Rate Of Receptor Loss Research Articles

The Regulation of Agonist-Stimulated Phospholipase D Activity in Swiss 3T3 Fibroblasts

The work in this thesis aimed to characterise the regulation of phospholipase D (PLD) activity by receptor occupancy in Swiss 3T3 fibroblasts. Bombesin-stimulated PLD activity had previously been found to be partially dependent on protein kinase C (PKC) suggesting that activation of PLD was to some extent down stream of the G-protein coupled receptor activated phosphatidylinositol 4,5, bisphosphate (PtdIns(4,5)P2) hydrolysis (Cook et al., 1992). Using a permeabilised cell system to introduce non-hydrolysable analogues of guanine-nucleotides, bombesin-stimulated PLD activity was found to be indirectly regulated by a G-protein. The phosphorylation of proteins on tyrosine was determined using immunoblotting with a monoclonal anti-phosphotyrosine antibody. The elevation of tyrosine phosphorylation using pervanadate, an inhibitor of tyrosine phosphatases, was found to activate PLD activity. PLD activity was found to be reduced by pretreatment with the tyrosine kinase inhibitor Genistein. Pretreatment with both Genistein and the selective PKC inhibitor Ro-31-8220 reduced bombesin-stimulated PLD activity to basal. Elevation of Ca2+ from intracellular pools using thapsigargin, an inhibitor of the Ca2+-ATPase on the intracellular stores was found to be insufficient to activate PLD alone nor did it affect bombesin-stimulated PLD activity. Elevation of cyclic AMP using forskolin and dibutyrylcyclicAMP did not affect bombesin-stimulated PLD activity nor did it alone affect the enzyme. A common event in cells exposed to agonist is a loss of responsiveness to the stimulant, referred to as desensitisation. Bombesin-stimulated PLD activity was found to desensitise rapidly with a complete loss in the response after a 40 second agonist exposure. Desensitisation was reversible and occurred within 4.5 minutes of agonist removal, however was never complete. In the continuous presence of agonist resensitisation of PLD activity occurred. The resensitised rate was reduced from that generated in response to the initial stimulus but activity continued over a period of an hour. The recovery of bombesin-stimulated total inositol phosphate generation commenced later than that of bombesin-stimulated PLD activation and reached completion within 11.5 minutes after removal of the desensitising stimulus. Radio-receptor binding studies showed that there was a transient loss of binding sites from the cell surface which was due to internalisation and recycling of the receptor. Although the rate of receptor loss and recovery paralleled that of the desensitisation and resensitisation of PLD activity, differences were observed in the extent of the two processes. Readdition of bombesin was essential for a stimulation of PLD activity to be observed on resensitisation of the enzyme. Hence degradation of the ligand was occurring before the receptor was recycled. Desensitisation and resensitisation was independent of PKC activation and a desensitising pretreatment with bombesin did not affect the PMA- stimulated PLD activity. A23187-stimulated PLD activity also underwent homologous desensitisation and could not stimulate enzyme activity after prior desensitisation by bombesin pretreatment. Thus desensitisation was not due to a decrease in intracellular Ca2+ levels. Bombesin also induced heterologous desensitisation. Pretreatment with bombesin or vasopressin reduced a subsequent stimulation with the other agonist by approximately 70%. A desensitising pretreatment with either agonist attenuated the GTP?S-stimulated PLD activity in permeabilised cells by approximately 50%. It was thus proposed that desensitisation not only affected the cell surface receptors but also occurred at or distal to the receptor-coupled G-protein. These findings support a role for PLD-derived PtdOH in mitogenic signalling, in view of the resensitisation and subsequent maintenance of PLD activity in an early mitogenic situation when the agonist is continually present. The kinetics of the agonist-stimulated PLD activity are discussed with relation to the regulatory pathways of PLD activation and mitogenic signalling.

Subcellular compartmentation of opioid receptors: modulation by enkephalin and alkaloids.

A subclone of NG108-15 neuroblastoma-glioma hybrid cells was used to study the intracellular distribution of opioid receptors. Subcellular organelles were separated on self-generating Percoll-sucrose gradients and the enzymes beta-glucuronidase, galactosyltransferase, 5'-nucleotidase, and glucose-6-phosphatase were used as markers to localize the various structures. Analysis of the receptor distribution from untreated cells shows that the plasma membranes contained the highest receptor density, but a significant portion of the opioid binding sites was unevenly distributed between the lysosomes, microsomes, and Golgi elements. The enzyme markers indicated that appearance of opioid receptors in these intracellular structures does not result merely from contamination with plasma membranes. About 11% of the receptors appeared in a fraction lighter than plasma membranes. The antilysosomal agent chloroquine altered the intracellular compartmentation of the receptors, possibly by blocking their translocation in the cells. Leu-enkephalin induced time-dependent loss of receptors from all four intracellular compartments examined, but a kinetic analysis showed that the rate of receptor loss in these fractions was not identical. Thus, the percent of receptors appearing in the lysosomal fraction that could still bind [3H]D-Ala2-D-Leu5-enkephalin in vitro was increased on treatment with Leu-enkephalin. As an additional approach to follow the intracellular fate of the receptors, cells were labeled with [3H]diprenorphine, chased with various unlabeled opiates, and the distribution of 3H-ligand-receptors in the cells was monitored. Leu-enkephalin and etorphine altered the distribution of receptor-bound [3H]diprenorphine between the plasma membranes, lysosomes, and Golgi elements, whereas morphine had no such effect. The study sheds light on the role of intracellular structures in the metabolism of opioid receptors in untreated and opioid-treated cells.

Dissociation of the triiodothyronine receptor from the nucleus induced by cations: evidence for divalent cation sensitive and insensitive nuclear sites.

Aliquots of purified rat liver nuclei were diluted at 0 degrees C in isotonic buffers containing monovalent (Na+) or divalent (Ca2, Mg2+) cations. At different times following dilution the nuclear suspensions were centrifuged and the T3 receptor was measured in KCl extracts of the nuclear pellets. The rate of receptor loss after dilution in EDTA was 0.0025 min-1. Dilution in the presence of cations caused a fast release of receptor during the first 10 min. This phase, which was not observed when the nuclei were diluted in EDTA without salt, was followed by a second phase where the receptor was released at the same rate as in EDTA. Receptor release was only dependent on the presence of cations in the dilution buffer during the first 10 min after dilution. The amounts of receptor remaining in the nuclei after the first 10 min of dilution were 51.8 +/- 9.2%, in the presence of Ca2+ and Mg2+, 38.6 +/- 8.9% in 0.15 M NaCl, and 18.0 +/- 4.8% in 0.15 M NaCl in the presence of Ca2+ and Mg2+. The release of receptor was not influenced by the integrity of the nuclear membrane. These results suggest the presence of divalent cation sensitive and insensitive nuclear sites for the T3 receptor, in amounts which could be estimated to be about 48 and 52%, respectively. Other interpretations are also possible, such as the presence of a high proportion of free receptors in the nucleosol, which could be released during the first phase of dilution if the negative charges in chromatin are blocked by cations to avoid redistribution of receptors immediately after dilution.

Insulin receptor degradation is accelerated in cultured lymphocytes from patients with genetic syndromes of extreme insulin resistance.

Previous studies of the insulin receptor in disease states have utilized primarily techniques of equilibrium binding and, to a limited extent structural, analysis. Though techniques have been developed to study receptor degradation in normal cells, they have not been applied to disease states. In the present study we have examined insulin receptor degradation rate in B lymphocytes that were obtained from peripheral blood of normal subjects and patients with several syndromes of extreme insulin resistance. B lymphocytes were established in culture from each patient's peripheral cells by transformation with Epstein-Barr virus. The insulin receptors were surface labeled using Na125I/lactoperoxidase and the cells were returned to incubate in growth media. After varying periods of incubation, aliquots of cells were solubilized and the cell content of labeled receptor subunits were measured by immunoprecipitation with anti-receptor antibodies and NaDodSO4/polyacrylamide gel electrophoresis. The fall in 125I-insulin receptor content approximated a single exponential and was quantitated as receptor subunit half-life (t1/2). In cell lines from four patients in whom the number of insulin receptors was reduced by greater than 90%, the rate of receptor loss was greater than normal (t1/2 equals 3.8 +/- 0.9 h vs. 6.5 +/- 1.2 h; mean +/- SD, P less than 0.01). However, a similar acceleration in receptor degradation was seen in cells from five patients with extreme insulin resistance but low-normal insulin receptor concentration (t1/2 equals 4.4 +/- 0.9 h). This group included cells from one patient with a qualitatively abnormal receptor. Thus, all the patients with genetic syndromes of insulin resistance had accelerated receptor degradation, regardless of their receptor concentration. By contrast, insulin receptors on cultured lymphocytes that were obtained from patients with extreme insulin resistance secondary to autoantibodies to the insulin receptor had normal receptor degradation (t1/2 equals 6.1 +/- 1.9 h). We conclude that (a) accelerated insulin receptor degradation is an additional feature of cells from patients with genetic forms of insulin resistance; (b) that accelerated insulin receptor degradation may explain the low-normal receptor concentrations that were seen in some patients with extreme insulin resistance; and (c) that accelerated degradation does not explain the decreased receptor concentration in patients with very low insulin receptor binding and, therefore, by inference, a defect in receptor synthesis must be present in this subgroup.

Temperature shifts induce the selective loss of alveolar-macrophage plasma membrane components.

A shift in the incubation temperature of rabbit alveolar macrophages (0 degree C leads to 37 degrees C leads to 0 degree C) resulted in a 40-60% reduction in the ability of cells to bind alphamacroglobulin. 125I-trypsin complexes (alphaM. 125I-T). The reduction in binding activity did not reflect a disruption of cell integrity since the levels of intracellular components (lactate dehydrogenase, beta-N-acetyl-hexosaminidase) or other plasma membrane components (alkaline phosphodiesterase) were unaltered. Analysis of receptor-ligand interaction indicated that the temperature shift effected a decline in receptor number rather than an alteration in ligand-receptor affinity. Studies indicated that a temperature shift resulted in the loss of unoccupied receptors, and that ligand bound to receptors was not lost. However, after ligand internalization, receptors were removed by the temperature shift. The rate of receptor loss was maximal when cells were incubated at temperatures greater than 24 degrees C. Receptor loss was not prevented by treatment of cells with colchicine, cytochalasin B, or N-ethylamaleimide, but was prevented by treatment with the cross-linking agent paraformaldehyde. Data indicate that the reduction in alphaM. 125I-T binding activity resulted from shedding of receptors into the media since media obtained from temperature-shifted cells contained material that competed with cell-bound receptors for alphaM. 125I-T. Additionally, binding of alphaM. 125I-T was diminished on membrane fragments obtained from temperature-shifted cells. Incubation with Triton X-100, of cells whose receptors were occupied with alphaM. 125I-T, led to the extraction of 40% of cell-bound activity. However, no radioactivity was extracted from cells labeled with alphaM. 125I-T after a temperature shift. Measurement of ligand accumulation by control and temperature-shifted cells incubated at 20 degrees C indicated that control cells exhibited a subpopulation of receptors capable of binding ligand but only slowly internalizing it. This subpopulation was not present on temperature-shifted cells. These results indicate that surface receptors for alphamacroglobulin . protease complexes are heterogeneous and that the temperature shift resulted in the selective loss of membrane components.

Regulation of insulin receptors in cultured human fibroblasts.

The ability of insulin to regulate its own receptor was investigated in cultured human fibroblasts. Physiological insulin concentrations led to a loss of insulin receptors, and this process was dependent on cellular energy and ongoing protein synthesis. The insulin concentration primarily affected the rate of receptor loss rather than the eventual cellular receptor concentration. Studies of receptor regeneration revealed that the absolute rate of insertion of new or recycled receptors into the plasma membrane was constant and independent of the amount of proceeding receptor loss or the time over which receptor loss occurred. These results are compatible with the concept that insulin-induced receptor loss in cultured human fibroblasts is a self-limited process mediated by an endocytotic internalization pathway which is dependent on new protein synthesis. Receptor regeneration is a distinct cellular process dependent on ongoing protein synthesis and does not imply represent the absence of ongoing receptor loss.

Characterization of insulin-induced receptor loss and evidence for internalization of the insulin receptor.

We studied the relationship between insulin-induced receptor loss and insulin internalization in isolated rat adipocytes. When cells were exposed to 100 ng/ml insulin at 37°C for times ranging from 10 min to 6 h, a monoexponential loss of insulin receptors was observed for up to 4 h, with a t½ of 75 min and a 75% maximal loss. The insulin-induced loss of receptors was dose-dependent with a % maximal effective insulin concentration of 7.6 and 1.3 ng/ml in 2- and 6-h insulin- pretreated cells, respectively. The ability of insulin to mediate receptor loss was inhibited by low temperature (16°C) and by the metabolic energy depleters dinitrophenol and NaF, suggesting that receptor loss is mediated by an 125I-insulin compared with control cells, and the relationship between insulin-induced receptor loss and insulin internalization was assessed by comparing these two processes over time and as a function of temperature. Using this approach, a significant linear relationship was observed between these partners at various temperatures (r = 0.99) and times (r = 0.94). These results suggest that the mechanism whereby insulin induces receptor loss in adipocytes is by internalization of insulin-receptor complexes. Insulin receptors remaining on 2-h insulin, pretreated cells had identical dissociation rates and exhibited similar negative cooperative interactions when compared with control cells. Furthermore, anti-insulinreceptor antibodies inhibited binding in both groups of cells to the same extent. In contrast to these similarities, insulin-induced receptor loss was accompanied by a progressive impairment in the ability of cells to accumulate intracellular insulin in the presence ofchloroquine, which we interpret as an insulin-induced slowing of insulin internalization. In conclusion, (1) insulin causes a time-, temperature-, dose-, and energy-dependent loss of cell-surface insulin receptors; (2) the rate of receptor loss and the extent of insulin internalization are closely correlated, suggesting that insulin-induced receptor loss in adipocytes is mediated by endocytosis of insulin-receptor complexes; and (3) insulin-induced receptor loss is accompanied by a reduced ability of residual receptors to internalize insulin.