Normal Receptor Function Research Articles

Aortic and coronary atheromatosis in a woman with severe hypercholesterolaemia without LDL receptor alterations

Familial hypercholesterolaemia (FH) is a monogenic disorder, with a strong family history, characterized by a deficiency in functional receptors for low density lipoproteins (LDL). The case of a patient with all the clinical traits of FH, including elevated cholesterol, xanthomas and early coronary and peripheral arterial lesions, but with a normal LDL receptor function, is described. In the patient the molecular weight and immunological properties of apolipoprotein (apo) B were normal; furthermore, autoantibodies to either LDL or to their receptor were also absent. The increased apo B/cholesterol ratio in LDL was compatible with the diagnosis of hyperapobetalipoproteinaemia. With the help of a turnover study using 131I homologous and 125I autologous LDL, it could be established that the patient had an almost three-fold increase in LDL-apo B biosynthesis, with, however, a fractional catabolic rate within normal limits. These findings pointed to the possibility of a genomic alteration in the region responsible for the control of apo B biosynthesis. However, extensive studies both at the cDNA level and in the 5' region of the apo B gene, failed to detect any significant alteration vs published nucleotide sequences. Although the exact mechanism for this unusual clinical presentation of an FH-like syndrome could not be uncovered, this case provides an extreme example of hypercholesterolaemia, with early and severe arterial disease, solely explained by an increased LDL biosynthesis.

Thyroid hormone resistance syndrome. Inhibition of normal receptor function by mutant thyroid hormone receptors.

Thyroid hormone (T3) resistance is inherited in most cases in an autosomal dominant manner. The disorder is characterized by elevated free thyroid hormone levels and partial resistance to thyroid hormone at the cellular level. Distinct single amino acid substitutions in the ligand binding domain of the beta form of the thyroid hormone receptor have been described in two kindreds with this disorder. We used transient expression assays to characterize the functional properties of these receptor mutants, one containing a Gly to Arg change at amino acid 340 (G340R) and the other a Pro to His change at amino acid 448 (P448H). A nine amino acid carboxy terminal deletion (delta 448-456), analogous to an alteration that occurs in v-erbA, was also studied for comparison with the mutations that occur in the T3 resistance syndrome. None of the receptor mutants were able to mediate thyroid hormone dependent activation (TreTKCAT) or repression (TSH alpha CAT) of reporter genes when compared with the wild type receptor. In addition, the mutants inhibited the activity of normal alpha and beta receptor isoforms when examined in coexpression assays. This activity, referred to as dominant negative inhibition, was manifest with respect to both the positively and negatively regulated reporter genes. Although mutant receptor binding to DNA was unaffected, ligand binding studies showed that the G340R and delta 448-456 mutants failed to bind T3, whereas the P448H mutant bound hormone with reduced affinity (approximately 10% of normal) compared to the wild type receptor. Consistent with this finding, the P448H mutant receptor was partially active at higher T3 concentrations. Furthermore, the dominant negative inhibition elicited by the P448H receptor mutant at higher T3 concentrations was reversed in the presence of high doses of T3. These findings indicate that mutant beta receptors in patients with thyroid hormone resistance have reduced affinity for T3 and are functionally deficient, but impair the activity of normal receptors, thereby providing a mechanism for the dominant mode of inheritance in this disorder.

Clinical signs of familial hypercholesterolemia in patients with familial defective apolipoprotein B-100 and normal low density lipoprotein receptor function.

In a previous study (Tybjaerg-Hansen et al, Atherosclerosis 1990;80:235-242), we identified nine patients heterozygous for the apolipoprotein B (apo B) arginine-to-glutamine (Arg3,500----Gln) mutation (familial defective apolipoprotein B-100 [FDB]). Six of these had been diagnosed clinically as familial hypercholesterolemic (FH) heterozygotes. We have since examined low density lipoprotein (LDL) receptor function in the FDB index patients and in three of their families. Skin fibroblasts from seven of seven unrelated FDB patients from whom cell lines were established exhibited normal high-affinity binding and degradation of normal LDL in vitro. In the three families, a raised plasma LDL concentration did not segregate with a haplotype of two polymorphic restriction sites at the LDL receptor locus. We conclude that the clinical and biochemical signs of classical FH can occur in the presence of the FDB mutation and a normal LDL receptor gene. In a four-generation family with 11 proven or presumed FDB heterozygotes, expression of the mutation ranged from normal plasma LDL concentrations and no clinical signs in two individuals, to hypercholesterolemia and death from myocardial infarction at age 31. Variable expression of the FDB mutation could not be explained conclusively by variation in diet, body mass index, smoking habit, apo E genotype, or plasma Lp(a) concentration.

Proton inhibition of N-methyl-D-aspartate receptors in cerebellar neurons

Mammalian neurons contain at least three types of excitatory amino-acid receptors, selectively activated by N-methyl-D-aspartate (NMDA) or aspartate, (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate ((S)-AMPA) and kainate. An important aspect of NMDA receptors is their regulation by a variety of factors such as glycine, Mg2+ and Zn2+ that are present in vivo. We show here that NMDA receptor responses are selectively inhibited by protons, with a 50% inhibitory concentration (IC50) that is close to physiological pH, implying that NMDA receptors are not fully active under normal conditions. (S)-AMPA and kainate responses remain unchanged at similar pH levels. Proton inhibition is voltage-insensitive and does not result either from fast channel block, a change in channel conductance, or an increase in the 50% excitatory concentration (EC50) of aspartate/NMDA or glycine. Instead, protons seem to decrease markedly the opening frequency of 30-50 pS NMDA channels, and reduce the relative proportion of longer bursts. This feature of NMDA receptors could be relevant to neurotoxic activation of NMDA receptors during ischaemia, as well as to seizure generation, as extracellular proton changes occur during both of these pathological situations. Furthermore, these results may have implications for normal NMDA receptor function as transient changes in extracellular protons occur during synaptic transmission.

Glucocorticoid-Dependent Maturation of Mouse Mammary Tumor Virus Glycoproteins in Mouse Lymphoma Cells: Isolation of Variants with Constitutive Viral Protein Maturation and Normal Glucocorticoid Receptor Function

The posttranslational maturation and cell surface localization of mouse mammary tumor virus (MMTV) envelope glycoproteins is regulated by glucocorticoid hormone in mouse T-lymphoma cell line W7MG1. Only when the cells are cultured with glucocorticoid is the MMTV envelope precursor, Pr74, converted efficiently to the two mature proteolytic products, gp52 and gp33. By immunological selection we have isolated protein-processing variants that express the mature viral proteins constitutively on the cell surface. The rate of synthesis of Pr74 is indistinguishable in variant and wild-type cells, but the variants efficiently convert Pr74 to gp52 and gp33 even when grown without the hormone. The variant phenotype persists when the variant cells are fused with uninfected wild-type cells to form somatic cell hybrids, indicating that the variant phenotype resulted from expression of a new or altered function that is not expressed in wild-type cells grown without glucocorticoid. Although the specific gene whose structure or regulation is altered in the variant has not yet been determined, some possibilities have been eliminated. First, the number and function of the glucocorticoid receptors in the variant cells was normal, suggesting that alterations in this protein were not responsible for the variant phenotype. Second, comparison by two-dimensional gel electrophoresis of gp52 produced in variant and wild-type cells revealed no differences in size or charge, indicating no gross differences in the processing of the viral proteins in the variant and wild-type cells.

Epidermal growth factor receptor defects in leprechaunism. A multiple growth factor-resistant syndrome.

Leprechaunism is a rare genetic disorder characterized by severe growth retardation and insulin resistance. Maximal epidermal growth factor (EGF) binding was reduced in fibroblasts from three unrelated patients with leprechaunism (Ark-1, Can-1, and Minn-1) compared with control (0.8-2.2%/mg protein vs. 5.5%/mg protein). This was due to a decrease in receptor affinity in Ark-1 and Can-1 and a decrease in receptor number in Minn-1. In all cell lines, EGF-stimulated receptor autophosphorylation was also decreased to 18-60% of control, whereas EGF internalization and degradation was normal. Sphingosine (40 microM), a protein kinase C inhibitor, increased EGF receptor affinity twofold in control cells and six- to nine-fold in cells of leprechaunism. However, sphingosine did not enhance EGF-stimulated receptor autophosphorylation in either the controls or the patients' cells. By contrast, only one of the three cell lines of patients with the type A syndrome demonstrated a decrease in EGF binding and all demonstrated normal or near normal EGF-stimulated receptor autophosphorylation. These data indicate that in patients with leprechaunism, there are functional abnormalities of the EGF receptor, as well as of the insulin receptor, that may contribute to the severity of the syndrome. These data also suggest a role for the insulin receptor in maintaining normal EGF receptor function in these cells.

Insulin receptors with defective tyrosine kinase inhibit normal receptor function at the level of substrate phosphorylation.

Rat 1 fibroblasts have been transfected with the cDNA encoding a kinase-defective mutant human insulin receptor (A/K1018). Expression of this cDNA results in a receptor that is not only biologically inactive but also inhibits normal insulin action through the normal endogenous rat receptors in this fibroblast line (McClain, D. A., Maegawa, H., Lee, J., Dull, T. J., Ullrich, A., and Olefsky, J. M. (1987) J. Biol. Chem. 262, 14663-14671). We have investigated the mechanism of this inhibition and show that: 1) rat receptors are expressed at normal to increased levels in two cell lines which also express A/K1018 receptors at low (A/K1018-A, 5700 total receptors) or high (A/K1018-B, 2.2 x 10(5) total receptors) levels. 2) The rat receptors in the A/K1018 lines can be normally autophosphorylated under the control of insulin in vitro. 3) A/K1018 receptors do not inhibit the kinase activity of normal receptors when mixed together in vitro. 4) In intact A/K1018-B cells, the ability of insulin to stimulate autophosphorylation of the rat receptor is unimpaired; furthermore, the autophosphorylated rat receptor becomes normally activated as a tyrosine kinase. 5) The expression of receptors for insulin-like growth factor I and stimulation of hexose uptake mediated by this receptor are unaffected in cells expressing inhibitory A/K1018 receptors. 6) Expression of the A/K1018 receptor inhibits insulin-stimulated phosphorylation of two endogenous protein substrates (pp220 and pp170) by the native rat receptors. We conclude that the inhibition of insulin action seen in the A/K1018 cells is not mediated at the levels of native receptor expression or activation, nor is the effector (hexose uptake) mechanism affected by the A/K1018 receptors. The expression of this kinase-defective receptor does, however, inhibit the phosphorylation of substrate molecules by the normally activated endogenous rat receptors.

Abnormalities of the human growth hormone gene and protooncogenes in some human pituitary adenomas.

Hypersecretion of human GH (hGH) or PRL by human pituitary adenomas is not under normal homeostatic control despite normal receptor function mediating the regulatory effects of hypothalamic peptides for these trophic hormones. This implies that the defects underlying hormonal hypersecretion may not reside at the plasma membrane of the adenoma cell; instead, dysregulation may reside at the hormone gene level. To investigate this, genomic DNA derived from a prolactinoma and a hGH-secreting adenoma were digested with the restriction endonuclease EcoRI and the methylation sensitive restriction endonuclease HpaII and hybridized with the 32P-labeled genomic hGH (2.6 kilobase) probe. Our data revealed hypomethylation of genes of the hGH family (hGH and chorionic somatomammotropin) in the absence of gross abnormalities such as gene translocation. In a similar analysis using a 32P-labeled probe consisting of the EcoRI-BamHI (500 base pair) fragment in the 5'-flanking region upstream of the first exon of the hGH gene, hypomethylation of this specific site of the hGH genes was observed. These results are consistent with the concept that hypomethylation of genes is involved in gene expression. At the same time, protooncogene abnormalities in these adenomas were investigated to delineate any genetic basis for their neoplastic growth. Genomic DNA of adenomas were subjected to Southern blotting analysis using a panel of protooncogene probes. Amplification of the v-fos gene was observed in one prolactinoma. The significance of this observation is discussed.

Sterol depletion reduces receptor-mediated low-density lipoprotein binding in NS-1 mouse myeloma cells

NS-1 mouse myeloma cells, a cholesterol auxotrophic cell line with a lesion in the cholesterol biosynthetic pathway at the demethylation of lanosterol to C-29 sterol, were depleted of cholesterol by incubation in cholesterol-free medium for 24 to 48 h. The low-density lipoprotein receptor activities in untreated and in cholesterol-depleted cells were then compared. The cholesterol-depleted NS-1 cells consistently exhibited a 75 to 90% reduction in receptor-mediated low-density lipoprotein binding compared to untreated cells. The decline of the low-density lipoprotein binding of cholesterol-free medium-incubated NS-1 cells was prevented by addition of free cholesterol or its biosynthetic intermediate, demosterol, to the medium. The addition of lanosterol, an intermediate upstream to the lesion site in the cholesterol biosynthetic pathway, was completely ineffective. The results indicate that proper membrane cholesterol content is necessary for the maintenance of normal low-density lipoprotein receptor function in NS-1 cells.

Direct anesthetic-like effects of forskolin on the nicotinic acetylcholine receptors of PC12 cells.

Forskolin is thought to be a highly specific activator of adenyl cyclase. However, when applied to rat pheochromocytoma (PC12) cells at concentrations of 1 microM or higher it caused an immediate, concentration-dependent inhibition of carbachol-stimulated uptake of 86Rb+ through the nicotinic receptors, which did not appear to be related to activation of adenyl cyclase. The inhibition of receptor activation occurred instantaneously whereas cellular cAMP content did not increase for a measureable period of time. Normal receptor function was recovered rapidly upon removal of forskolin. Additional evidence that this effect of forskolin was not related to cAMP was obtained when 1,9-dideoxyforskolin (an analog of forskolin which does not activate adenyl cyclase) also caused a rapid, concentration-dependent, rapidly reversible inhibition of receptor-mediated influx of 86Rb+ into the cells. An examination of the effect of forskolin on 86Rb+ uptake at various concentrations of carbachol showed that forskolin was not acting by competing with carbachol for the receptor activation site. Given the lipophilic nature of forskolin, it probably acts like a general anesthetic to perturb the plasma membrane lipid structure and alter the function of the nicotinic acetylcholine receptors, possibly by increasing the rate of closure of open channels.

Anticomplement receptor activity in the serum of patients with primary biliary cirrhosis.

Patients with primary biliary cirrhosis have a defect in the receptor mediated clearance of complement coated erythrocytes by fixed macrophages of the reticuloendothelial system. To investigate the probable mechanism of this defect peripheral blood monocytes were isolated from nine patients with primary biliary cirrhosis and seven control subjects and the ability of these cells to form rosettes with complement coated, IgM-sensitised sheep erythrocytes was assessed. Primary biliary cirrhosis peripheral blood monocytes formed rosettes to the same extent as control peripheral blood monocytes (71.0 +/- 7.1% [SEM] versus 73.3 +/- 4.3%) suggesting normal complement receptor function of primary biliary cirrhosis peripheral blood monocytes. When primary biliary cirrhosis or control peripheral blood monocytes were preincubated with primary biliary cirrhosis serum, however, the per cent of peripheral blood monocytes that formed rosettes was decreased: 2.4 +/- 0.8 and 3.1 +/- 1.3 fold respectively. To study this phenomenon further, fractions containing IgG or IgM synthesised by cultures of control or primary biliary cirrhosis lymphocytes were prepared. Rosette formation was not affected by exposure to fractions containing control or primary biliary cirrhosis IgG or control IgM, but was markedly inhibited (6.0 +/- 4.8 fold) by exposure to fractions containing primary biliary cirrhosis IgM. Similar results were obtained when freshly isolated peripheral blood monocytes or peripheral blood monocytes that had been cultured for 7-10 days--that is, macrophages, were used. Assuming that one can draw inferences concerning the status of fixed macrophages from data obtained using peripheral blood monocytes, the results of this study suggest that the complement specific defect in reticuloendothelial system clearance function in primary biliary cirrhosis is not caused by abnormality in the functional status of complement receptors on fixed macrophages but rather by a factor present in the serum of patients with primary biliary cirrhosis that has the capacity to inhibit the adherence of complement coated erythrocytes to complement receptors present on the surface of fixed macrophages. This serum factor does not appear to be a complement component but rather a product of peripheral blood mononuclear cells, other than IgG.

A distinct glucocorticoid hormone response regulates phosphoprotein maturation in rat hepatoma cells.

Glucocorticoid hormone-dependent maturation of the mouse mammary tumor virus (MMTV) phosphorylated polyprotein (Pr74) allows experimental access to certain posttranslational regulatory circuits under steroid control in M1.54 cells, an MMTV-infected rat hepatoma cell line. Pulse-chase experiments revealed that [35S]methionine-labeled Pr74 synthesized in uninduced cells could be converted posttranslationally into p24, a stable phosphorylated maturation product, only after 4 h of exposure to 1 microM dexamethasone, a synthetic glucocorticoid. This regulated processing could be prevented by prior exposure, during the chase period, to inhibitors of RNA (actinomycin D) or protein (cycloheximide or puromycin) synthesis. Moreover, half-maximal production of p24 occurred at 10 nM dexamethasone, a concentration that approximated half-maximal receptor binding and stimulation of MMTV transcript synthesis. Kinetic, hormonal, and genetic evidence suggest that p24 expression did not require or result from the overall glucocorticoid-dependent increase in polyprotein concentration. First, 20 h after dexamethasone withdrawal, Pr74 maturation was completely deinduced, whereas the absolute level of this MMTV precursor remained 10-fold over its basal level. Second, progesterone, which competes with dexamethasone for receptor binding, facilitated the regulated production of p24 but prevented the steroid-mediated accumulation of functional MMTV mRNA. Lastly, certain glucocorticoid-responsive variants, derived from M1.54 cells by resistance to complement cytolysis, expressed p24 in the presence or absence of glucocorticoid-induced levels of Pr74. Taken together, our results suggest that the glucocorticoid-regulated maturation of MMTV phosphopolyproteins resulted from an independent hormone response that required normal receptor function and de novo RNA and protein synthesis.

A distinct glucocorticoid hormone response regulates phosphoprotein maturation in rat hepatoma cells.

Glucocorticoid hormone-dependent maturation of the mouse mammary tumor virus (MMTV) phosphorylated polyprotein (Pr74) allows experimental access to certain posttranslational regulatory circuits under steroid control in M1.54 cells, an MMTV-infected rat hepatoma cell line. Pulse-chase experiments revealed that [35S]methionine-labeled Pr74 synthesized in uninduced cells could be converted posttranslationally into p24, a stable phosphorylated maturation product, only after 4 h of exposure to 1 microM dexamethasone, a synthetic glucocorticoid. This regulated processing could be prevented by prior exposure, during the chase period, to inhibitors of RNA (actinomycin D) or protein (cycloheximide or puromycin) synthesis. Moreover, half-maximal production of p24 occurred at 10 nM dexamethasone, a concentration that approximated half-maximal receptor binding and stimulation of MMTV transcript synthesis. Kinetic, hormonal, and genetic evidence suggest that p24 expression did not require or result from the overall glucocorticoid-dependent increase in polyprotein concentration. First, 20 h after dexamethasone withdrawal, Pr74 maturation was completely deinduced, whereas the absolute level of this MMTV precursor remained 10-fold over its basal level. Second, progesterone, which competes with dexamethasone for receptor binding, facilitated the regulated production of p24 but prevented the steroid-mediated accumulation of functional MMTV mRNA. Lastly, certain glucocorticoid-responsive variants, derived from M1.54 cells by resistance to complement cytolysis, expressed p24 in the presence or absence of glucocorticoid-induced levels of Pr74. Taken together, our results suggest that the glucocorticoid-regulated maturation of MMTV phosphopolyproteins resulted from an independent hormone response that required normal receptor function and de novo RNA and protein synthesis.

Dopamine agonist activity of EMD 23,448.

EMD 23,448 was examined in tests of dopaminergic function and was found to be an atypical dopamine (DA) agonist. EMD 23,448 was a weak or inactive DA agonist when examined in tests of normal postsynaptic DA receptor function: production of stereotypy in the rat (ED50 greater than 5.0 mg/kg i.p.); production of emesis in beagles (minimum effective dose = 81 micrograms/kg i.v.); and, enhanced locomotor activity of the mouse (no excitation in doses less than or equal to 50 mg/i.p.). Moreover, EMD 23,448 was relatively weak in competing for [3H]-apomorphine binding to rat striatal membranes (Ki, 205 nM). On the other hand, this indolyl-3-butylamine did activate supersensitive postsynaptic DA receptors. Specifically, it elicited contralateral turning in rats with a unilateral 6-hydroxydopamine lesion of the substantia nigra (ED50 value = 0.9 mg/kg) and did elicit stereotypy in rats given chronic daily haloperidol treatments. EMD 23,448 also exerted pharmacological effects in tests designed to measure activation of dopamine autoreceptors. It inhibited the gamma-butyrolactone-induced increase in striatal dopa levels (ED50 = 1 mg/kg i.p.) and produced a dose-related fall in the locomotor activity of the mouse. The results are discussed and contrasted with data derived for apomorphine and the putatively selective autoreceptor agonist (+/-)-3-PPP.

Defective natural killer cytotoxicity and polymorphonuclear leukocyte antibody-dependent cellular cytotoxicity in patients with LFA-1/OKM-1 deficiency.

Four children with an immunodeficiency involving the absence of leukocyte membrane glycoproteins reacting with anti-LFA-1 and OKM-1 monoclonal antibodies were unable to mediate adherence-dependent leukocyte functions. Even with normal Fc receptor function, their PMN-ADCC and MC-NKC were markedly deficient. Single cell analysis demonstrated deficient antibody-mediated PMN-target cell adherence. Monoclonal antibodies against LFA-1 and OKM-1 reproduced this immunodeficiency in leukocytes from normal adults. LFA-1/OKM-1 mediates a PMN-target cell adhesive step.

Mutations that affect membrane receptor for LDL are useful for studying normal receptor function.

Low-density lipoprotein (LDL), the major plasma cholesterol transport protein, is taken up by cells through a receptor-mediated process. After internalization through specialized segments of the cell surface called coated pits, the LDL is degraded in the lysosome and the released cholesterol is used by cells to meet various metabolic needs. The discovery of the LDL receptor and the studies of its function have provided new insights into both the biochemical aspects of cholesterol metabolism and the cell biology of receptor-mediated endocytosis. Of paramount importance in all of these studies has been the availability of human cells that express one or more allelic mutations that affect the function of the LDL receptor. These mutations have been valuable for assessing normal receptor function. Just as important, these mutations have been used as a reference point in the development of various cytochemical and biochemical techniques for studying receptor activity.

Effect of sodium, potassium, and calcium ions on electroreceptor function in catfish

Impulses from single electroreceptors (small pit organs) of catfish (Ictalurus nebulosus) were recorded during stimulation by square pulses. Solutions with different concentrations of potassium, sodium, and calcium ions were applied to the pore of the receptor. Solutions with a low CaCl2 concentration did not alter the responses of the receptor. Calcium ions in concentrations of over 5 mM increased the threshold of the response to electrical stimulation. The threshold to anodal stimulation was increased in solutions of 2 mM sodium and potassium and no response was given to a cathodal stimulus. The effect of 2 mM solutions of NaCl and KCl was abolished by the addition of 0.4 mM CaCl2 or by application of a long anodal stimulus of high intensity (10−8∓10−7 A/mm2). Increasing the potassium ion concentration to 10–20 mM restored normal receptor function but a further increase led to elevation of the threshold. The action of an electric current is compared with the action of the ions.

Recovery of structure and function following transection of the primary olfactory nerves in pigeons.

Preliminary experimentation on ring doves to ascertain whether they might regulate any aspects of their reproductive behavior in terms of olfactory cues was vitiated by the discovery that their sectioned olfactory nerves had apparently regenerated. Concurrent work on frogs has shown that the olfactory receptors degenerate after axotomy and are replaced by new ones. This phenomenon was studied in pigeons. All transected nerves were found to be healed. Electrical recording from the regenerated nerves revealed apparently normal receptor function and, indirectly, autonomic reflex responsiveness. Previously untrained pigeons learned an olfactory discrimination after reconstitution of the peripheral olfactory system using a conditioned suppression procedure. The olfactory nerves of trained pigeons were sectioned and the behavioral response recovered within 16-82 days. The gross sizes of primary olfactory nerves and olfactory bulbs were frequently much less than those of controls, but on the ultrastructural level there was no recognizable morphological deficiency in the receptor cellular organelles or terminal synaptic contacts in bulbar glomeruli. Recent results by other workers indicate that pigeons utilize olfactory cues in homing performance.