scholarly journals Uncoupling between the insulin-receptor cycle and the cellular degradation of the hormone in cultured foetal hepatocytes. Effect of drugs and temperature that inhibit insulin degradation

1987 ◽  
Vol 246 (3) ◽  
pp. 567-573 ◽  
Author(s):  
P Soubigou ◽  
M Ali ◽  
C Plas
Keyword(s):  
Cell Surface ◽  
Insulin Receptor ◽  
Rat Hepatocytes ◽  
Receptor Internalization ◽  
Cell Surface Receptor ◽  
Insulin Degradation ◽  
Receptor Recycling ◽  
Partial Restoration ◽  
Receptor Sites ◽  
Surface Receptor

Sequential changes in the numbers of cell-surface receptors induced by a transitory exposure to insulin in cultured 18-day foetal-rat hepatocytes were investigated in the presence of drugs and at a temperature of 22 degrees C, which inhibit cellular insulin degradation. Chloroquine (70 microM) and monensin (3 microM) did not greatly change the initial rate of internalization of cell-surface receptor sites after exposure to 10 nM-insulin, but led to a steady state after 20 min, which represented 40% of the initial binding, compared with 5 min and 60% in the absence of the drug. Moreover, these drugs strongly decreased the proportion of receptor sites recovered at the cell surface after subsequent removal of the hormone. They were ineffective when insulin was not present. The removal of monensin together with the hormone allowed partial restoration of cell-surface receptor sites and degradation of cell-associated insulin to start again at the initial speed, indicating a reversible effect of the drug. During this phase, the drug concentration-dependence for the two effects showed that receptor recycling was restored with concentrations of monensin not as low as for insulin degradation. The effect of vinblastine (50-100 microM) was similar to that of chloroquine and monensin, whereas no modification in the internalization and recovery processes was observed in the presence of bacitracin concentrations (1-3 mM) that inhibit insulin degradation by 70%. A temperature of 22 degrees C did not prevent the receptor internalization, but had a slowing effect on the recycling process, which appeared to vary in experiments where insulin degradation remained inhibited. The present study shows that the process of insulin degradation mediated by receptor endocytosis is not a prerequisite for insulin-receptor recycling in cultured foetal hepatocytes.

scholarly journals Evidence that the thyrotropin-releasing hormone receptor and its ligand are recycled dissociated from each other

1995 ◽  
Vol 306 (1) ◽  
pp. 107-113 ◽  
Author(s):  
C P Petrou ◽  
A H Tashjian
Keyword(s):  
Cell Surface ◽  
Hormone Receptor ◽  
Thyrotropin Releasing Hormone ◽  
Cell Surface Receptor ◽  
Acidic Ph ◽  
Receptor Recycling ◽  
Bafilomycin A1 ◽  
Receptor Ligand ◽  
Surface Receptor ◽  
Receptor Pool

We have examined the trafficking of the thyrotropin-releasing hormone receptor (TRHR) and its ligand, after TRHR-TRH internalization in rat pituitary GH4C1 cells. After rapid ligand-induced receptor sequestration, the cell surface receptor pool was replenished. Replenishment was insensitive to inhibition of protein synthesis and was dependent on the duration of internalization; therefore, the replenished receptors were not newly synthesized but recycled. The total amount of recycled receptors decreased with increasing internalization time, resulting in only partial replenishment of the cell-surface receptor pool after prolonged incubation with ligand. Thus, in addition to a receptor recycling pathway, a non-cycling route exists for TRHR sorting; this route became dominant with increasing internalization periods. TRHR entry into these pathways was not determined by the affinity of the receptor-ligand interaction, because the extent of receptor recycling was similar after TRH- and methyl-TRH (MeTRH)-induced internalization. Unlike results with the TRHR, the TRH recycling pool was not depleted by the noncycling pathway. After multiple rounds of [3H]MeTRH internalization, the amount of cell-associated radioactivity increased with increasing internalization time due to accumulation of the ligand or its metabolites in a non-cycling pathway, but the absolute amount of recycled ligand remained constant after short or long internalization times. The difference in the proportion of TRHR and MeTRH that were diverted into a noncycling pathway indicated intracellular dissociation of the internalized TRHR-TRH complex. Dissociation of the internalized TRHR-TRH complex was dependent on the acidic pH in an intracellular compartment. Although extracellular acidic pH did not enhance cell-surface receptor-ligand (RL) dissociation, bafilomycin A1 inhibited both receptor and ligand recycling. We conclude that the TRHR-TRH system is unique among recycling receptors because, after RL sequestration, the TRHR-TRH complex becomes dissociated intracellularly via a bafilomycin A1-sensitive, acidic pH-dependent mechanism, and both the unoccupied TRHR and TRH recycle disassociated from each other.

scholarly journals THE EFFECTS OF THROMBIN ON PHYTOHEMAGGLUTININ RECEPTOR SITES IN HUMAN PLATELETS

1974 ◽  
Vol 60 (3) ◽  
pp. 541-553 ◽  
Author(s):  
John R. Feagler ◽  
Thomas W. Tillack ◽  
David D. Chaplin ◽  
Philip W. Majerus
Keyword(s):  
Cell Surface ◽  
Affinity Chromatography ◽  
Human Platelet ◽  
Human Platelets ◽  
Cell Surface Receptor ◽  
Platelet Surface ◽  
Release Reaction ◽  
Intramembranous Particles ◽  
Receptor Sites ◽  
Surface Receptor

We have previously demonstrated that lentil phytohemagglutinin (lentil-PHA) binds to human platelet membranes without causing either aggregation or the release reaction. When platelets are treated with thrombin, there is an increase in lentil-PHA binding suggesting the appearance of new receptor sites on the cell surface. We prepared a lentil-PHA-ferritin conjugate using affinity chromatography which was used to saturate cell surface receptor sites. Studies using this conjugate suggest that thrombin causes a complex change in the platelet surface involving a decrease in the number of lentil-PHA receptor sites on the external platelet surface with a marked increase in sites within the center of the canalicular system. These increased sites may result from fusion of granule membranes with the canalicular membranes during the secretion process. There is no obvious relationship between lentil-PHA receptor sites and intramembranous particles.

scholarly journals Chymotrypsin substrate analogues inhibit endocytosis of insulin and insulin receptors in adipocytes.

1986 ◽  
Vol 103 (5) ◽  
pp. 1807-1816 ◽  
Author(s):  
A L Jochen ◽  
P Berhanu
Keyword(s):  
Cell Surface ◽  
Ethyl Ester ◽  
Insulin Receptors ◽  
Cell Surface Receptor ◽  
Insulin Degradation ◽  
Receptor Mediated Endocytosis ◽  
Receptor Complexes ◽  
Related Substances ◽  
Substrate Analogues ◽  
Surface Receptor

To explore the possible role of proteolytic step(s) in receptor-mediated endocytosis of insulin, the effects of inhibitors of various classes of proteases on the internalization process were studied in isolated rat adipocytes. Intracellular accumulation of receptor-bound 125I-insulin at 37 degrees C was quantitated after rapidly dissociating surface-bound insulin with an acidic buffer (pH 3.0). Of the 23 protease inhibitors tested, only chymotrypsin substrate analogues inhibited insulin internalization. Internalization was decreased 62-90% by five different chymotrypsin substrate analogues: N-acetyl-Tyr ethyl ester, N-acetyl-Phe ethyl ester, N-acetyl-Trp ethyl ester, benzoyl-Tyr ethyl ester, and benzoyl-Tyr amide. The effect of the substrate analogues in inhibiting insulin internalization was dose-dependent, reversible, and required the full structural complement of a chymotrypsin substrate analogue. Cell surface receptor number was unaltered at 12 degrees C. However, concomitant with their inhibition of insulin internalization at 37 degrees C, the chymotrypsin substrate analogues caused a marked increase (160-380%) in surface-bound insulin, indicating trapping of insulin-receptor complexes on the cell surface. Additionally, 1 mM N-acetyl-Tyr ethyl ester decreased overall insulin degradation by 15-20% and also prevented the chloroquine-mediated increase in intracellular insulin, further indicating that surface-bound insulin was prevented from reaching intracellular chloroquine-sensitive degradation sites. The internalization of insulin receptors that were photoaffinity labeled on the cell surface with B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin was also inhibited 70-90% by the five chymotrypsin substrate analogues, as determined by the effects of the analogues on the accumulation of trypsin-insensitive (intracellular) 440-kD intact labeled receptors. In summary, these results show that chymotrypsin substrate analogues efficiently inhibit the internalization of insulin and insulin receptors in adipocytes and implicate a possible role for endogenous chymotrypsin-like enzyme(s) or related substances in receptor-mediated endocytosis of insulin.

scholarly journals The Glucagon-like Peptide-2 Receptor C Terminus Modulates β-Arrestin-2 Association but Is Dispensable for Ligand-induced Desensitization, Endocytosis, and G-protein-dependent Effector Activation

2005 ◽  
Vol 280 (23) ◽  
pp. 22124-22134 ◽  
Author(s):  
Jennifer L. Estall ◽  
Jacqueline A. Koehler ◽  
Bernardo Yusta ◽  
Daniel J. Drucker
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Receptor Expression ◽  
Receptor Internalization ◽  
Cell Surface Receptor ◽  
Receptor Desensitization ◽  
C Terminus ◽  
The Family ◽  
Surface Receptor ◽  
Glucagon Like Peptide

Classic models of receptor desensitization and internalization have been largely based on the behavior of Family A G-protein-coupled receptors (GPCRs). The glucagon-like peptide-2 receptor (GLP-2R) is a member of the Family B glucagon-secretin GPCR family, which exhibit significant sequence and structural differences from the Family A receptors in their intracellular and extracellular domains. To identify structural motifs that regulate GLP-2R signaling and cell surface receptor expression, we analyzed the functional properties of a series of mutant GLP-2Rs. The majority of the C-terminal receptor tail was dispensable for GLP-2-induced cAMP accumulation, ERK1/2 activation, and endocytosis in transfected cells. However, progressive truncation of the C terminus reduced cell surface receptor expression, altered agonist-induced GLP-2R trafficking, and abrogated protein kinase A-mediated heterologous receptor desensitization. Elimination of the distal 21 amino acids of the receptor was sufficient to promote constitutive receptor internalization and prevent agonist-induced recruitment of β-arrestin-2. Site-directed mutagenesis identified specific amino acid residues within the distal GLP-2R C terminus that mediate the stable association with β-arrestin-2. Surprisingly, although the truncated mutant receptors failed to interact with β-arrestin-2, they underwent homologous desensitization and subsequent resensitization with kinetics similar to that observed with the wild-type GLP-2R. Our data suggest that, although the GLP-2R C terminus is not required for coupling to cellular machinery regulating signaling or desensitization, it may serve as a sorting signal for intracellular trafficking. Taken together with the previously demonstrated clathrin and dynamin-independent, lipid-raft-dependent pathways for internalization, our data suggest that GLP-2 receptor signaling has evolved unique structural and functional mechanisms for control of receptor trafficking, desensitization, and resensitization.

scholarly journals Quantitative studies of the rate of insulin internalization in isolated rat hepatocytes

1984 ◽  
Vol 218 (2) ◽  
pp. 307-312 ◽  
Author(s):  
B Draznin ◽  
M Trowbridge ◽  
L Ferguson
Keyword(s):  
Cell Surface ◽  
Insulin Receptor ◽  
Insulin Receptors ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Receptor Internalization ◽  
Insulin Concentration ◽  
Receptor Complex ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat

We studied internalization of 125I-labelled insulin in isolated rat hepatocytes. Using the acidification technique, we were able to dissociate the ligand from its cell-surface receptors, and thus to separate internalized from surface-bound insulin. Because during the first 5 min of incubation of 125I-labelled insulin with freshly isolated hepatocytes there is no loss of internalized label, the ratio of the amount of internalized ligand to the amount of cell-surface-bound ligand may serve as an index of insulin internalization. Within the first 10 min of insulin's interaction with hepatocytes, the plot of the above ratio as a function of time yields a straight line. The slope of this line is referred to as the endocytic rate constant (Ke) for insulin and denotes the probability with which the insulin-receptor complex is internalized in 1 min. At the insulin concentration of 0.295 ng/ml, the Ke is 0.049 min-1. It is independent of insulin concentration until the latter exceeds 1 ng/ml. At the insulin concentration of 3.2 ng/ml, the Ke accelerates to 0.131 min-1. With the Ke being the probability of insulin-receptor-complex internalization, 4.9% of occupied insulin receptors will be internalized in 1 min at an insulin concentration of 0.295 ng/ml, and 13.1% of occupied insulin receptors will be internalized in 1 min at 3.2 ng/ml. When the insulin concentration decreases from 3.2 to 0.3 ng/ml, the Ke decreases accordingly. The half-time of occupied receptor internalization is 15.4 min at the lower insulin concentration and 5.3 min at the higher insulin concentration.

Expression, function, and localization of the REG cell surface receptor

2001 ◽  
Vol 120 (5) ◽  
pp. A18-A19
Author(s):  
B DIECKGRAEFE ◽  
C HOUCHEN ◽  
H ZHANG
Keyword(s):  
Cell Surface ◽  
Cell Surface Receptor ◽  
Surface Receptor
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