Rapid, reversible internalization of cell surface insulin receptors. Correlation with insulin-induced down-regulation

Chronic treatment of 3T3-C2 fibroblasts with insulin causes the slow (t1/2 = 3-4 h) down-regulation of cellular insulin receptor to a new steady state level by accelerating receptor decay (Knutson, V.P., Ronnett, G.V., and Lane, M.D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2822-2826). In the present investigation, the synthesis and turnover of the receptor during the transition to the down-regulated state was examined by the heavy isotope density-shift method. It was observed that within two h after insulin addition, receptor decay increased abruptly for several hours then gradually declined until the "down-regulated" rate was achieved. The abrupt increase in receptor decay induced by insulin was preceded by a more rapid (t1/2 less than or equal to 10 min) translocation of cell surface receptor to an "intracellular" trypsin-resistant compartment. Thus, upon exposure to ligand, insulin receptor rapidly redistributes from the cell surface to an intracellular compartment, without an initial net loss of cellular receptors. The translocation process was rapidly reversed (t1/2 less than or equal to 20 min) upon removal of insulin. With prolonged exposure to insulin, the initial rapid translocation of receptor was followed by a slower inactivation of receptor apparently in the intracellular compartment. Cycloheximide, which lengthens receptor half-life by blocking a step in receptor inactivation, had no effect on receptor internalization. Internalization of insulin receptor and its bound ligand were, however, rapidly (less than 10 min) blocked by phenylarsine oxide. These results support the following sequence of events. Upon exposure to ligand, insulin receptors are translocated from the cell surface to an intracellular site which results in accelerated receptor decay and ultimately to a lower steady state cellular receptor level.     

Short-term disappearance of muscarinic cell surface receptors in carbachol-induced desensitization

N6-Phenylisopropyladenosine was employed in the absence of endogenous adenosine to explore the influence exerted by the R-site over the antagonistic interaction of insulin and catecholamines on several parameters of fat cell metabolism. When no hormones were present, N6-phenylisopropyladenosine had little or no effect; however, the nucleoside potentiated insulin inhibition of catecholamine-stimulated events, such as lipolysis, and, conversely, diminished or blocked catecholamine inhibition of insulin-stimulated processes, such as 2-deoxyglucose uptake, glucose oxidation and esterification, even under conditions where N6-phenylisopropyladenosine, alone, was ineffective in reversing catecholamine actions.     

Cyclic AMP-induced reversible decrease in cAMP-binding to cell surface receptors of Dictyostelium discoideum

Abstract Adaptation may be the result of a change in affinity and/or number of cAMP-binding sites at the cell surface. To test this possibility we used agip 53, a mutant that does not synthesize cAMP in response to cAMP stimulation. cAMP induced a fast decrease in cAMP-binding to aggregation-competent cells, which reached a maximum at 10–20 s and was reversible with a t _0.5 of about 70 s. The decrease in cAMP-binding involved 46000 sites per cell and was mainly due to a reduction in the apparent affinity for cAMP-binding and to a smaller extent to slowly dissociating cAMP. Our results suggest that under these conditions only a fraction of the cAMP-binding sites at the cell surface are involved in transmembrane signalling, which is indeed observed for many of the physiological responses in Dictyostelium discoideum .     

Rapid recycling of beta-adrenergic receptors is dependent on the actin cytoskeleton and myosin Vb

For the beta(2)-adrenergic receptor (beta(2)AR), published evidence suggests that an intact actin cytoskeleton is required for the endocytosis of receptors and their proper sorting to the rapid recycling pathway. We have characterized the role of the actin cytoskeleton in the regulation of beta(2)AR trafficking in human embryonic kidney 293 (HEK293) cells using two distinct actin filament disrupting compounds, cytochalasin D and latrunculin B (LB). In cells pretreated with either drug, beta(2)AR internalization into transferrin-positive vesicles was not altered but both agents significantly decreased the rate at which beta(2)ARs recycled to the cell surface. In LB-treated cells, nonrecycled beta(2)ARs were localized to early embryonic antigen 1-positive endosomes and also accumulated in the recycling endosome (RE), but only a small fraction of receptors localized to LAMP-positive late endosomes and lysosomes. Treatment with LB also markedly enhanced the inhibitory effect of rab11 overexpression on receptor recycling. Dissociating receptors from actin by expression of the myosin Vb tail fragment resulted in missorting of beta(2)ARs to the RE, while the expression of various CART fragments or the depletion of actinin-4 had no detectable effect on beta(2)AR sorting. These results indicate that the actin cytoskeleton is required for the efficient recycling of beta(2)ARs, a process that likely is dependent on myosin Vb.     

Rapid disappearance of translatable actin mRNA during cell differentiation in Naegleria

Actin, the major protein of Naegleria gruberi, is selectively not synthesized during the differentiation of amebae to flagellates. When RNA extracted from cells at intervals during differentiation is translated in the wheat germ cell-free system, a major translation product with the electrophoretic mobility of actin is seen to disappear with time during differentiation. This translation product is shown to be actin by its electrophoretic mobility, copolymerization with rabbit actin, peptide map, and immunoprecipitation by antibodies specific to Naegleria actin. Multiple isoforms of actin are synthesized in the cell-free system. Quantitative immunoprecipitation of translation products was employed to measure the relative amount of actin mRNA. Translatable actin mRNA begins to decrease in abundance within 7 min after the initiation of differentiation and thereafter decreases with a half-life of about 25 min. The selective disappearance of this major translatable mRNA provides a favorable opportunity to dissect the rules governing the half-life of a specific mRNA.     

Evaluation of the negative cooperativity model for fat cell beta-adrenergic receptors

Cooperative site-to-site interactions among beta-adrenergic receptors of fat cell membranes are probed with the potent beta-adrenergic antagonist (?)-[³H]-dihydroalprenolol according to the kinetic method of De Meyts et al. (De Meyts, P., Roth, J., Neville, Jr., D.M., Gavin, III, J.R. and Lesniak, M.A. (1973) Biochem. Biophys. Res. Commun. 55, 154–161). Dissociation of specific (?)-[³H]dihydroalprenolol binding from fat cell membranes following a 100-fold dilution was rapid at 37°C; only 40% of the initial equilibrium binding remained 30 s after dilution. Dissociation of (?)-[³H]dihydroalprenolol bound under conditions yielding approximately 20% initial occupancy was performed in the absence and in the presence of a large molar excess of beta-adrenergic agonist ((?)-isoproterenol) or beta-adrenergic antagonist ((?)-alprenolol or(?)-propanalol). Neither agonists nor antagonists influenced the rate of (?)-[³H]dihydroalprenolol dissociation from fat cell membranes performed at 4, 22 or 37°C. Although analysis of the steady-state binding of (?)-[³H]-dihydroalprenolol to fat cell membranes yields Hill coefficients, n_H, less than 1.0, the present study indicates that these fat cell beta-adrenergic receptors display no cooperative site-to-site interactions.     

Heterogeneity of cell surface endothelin receptors

Two distinct cell surface endothelin receptors were identified, namely a 73-kDa protein referred to as ET-R1 and a 60-kDa protein named ET-R2. ET-R1 was expressed as the sole endothelin receptor on rat A10 vascular smooth muscle cells and C6 glial cells. Binding of 125I-ET-1 to these cells was inhibited by 50-200 pM endothelin-1 and -2, whereas endothelin-3 did not compete for this receptor subtype. Binding of 125I-ET-1 to intact A10 and C6 cells was reversible, indicating that ET-R1 is located on the cell surface. Affinity labelling of a single 73-kDa band on sodium dodecyl sulfate-polyacrylamide gels by 125I-ET-1 in A10 and C6 cells was inhibited by endothelin-1 but not by endothelin-3. In A10 cells, endothelin-1 but not endothelin-3 elicited a concentration-dependent increase in intracellular inositol trisphosphate levels. ET-R1 was also expressed in cultured rat glomerular mesangial cells based on findings of a subset of receptors with an apparent molecular mass of 73 kDa that bound 125I-ET-1 displacable by endothelin-1 and endothelin-2 but not by endothelin-3. These cells also expressed the ET-R2 receptor subtype, based on findings of a 60-kDa binding site that could be labeled by both 125I-ET-1 and 125I-ET-3. Labeling of ET-R2 by the radioactive endothelins-1 and -3 was inhibited competitively by endothelins-1, -2, and -3. Furthermore, ET-R2 was shown to be a functional receptor, as endothelin-3 caused inositol trisphosphate levels to rise in mesangial cells. An endothelin binding site with high affinity for endothelin-3 was also identified on rat PC12 pheochromocytoma cells, although the apparent molecular mass of this receptor could not be verified by cross-linking studies. Since endothelin-1 or -3 failed to augment inositol trisphosphate levels in these cells, this binding site could represent a third endothelin receptor subtype. Thus, two distinct functional receptors for endothelins were identified on rat cells, namely the 73-kDa ET-R1 which has an exceedingly low affinity for endothelin-3 and the 60-kDa ET-R2 which binds endothelin-3 with high affinity. Whether an additional endothelin receptor subtype exists in PC12 cells remains to be shown with certainty.     

Structural analysis of purified beta-adrenergic receptors

We have characterized the structure of purified beta-adrenergic receptors by a combination of photoaffinity labeling, high performance liquid chromatography (HPLC)-tryptic mapping, CNBr fragmentation, target size analysis, and electron microscopy of purified receptor molecules. Guinea pig lung beta-adrenergic receptors purified by affinity chromatography, ion exchange chromatography, and HPLC size exclusion chromatography or photoaffinity labeled with [125]-iodocyanopindolol diazirine displayed mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that corresponded to Mr = 68,000. Purified, radioiodinated guinea pig lung beta-receptors were subjected to complete trypsin digestion and subsequent reverse-phase HPLC analysis, which revealed nine peptides. Active site labeling and tryptic digestion of partially purified hamster lung beta-receptors produced one peptide, whereas CNBr digestion of the same material produced two labeled fragments, yielding information about the location of the active site within the primary sequence. Purified guinea pig lung receptors were examined with transmission electron microscopy. Electron micrographs revealed slightly asymmetric, rod-shaped structures with an average length of 13 nm and width of 3.4 nm. Many receptors were arranged as apparent dimeric structures. These findings confirm data obtained from target size analysis of guinea pig lung beta-receptors in situ which suggest that receptors may exist as oligomeric arrays in the native membrane. Taken together, these data provide information about putative functional domains of the beta-adrenergic receptor and its quaternary structure.     

Functional integrity of desensitized beta-adrenergic receptors

The adenylate cyclase-coupled beta 2-adrenergic receptor of the frog erythrocyte has served as a useful model system for elucidating the mechanisms of catecholamine-induced densensitization. In this system, it has been previously demonstrated that agonist-induced refractoriness is associated with sequestration of the beta-adrenergic receptors in vesicles away from the cell surface and from their effector unit, the adenylate cyclase system (Stadel, J.M., Strulovici, B., Nambi, P., Lavin, T.N., Briggs, M.M., Caron, M.G., and Lefkowitz, R.J. (1983) J. Biol. Chem. 258, 3032-3038). These internalized beta-adrenergic receptors appear to be structurally intact as assessed by photoaffinity labeling, but their functional status has previously been unknown. In the present studies, we sought to assess the functionality of the sequestered vesicular receptors by fusing them to Xenopus laevis erythrocytes. This cell is suitable for such studies, since it has almost no detectable beta-adrenergic receptor or catecholamine-sensitive adenylate cyclase, but contains prostaglandin E1-stimulable adenylate cyclase. Fusion of beta-adrenergic receptor-containing vesicles from desensitized frog erythrocytes with X. laevis erythrocytes results in a 30-fold stimulation of the hybrid adenylate cyclase by the beta-adrenergic agonist isoproterenol. This effect was entirely blocked by the beta-antagonist propranolol. The catecholamine-sensitive adenylate cyclase activity established in the vesicle-Xenopus hybrids showed the characteristic agonist potency series of the donor frog erythrocyte beta 2-adrenergic receptor. Fusion of vesicles from desensitized frog erythrocytes in which the beta-adrenergic receptors had been inactivated with the group specific reagent dicyclohexylcarbodiimide, or of vesicles derived from control frog erythrocytes, which contain low amounts of beta-adrenergic receptor, did not establish catecholamine-sensitive adenylate cyclase activity in the hybrids. These data demonstrate that beta-adrenergic receptors internalized during desensitization retain their functionality when recoupled to an adenylate cyclase system from a different source. The functional uncoupling of these receptors during desensitization is thus more likely due to their sequestration away from the other components of the adenylate cyclase than to any alterations in the receptors themselves.     

Human fat cell beta-adrenergic receptors: beta-agonist-dependent lipolytic responses and characterization of beta-adrenergic binding sites on human fat cell membranes with highly selective beta 1-antagonists

Beta-adrenergic receptors were characterized in human fat cell membranes using 125I-labeled cyanopindolol (125I-labeled CYP) and highly selective beta 1-antagonists. The iodinated radioligand bound saturably and specifically to a single class of high affinity binding sites. The number of binding sites determined with 125I-labeled CYP closely agreed with that determined with two other tritiated radioligands: [3H]dihydroalprenolol and [3H]CGP-12,177. Since 125I-labeled CYP does not discriminate between beta 1- and beta 2-adrenoceptors, the densities of the two receptor subtypes were determined from the competition curves of 125I-labeled CYP by highly selective beta 1-antagonists (bisoprolol, ICI-89,406, CGP-20,712A, and LK-204,545). Moreover, in order to enable correlation with binding data, the regulation of adenylate cyclase activity and of lipolysis was tested with various beta-agonist and antagonist compounds. The results obtained on fat cell membranes from abdominal subcutaneous adipose tissue demonstrated the following. 1) 125I-labeled CYP represents a valuable tool for the quantification and the delineation of beta-receptor subtypes. 2) The presence of sodium ions in binding buffers causes a modification of the affinity of beta-sites for some beta-antagonists. 3) The human fat cell beta adrenergic receptor population defined by nonselective radioligands is composed of two subtypes that can be interpreted in terms of classic beta 1- and beta 2-adrenergic receptor subtypes as assessed by competition studies with highly selective antagonists; beta 2-sites are predominant (60-70% of 125I-labeled CYP sites) in the adipocytes of slightly overweight women. 4) Results support the idea that beta 1- as well as beta 2-adrenergic receptors are coupled with adenylate cyclase and involved in the induction of lipolysis. 5) The results focus on the interest in some beta 2-agonist drugs (zinterol, clenbuterol) as partial inductors of lipolysis, with the lipolytic efficacies of these compounds being well correlated with their efficacies at 125I-labeled CYP sites.     

Rapid small-scale preparation method of cell surface polysaccharides

A rapid small-scale method of extraction of lipopolysaccharide (LPS) and capsular polysaccharides was developed for the purpose of identification of chemotypes of LPS and serotypes of capsular antigens. Cell surface polysaccharides were prepared within less than 2 hr from 1.5 ml of broth or suspension of colonies cultured overnight. The preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for LPS, and by double diffusion gel precipitation (Ouchterlony) test and blotting to nitrocellulose membrane for capsular polysaccharide. The analyses with the preparations obtained by the method could provide adequate results capable of identifying chemotypes of LPS and serotypes of capsular antigens.     

Role of specific cell surface receptors in thrombin-stimulated cell division

Previous studies have shown that thrombin action at the cell surface is sufficient to bring about division of cultured fibroblast-like cells (Carney and Cunningham, 1978). This prompted the present binding experiments with ¹²⁵I-thrombin which led to the Identification of a thrombin receptor on the surface of mouse embryo cells. Scatchard plots of binding data at 4, 22 and 37°C were linear over a broad range of thrombin concentrations, indicating a single affinity class of receptors. The association constant was about 1 × 10⁹ M^?1 and there were approximately 2 × 10⁵ receptors per cell. Neither insulin, epidermal growth factor nor prothrombin competed for thrombin binding to its receptor, indicating that It was unique for thrombin. Comparisons of thrombin binding and the amount of cell division produced by various concentrations of thrombin indicated that there was a relationship between receptor occupancy and increase in cell number. Low concentrations of serum (0.1%) inhibited both the mitogenic action of thrombin and the specific binding of thrombin to its receptor. It did not, however, inhibit nonspecific association of ¹²⁵I-thrombin with the cells. Experiments showed that this inhibition by serum resulted from a masking of thrombin receptors on the cells and not from binding of thrombin by serum factors. Together these studies suggest that thrombin must bind to Its surface receptor to stimulate cell division.