutant receptors, which did not form oligomers, did not enter coated pits and were not rapidly internalized by cells. However, the mutant receptor that terminates at position 812 was internalized normally even though oligomer formation was greatly reduced. Moreover, a mutant receptor with a cysteine substituted for a tyrosine at position 807, which internalized very slowly, showed a normal susceptibility to chemical cross-linking. Deletion of external domains of the LDL receptor, including the epidermal growth factor homology region and the O-linked sugar domain, did not alter susceptibility to chemical cross-linking. We conclude that the cytoplasmic domain of the LDL receptor is responsible both for self-association into oligomers and for clustering in coated pits, but the available data do not establish a causal connection between these two events.

Adrenal Cortex↗
PubMed1987

The low density lipoprotein receptor. Identification of amino acids in cytoplasmic domain required for rapid endocytosis.

The 50-residue cytoplasmic domain of the low density lipoprotein receptor (amino acids 790-839) directs the receptor to coated pits, thereby facilitating rapid endocytosis of bound low density lipoprotein. To determine the structural features required for this targeting, we produced 24 mutations in the cytoplasmic domain through use of oligonucleotide-directed mutagenesis. The first 22 amino acids of the cytoplasmic domain (residues 790-811) are sufficient for rapid internalization. The amino acid at position 807 is especially critical. Aromatic residues (tyrosine, phenylalanine, or tryptophan) at this position allow rapid internalization. Charged or uncharged aliphatic residues do not substitute. Although the requirements at the neighboring positions (806 and 808) are less stringent, the insertion of proline at position 806 is detrimental. These specificities suggest that the juxtamembranous region of the cytoplasmic domain participates in protein:protein interactions that allow the low density lipoprotein receptor to cluster in coated pits.

Amino Acid Sequence↗
PubMed1987

The Lebanese allele at the low density lipoprotein receptor locus. Nonsense mutation produces truncated receptor that is retained in endoplasmic reticulum.

We here describe a mutant low density lipoprotein receptor gene that produces a shortened receptor protein lacking three domains: the region of clustered O-linked carbohydrates, the membrane-spanning region, and the cytoplasmic tail. The defect is attributable to a single nucleotide substitution that creates a premature termination codon at amino acid 660, eliminating 180 residues from the mature protein. The truncated protein retains only two domains: a complete ligand-binding region (residues 1-292) and a partial epidermal growth factor precursor homology region (residues 293-659). The termination codon occurs in the middle of a cysteine-rich sequence that is part of the epidermal growth factor precursor homology domain. The mutant protein is present in markedly reduced amounts and may be translated at a reduced rate. After synthesis, most of the receptor remains within the cell for several hours with its N-linked carbohydrate in an unprocessed endoglycosidase H-sensitive form. This finding suggests that the shortened receptor leaves the endoplasmic reticulum at an abnormally slow rate, which is likely attributable to abnormal folding of the truncated protein. The mutation creates a new restriction site for the enzyme HinfI, thus permitting diagnosis by Southern blotting of genomic DNA. Two copies of this mutant gene were present in each of four unrelated Arab patients with homozygous familial hypercholesterolemia (three from Lebanon and one from Syria). We believe that this mutation, hereafter referred to as the "Lebanese allele," is responsible for the extraordinarily high incidence of familial hypercholesterolemia in Lebanon.

Base Sequence↗
PubMed1987

The LDL receptor: oligonucleotide-directed mutagenesis of the cytoplasmic domain.

Mutations at many different sites in the gene for the low density lipoprotein (LDL) receptor can cause the disease familial hypercholesterolaemia. A particularly interesting class of mutations includes those producing 'internalization-defective' receptors-receptors which are expressed on the cell surface and which bind LDL normally, but which fail to cluster in coated pits. This defect was first observed in fibroblasts from patient J.D. Cloning and sequencing of the terminal exons of J.D.'s internalization-defective LDL receptor gene revealed a single point mutation which caused the substitution of cysteine for tyrosine at residue 807. On the basis of this observation, we have used techniques of oligonucleotide-directed mutagenesis to make an extensive series of mutations in the full-length LDL receptor cDNA. Stable cell-lines expressing these mutant receptors have been analysed for receptor function. Of 13 different amino acids expressed at residue 807, only the aromatics tyrosine, phenylalanine and tryptophan allowed rapid internalization. Position 807 seems to be a particularly sensitive site, since neither substitution of a cysteine for residue 806 or 808 nor deletion of two triplets downstream had any effect on receptor internalization. In addition, a series of truncations localize the signals for internalization to the membrane-proximal 22 amino acid residues.

Amino Acid Sequence↗
PubMed1986

The J.D. mutation in familial hypercholesterolemia: amino acid substitution in cytoplasmic domain impedes internalization of LDL receptors.

Genomic DNA encompassing the terminal exons of the gene for the low density lipoprotein (LDL) receptor was isolated from J.D., a patient with familial hypercholesterolemia whose receptor fails to cluster in coated pits. The DNA sequence revealed a substitution of a cysteine codon for a tyrosine codon at residue 807 in the cytoplasmic domain of the receptor. We reproduced this substitution through oligonucleotide-directed mutagenesis of the normal human receptor cDNA. Upon transfection into receptor-deficient hamster cells, the cDNA specified a receptor that bound LDL normally, but entered the cell slowly. Electron microscopy showed that this receptor was distributed diffusely over the cell surface, whereas the receptor produced by the normal cDNA was concentrated in coated pits. These results support the hypothesis that cytoplasmic domains direct receptors to coated pits, thereby determining the high rate of receptor internalization in animal cells.

Alleles↗
PubMed1986

Deletion of clustered O-linked carbohydrates does not impair function of low density lipoprotein receptor in transfected fibroblasts.

A single exon in the gene for the receptor for plasma low density lipoprotein (LDL) encodes a region of clustered serine and threonine residues that is immediately external to the membrane-spanning sequence. This region has been proposed as the site of clustered O-linked carbohydrate chains. In the current studies we have deleted the 144 base pairs (48 amino acids) that encode this serine- and threonine-rich region from the cDNA for the human LDL receptor. Upon transfection into receptor-deficient hamster fibroblasts, this mutated cDNA encoded a shortened receptor that no longer showed an anomalously high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling with [3H]glucosamine confirmed the lack of clustered O-linked sugars and further revealed that the shortened receptor and the normal receptor both contained isolated O-linked carbohydrate chains attached to the NH2-terminal portion of the protein. The ratio of clustered to isolated O-linked sugar chains in the normal receptor was estimated to be approximately 4-6 to 1. Despite the loss of clustered O-linked carbohydrate, the LDL receptor encoded by the deletion-bearing cDNA bound and internalized LDL normally. It also recycled normally and exhibited a normal half-life. We conclude that: 1) the serine- and threonine-rich region of the LDL receptor is the site for addition of clustered O-linked carbohydrates; 2) the receptor contains a small number of isolated chains of O-linked carbohydrates in addition to the clustered chains; and 3) the clustered O-linked carbohydrates are not essential for LDL receptor function in cultured hamster fibroblasts.

Amino Acid Sequence↗
PubMed1986

The LDL receptor in familial hypercholesterolemia: use of human mutations to dissect a membrane protein.

Since the discovery of the LDL receptor 13 years ago, a multidisciplinary approach to its study has revealed much about this important cell-surface protein. Most recently, we have developed tools in the form of full-length cDNAs and cloned genomic DNAs necessary to understand the molecular genetics of this locus. The frequent occurrence of mutations in the LDL receptor gene in patients with FH provides a fertile ground on which to explore the parts of the receptor that are necessary for its function. The analysis of four large deletions has revealed an unexpectedly universal involvement of Alu repeats in their generation. These studies indicate that repetitive DNAs can destabilize a gene through homologous recombination. Inasmuch as the LDL receptor gene is a mosaic of exons shared with at least five other proteins, it is possible that early exon-shuffling events involved recombination between these repetitive elements. Is it possible that the very plasticity that permitted evolution of the LDL receptor also accounts for its frequent disruption by mutation? Further study may help to answer this question. Mutations that disrupt the structure of the protein have been identified. The biochemical and cellular consequences of these mutations reveal crucial aspects of receptor structure. The receptor is clearly divided into quasi-independent domains with discrete functions. Mutations that disrupt the cytoplasmic domain alter the ability of the LDL receptor to cluster in coated pits, but they do not disrupt ligand binding or produce major effects on intracellular transport. Some of the mutations in the external domain disrupt binding but do not affect transport or internalization.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1984

The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA.

The nucleotide sequence of a cloned 5.3 kilobase cDNA for the human low density lipoprotein receptor revealed five domains in the 839 amino acid protein: 322 NH2-terminal amino acids, extremely rich in disulfide-bonded cysteine residues (15%) and including an 8-fold repeat of 40 residues that may contain the LDL binding site; 350 residues homologous to the precursor of mouse epidermal growth factor; a region immediately outside the plasma membrane, rich in serine and threonine and the site of O-linked glycosylation; 22 hydrophobic amino acids, spanning the plasma membrane; and 50 COOH-terminal amino acids, projecting into the cytoplasm. The mRNA for the receptor contains a 3' untranslated region of 2.5 kilobases that includes multiple copies of the Alu family of repetitive DNAs. Transfection of simian COS cells with the human LDL receptor cDNA linked to the SV40 early promoter resulted in expression of functional cell surface receptors.

Amino Acid Sequence↗
PubMed1984

Food frequency related to folacin status in adolescents.

Food frequency data for 372 adolescents from urban and rural low-income households were evaluated. The urban group consisted of blacks (N = 161) and Hispanics (N = 32); blacks (N = 58) and whites (N = 121) composed the rural group. A food frequency questionnaire with 24 food groups categorized according to nutrient contribution was completed for each subject by a trained interviewer. Urban blacks selected folacin-dense foods more frequently than urban Hispanics or rural blacks. This difference corresponded with a higher prevalence of poor folacin status in urban Hispanics vs. urban blacks and in rural blacks vs. urban blacks. Rural whites also consumed a higher frequency of folacin-dense food groups than rural blacks, which, again, corresponded with differences in folacin status. The infrequent consumption of vegetables and fruits, particularly by rural black and urban Hispanic adolescents, provides an explanation for the poor folacin status of the adolescents.

Adolescent↗
PubMed1983

Activation of acetylcholine receptors causes the partition of hydrophobic cations into postsynaptic membrane vesicles.

In the continued presence of cholinergic ligands, the acetylcholine receptor-channel complex (AChR) in postsynaptic membranes undergoes a sequence of conformational changes. On addition of the ligand, the receptor rapidly changes from a closed channel to an open channel conformation, then slowly changes to a nonconducting state termed desensitization. The lifetime of the open channel conformation and the rate of desensitization are both dependent on the magnitude of the membrane potential, suggesting that the ligand-induced conformational changes in AChR may involve the movement of electrical charges within the membrane. Measurements of charge redistribution in AChR-containing membranes following ligand binding have not been reported. Recently, measurements of changes in the membrane partition coefficient of hydrophobic ions have been used to detect electrostatic changes in both biological and model membranes. We report here that cholinergic ligands induce changes in the partition coefficient of the hydrophobic cation tetraphenylphosphonium (TPP) into AChR-enriched membranes. The extent and time course of these changes in TPP partition coefficient are accounted for in a kinetic model. We conclude that TPP movement is a monitor of a molecular event which may be associated with the slow component of AChR desensitization.

Animals↗
PubMed1982

Specificity and localization of the acetylcholine receptor kinase.

Acetylcholine receptor-phosphorylation has been compared in sealed and lysed right-side-out membrane vesicles prepared form Torpedo californica electric organ. Phosphorylation was increased 5- to 12-fold in hypotonically lysed vesicles as compared with untreated vesicles. Control experiments confirm that this enhancement is a result of increased permeability of the membrane to ATP. These data suggest that the acetylcholine receptor kinase is located on the cytoplasmic side of the plasma membrane. Results with detergent lysis support this conclusion. Although the acetylcholine receptor constitutes less than 10% of the total protein in these membranes, the kinase was found to be highly specific for polypeptides corresponding in molecular weight to acetylcholine receptor subunits.

Adenosine Triphosphate↗
PubMed1982

Folacin and iron status and hematological findings in black and Spanish-American adolescents from urban low-income households.

The folacin and iron status of 193 adolescents from urban low-income households was evaluated. Red blood cell folacin concentrations were less than 140 ng/ml in 42% of the subjects and 140 to 159 ng/ml in 13%. Of the serum folacin values, 45% were less than 6 ng/ml, and 15% were below 3 ng/ml. Serum folacin levels decreased with increasing age (p less than 0.01) and sexual maturity (p less than 0.05). Transferrin saturation was low (less than 16%) in 12% of the females and 2% of the males. Transferrin saturation levels for females declined as age increased in contrast to an increase over age in males (p less than 0.01). Eleven percent of the females and 3% of the males were classified as anemic (less than 12 g/dl). Mean cell Hb concentration was low (less than 32%) in 24% of the females and 7% of the males. Of all subjects, 17% had low mean cell volumes (less than 81 mum 3). These findings demonstrate folacin and iron status is less than adequate in a significant proportion of this adolescent population group.

Adolescent↗
PubMed1981

A hearing survey in diabetes mellitus.

A sample of 50 diabetics was compared with a control group matched for age and sex. Twenty-two of the diabetics were insulin-dependent. Subjects and controls were examined otologically and then tested audiometrically using pure tone audiometry, speech audiometry and tone decay after the method of Owens. No significant difference was found between the two groups on pure tone audiometry and speech testing showed all diabetics and controls to be normal. A statistically significant incidence of type-2 tone decay was found in the overall group of diabetics at 2000 Hz. This may reflect early presbycusis.

Adult↗
PubMed1980

Membrane-bound protein kinase activity in acetylcholine receptor-enriched membranes.

Membrane protein phosphorylation may be a general regulatory mechanism mediating the response of cells to exogenous metabolic and physical signals. We have determined that the membrane-bound acetylcholine receptor is the major substrate phosphorylated in situ by a nearby membrane protein kinase. Moreover, these same membranes also contain phosphoprotein phosphatase activity which dephosphorylates the membrane-bound receptor. These findings suggest that reversible phosphorylation of the actylcholine receptor may be critical for receptor function at the synapse. Therefore, it is necessary to define the properties of the enzymes which mediate this phosphorylation-dephosphorylation mechanism. In this report we describe the properties of the first component of this system, the membrane-bound protein kinase in receptor-enriched membranes from the electric organ of Torpedo californica. Only ATP is effective as a phosphate donor for this cyclic AMP-independent membrane kinase; GTP does not support phosphorylation of the receptor. Both casein and histone can also be phosphorylated by the membrane protein kinase, but casein is a better substrate. Although phosphorylation of the receptor appears to be regulated by cholinergic ligands and K+, casein phosphorylation is not specifically affected by these agents. Moreover, while phosphorylation of the acetylcholine receptor is maximal in receptor=enriched membranes, casein phosphorylation is similar in all membrane fractions prepared from the electric organ. Taken together, these findings suggest that the membrane protein kinase activity in receptor-enriched membranes is similar to most other membrane kinases. Therefore, the unique characteristics of membrane-bound acetylcholine receptor phosphorylation appear to be determined by the receptor and its availability as a substrate for the membrane kinase.

Acetylcholine↗
PubMed1980

Zinc status of elderly black Americans from urban low-income households.

The zinc status of 135 elderly blacks, aged 60 to 87 years, from urban low-income households was evaluated based on the zinc content of hair and/or serum. The mean (+/- SD) hair zinc concentration was 142 +/- 77 microgram/g and the mean (+/- SD) serum zinc concentration was 93 +/- 15 microgram/dl. Of the study population 39% had a hair zinc concentration less than or equal to 100 microgram/g and/or a serum zinc concentration less than or equal to 80 microgram/dl. Eleven percent had a hair zinc concentration less than or equal to 70 microgram/g and/or a serum zinc concentration less than or equal to 70 microgram/dl. These findings suggest that the zinc status of this elderly population may be less than ideal.

Aged↗
PubMed1980

Vitamin B12 status of elderly persons from urban low-income households.

Vitamin B12 status was evaluated in 111 noninstitutional elderly persons (age range, 60-87 years) living in an urban poverty area. The sample was predominantly black (90 subjects); the rest were Spanish Americans. Serum vitamin B12 levels were all normal (greater than 200 pg/ml) and ranged from 226 to 1200 pg/ml (mean +/- SD = 700 +/- 191 pg/ml). The findings indicate that vitamin B12 deficiency was not a problem in this elderly population.

Aged↗