Rate Of Receptor Internalization Research Articles

Heterodimerization and functional interaction between EGF receptor family members: a new signaling paradigm with implications for breast cancer research.

The EGF receptor (EGFR) and HER2 are members of a growth factor receptor family. Overexpression of either protein in advanced breast cancer correlates with poor prognosis. EGF stimulates growth by binding to EGFR, activating the receptor's intracellular tyrosine kinase. The initial consequence is phosphorylation of specific tyrosine-containing sequences in the receptor's carboxyl terminus. These phosphotyrosines serves as high affinity recognition sites for proteins that, in turn, transmit the growth signal inside the cell. Mechanistic studies suggest that EGF binds to a single EGFR, triggering dimerization with another like receptor molecule. This dimerization is thought to initiate the tyrosine kinase activation. The EGF receptor family was recently expanded with the sequencing of HER3 and HER4. Each of the four family members was postulated to regulate a unique growth or differentiation signaling repertoire when activated by a receptor-specific ligand. However, new data from numerous laboratories suggest that EGFR family members may play a complex and ultimately more flexible role in signaling by forming heterodimers between family members, e.g. EGFR:HER2 or HER4:HER2. These heterodimers may form even when only one member of the pair binds its ligand. This review summarizes current work on heterodimerization and attempts to predict the consequences for downstream signaling. In brief, when compared to ligand-dependent receptor homodimers comprised of two proteins with the same internalization sequence and phosphorylated tyrosine residues, heterodimers are likely to: i) expand substrate selection and downstream signaling pathway activation; ii) promote interaction between sets of substrates in the mixed receptor complexes that would not ordinarily be physically juxtaposed; iii) alter the duration of receptor signaling by changing rates of receptor internalization, ligand loss, kinase inactivation, recycling, etc.; and iv) alter rates of receptor and substrate dephosphorylation. In addition to understanding interactions of heterodimers with the internalization machinery, identification of receptor-specific substrates and binding proteins for each EGFR family member will be necessary to explicate the role of heterodimers in growth and differentiation.

An irregularity in the transmembrane domain helix correlates with the rate of insulin receptor internalization.

Internalization of insulin and its receptor via receptor-mediated endocytosis is an important step in insulin-induced signal transduction. To investigate the structural determinants underlying the enhanced internalization rate observed for the insulin receptor transmembrane mutant Gly933-Pro934-->Ala-Ala (GP-->AA), we have designed and chemically synthesized two peptides, IR(TM)-GP and IR-(TM)-AA, corresponding respectively to the N-terminal portion of the wild-type and the mutant insulin receptor TM segment containing these sites. Conformational studies by circular dichroism (CD) spectroscopy on these two peptides in their monomeric states revealed that peptide IR(TM)-GP forms an irregular helix in the membrane-mimetic environments of sodium dodecyl sulfate (SDS) micelles with a possible "kink" in the helix imposed by its Gly-Pro sequence, while peptide IR(TM)-AA assumes largely classical alpha-helical structure under corresponding conditions. The helical pattern of peptide IR(TM)-AA was maintained at elevated temperatures, while the shape of the CD curve for peptide IR(TM)-GP was found to alter as a function of temperature. At higher concentrations, both peptides formed high molecular weight aggregates in SDS micelles, as demonstrated by SDS-PAGE gels, but peptide IR(TM)-AA was shown to aggregate more readily and more extensively than peptide IR(TM)-GP. Fluorescent dye-leakage experiments indicated that peptide IR(TM)-GP produces an enhanced disruption of the membrane bilayer in phosphatidylglycerol vesicles vs that induced by IR(TM)-AA.(ABSTRACT TRUNCATED AT 250 WORDS)

Polycythaemia vera. IV. Specific binding of stem cell factor to normal and polycythaemia vera highly purified erythroid progenitor cells.

Polycythaemia vera (PV) patients' blood burst-forming units-erythroid (BFU-E) have an enhanced sensitivity to stem cell factor (SCF) compared to normal BFU-E. To characterize SCF receptors on erythroid progenitors from normal individuals and PV patients, we performed binding experiments using radioiodinated recombinant SCF (rSCF), day 1 BFU-E and day 8 erythroid colony-forming cells (ECFC), which are mostly colony-forming units-erythroid (CFU-E). 125I-rSCF binds to a single class of cell surface receptors (23,000/ECFC) at 0 degrees C with a high-binding affinity (Kd = 17 pM). Saturation occurred at 0.5 nM (10 ng/ml) which produces a nearly maximum biological effect. One half of the radiolabelled rSCF was internalized by the cells after 30 min at 37 degrees C. No significant differences in the receptor number, dissociation constant, or internalization rate were found between normal and PV ECFC. Autoradiographic analysis of 125I-rSCF binding to normal BFU-E and ECFC showed that no differences were present in either the percentage of positive cells or the number of radioactive grains/cell between the normal and PV erythroid progenitors. The enhanced sensitivity of PV BFU-E and CFU-E to SCF does not appear to be related to changes in SCF receptor number, binding affinity or internalization and the hypersensitivity of PV erythroid progenitors to SCF must reside in a further internal cellular abnormality.

Amino-terminal polymorphisms of the human beta 2-adrenergic receptor impart distinct agonist-promoted regulatory properties.

We have recently delineated three naturally occurring polymorphisms of the human beta 2-adrenergic receptor caused by missense mutations encoding for amino acids 16 and 27 of the extracellular N-terminus of the receptor. We have studied the functional consequences of these polymorphisms by site-directed mutagenesis and the recombinant expression of these receptors in Chinese hamster fibroblasts. The polymorphisms consist of substitutions of Gly for Arg at amino acid 16 (Arg16-->Gly), Glu for Gln at amino acid 27 (Gln27-->Glu), and a combination of both substitutions. All three mutated receptors displayed normal agonist binding and functional coupling to Gs, resulting in the stimulation of adenylyl cyclase activity. However, these mutations markedly altered the degree of agonist-promoted downregulation of receptor expression. After 24-h exposure to 10 microM isoproterenol, wild-type beta 2AR underwent a 26 +/- 3% reduction in receptor density. In contrast, Arg16-->Gly underwent a 41 +/- 3% reduction. Gln27-->Glu, on the other hand, was found to be completely resistant to downregulation. Arg16-->Gly+Gln27-->Glu also underwent an increased downregulation compared to wild-type beta 2AR (39 +/- 4%). The rates of new receptor synthesis after irreversible alkylation were not different between these receptors, nor were the rates of agonist-promoted receptor internalization to the intracellular pool. Gln27-->Glu cellular mRNA minimally increased during agonist exposure, and wild-type beta 2AR and the other mutated receptor mRNAs did not change, which infer that the aberrant downregulation patterns of these polymorphisms may be due to the altered degradation of receptor protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Deletion analysis of the m4 muscarinic acetylcholine receptor

In order to investigate whether coupling to and/or activation of guanine-nucleotide-binding proteins (G proteins) is involved in agonist-induced internalization of m4 muscarinic acetylcholine receptors (mAChRs), a deletion mutant [des-(264-394)mAChR] was constructed that lacks a substantial portion of the putative third intracellular loop. The wild-type receptor and des-(264-394)mAChR stably expressed in Chinese hamster ovary cells in essentially comparable amounts, exhibited identical antagonist-binding affinities. Consistent with the reported importance of the third cytoplasmic loop for Gi protein activation, the des-(264-394)mAChR showed a drastically reduced potential to mediate agonist-induced inhibition of adenylyl cyclase. In contrast, the ability of the mutant receptor to couple to Gi proteins was not impaired, as demonstrated by a similar guanine-nucleotide-sensitive and pertussis-toxin-sensitive high-affinity agonist-receptor binding for both mAChRs. In contrast, des-(264-394)mAChR was hardly able to stimulate the GTPase activity of G proteins, suggesting impaired activation of Gi proteins rather than ineffective coupling to Gi proteins. Internalization of wild-type receptor and des-(264-394)mAChR was observed with similar agonist concentrations and showed similar maximal values. However, des-(264-394)mAChR displayed a significantly reduced rate of receptor internalization. A similar attenuation of wild-type mAChR internalization was obtained upon pertussis toxin treatment. In conclusion, our data provide evidence that the molecular determinants of the m4 mAChR involved in Gi-protein coupling and activation are not identical and that activation of, but not coupling to, Gi proteins regulates m4 mAChR internalization.

Human insulin-like growth factor I receptor internalization. Role of the juxtamembrane domain.

Cytoplasmic submembrane domains of the insulin-like growth factor I (IGF-1) receptor ranging from glycine 940 to proline 959 were investigated for their role in endocytosis of the IGF-1 ligand in rat pituitary GC cells stably expressing mutant human IGF-1 receptors. Replacement of each of three tyrosine residues within the juxtamembrane domain reduced the internalization rate (Ke) by 33% (943Y-->A), 47% (950Y-->A), and 41% (957Y-->A), respectively. Other substitutions within the submembrane region variably retarded receptor-mediated IGF-1 ligand endocytosis. Thus, each amino acid appears to independently contribute to the overall ligand-stimulated IGF-1 receptor internalization rate. The effect of receptor occupancy on IGF-1 receptor endocytosis was also tested. The rate of 125I-labeled IGF-1 internalization by transfectants overexpressing the wild type IGF-1 receptor was rapid but decreased markedly in the presence of increasing unlabeled IGF-1 (ED50 = 44,000 receptors/cell). Substitution by alanine for any of the three submembrane tyrosines increased the ED50 to > 56,000 receptors/cell, decreased the slope (Kic), and had a variable effect on Vmax in the presence of increasing unlabeled IGF-1. In contrast, 125I-labeled IGF-1 endocytosis by the mutant kinase-deficient 952STOP cells was slow and not further retarded by unlabeled IGF-1. These results suggest that ligand-mediated internalization of the human IGF-I is consistent with saturable interactions between the IGF-I receptor juxtamembrane region (glycine 940-tyrosine 957) and components of the endocytic apparatus.

A tyrosine residue in the juxtamembrane segment of the platelet-derived growth factor beta-receptor is critical for ligand-mediated endocytosis.

The importance of tyrosine residues in ligand-mediated endocytosis of the platelet-derived growth factor beta-receptor was analyzed using a series of tyrosine residue-mutated beta-receptors, which together cover all of the tyrosine residues in the juxtamembrane segment, the kinase insert, and the carboxyl-terminal tail; also certain of the tyrosine residues within the first and second parts of the kinase domain were examined. Of all of these tyrosine residues, only Tyr-579 seemed to be important for internalization, since mutation of this residue resulted in substantial reduction in the rate of ligand-induced receptor internalization (approximately 60% of the wild-type level). Replacement of Tyr-579 by either an aromatic (Phe) or a nonaromatic (Asp) residue reduced the efficiency of the mutant receptors in internalization to the same extent, suggesting that the role of Tyr-579 in the beta-receptor is different from that of the previously described tyrosine-based internalization motifs, which were first determined for the low density lipoprotein receptor. Tyr-579 has been found to be an autophosphorylation site in the beta-receptor. Moreover, the internalization rate of a kinase negative receptor mutant was not altered by the additional mutation of Tyr-579. Thus, it is likely that phosphorylation of Tyr-579 is important for ligand-induced internalization of the beta-receptor.

Optimizing transmembrane domain helicity accelerates insulin receptor internalization and lateral mobility.

Transmembrane (TM) domains of integral membrane proteins are generally thought to be helical. However, a Gly-Pro sequence within the TM domain of the insulin receptor is predicted to act as a helix breaker. CD analyses of model TM peptides in a lipid-like environment show that substitution of Gly and Pro by Ala enhances helicity. On this basis, Gly933 and Pro934 within the TM domain of the intact human insulin receptor were mutated to Ala (G-->A, P-->A, GP-->AA) to assess effects of altered helicity on receptor functions. Mutated and wild-type receptors, expressed stably in cultured CHO cells at equivalent levels, were properly assembled, biosynthetically processed, and exhibited similar affinities for insulin. Receptor autophosphorylation and substrate kinase activity in intact cells and soluble receptor preparations were indistinguishable. In contrast, insulin-stimulated receptor internalization was accelerated 2-fold for the GP-->AA mutant, compared to a wild-type control or the G-->A and P-->A mutants. Insulin degradation, which occurs during receptor endocytosis and recycling, was similarly elevated in cells transfected with GP-->AA mutant receptors. Fluorescence photobleaching recovery measurements showed that the lateral mobility of GP-->AA mutant receptors was also increased 2- to 3-fold. These results suggest that lateral mobility directly influences rates of insulin-mediated receptor endocytosis and that rates of endocytosis and lateral mobility are retarded by a kinked TM domain in the wild-type receptor. Invariance of Gly-Pro within insulin receptor TM domain sequences suggests a physiologic advantage for submaximal rates of receptor internalization.

Variation in EGF-induced EGF receptor downregulation in human hepatoma-derived cell lines expressing different amounts of EGF receptor

The effect of epidermal growth factor (EGF) receptor overexpression on ligand-induced EGF receptor downregulation was examined using a hepatoma-derived cell line, PLC/PRF/5, which expresses normal amounts of the EGF receptor, and a subline, NPLC/PRF/5, which expresses 10-fold more receptors at its cell surface. PLC/PRF/5 cells efficiently downregulated surface receptor levels upon exposure to saturating and subsaturating concentrations of EGF; the rate of receptor downregulation corresponded to that of ligand-receptor internalization. Upon internalization, EGF receptors were degraded and receptor biosynthesis remained at basal levels. EGF surface receptor remained downregulated for as long as cells were exposed to EGF. By contrast, surface EGF receptor abundance in NPLC/PRF/5 cells decreased by only 5–15% after 1–4 h incubation with subsaturating doses of EGF and actually increased by 67% within 20 h. Exposure of these cells to saturating concentrations of EGF induced modest decreases in surface receptor abundance during the initial 12 h incubation, followed by a progressive decline to 30% of initial values by 24 h. Relative ligand-receptor internalization rates in NPLC/PRF/5 cells were lower than those in PLC/PRF/5, although their surface receptor population was even higher than that predicted by the decreased internalization rates. EGF receptor degradation in NPLC/PRF/5 cells was also inhibited; exposure to saturating levels of EGF for more than 16 h was necessary before significant degradation occurred. Receptor protein and mRNA biosynthesis in NPLC/PRF/5 were stimulated by 8 h exposure to EGF but when saturating concentrations of EGF were present for 16 h, receptor biosynthesis was inhibited. EGF receptor overexpression circumvents the downregulatory effect of EGF by decreasing the rate of receptor internalization, inhibiting degradation of the internalized receptor pool, and stimulating EGF receptor biosynthesis. Conversely, receptor downregulation becomes pronounced at late times when receptor degradation is high and biosynthesis is inhibited.

Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and internalization. Contrasting significance of tyrosine 992 in the native and truncated receptors.

The role of epidermal growth factor (EGF) receptor autophosphorylation sites in the regulation of receptor functions has been studied using cells transfected with mutant EGF receptors. Simultaneous point mutation of 4 tyrosines (Y1068, Y1086, Y1148, Y1173) to phenylalanine, as well as removal of these sites by truncation of the carboxyl-terminal 123 amino acid residues, resulted in reduced receptor phosphorylation of an in vivo specific substrate phospholipase C-gamma 1 to less than 50% compared to the wild-type receptor. The internalization rate constant Ke was also significantly lower in these mutants (0.15/min) compared to cells transfected with wild-type receptor (0.27/min). Additional mutation of tyrosine 992 to phenylalanine in the truncated receptor mutant (Dc-123F) further decreased the receptor internalization rate to a minimal level (ke = 0.07-0.10/min), equivalent to the ke measured for cells expressing kinase-negative receptor (A721). Moreover, tyrosine kinase activity of the Dc-123F receptor toward phospholipase C-gamma 1, compared to wild-type receptor, was reduced by 90%. Taken together, these results show that EGF receptor lacking five autophosphorylation sites functions similar to a kinase-negative receptor. Mutation of tyrosine residue Y992 alone in the context of full length EGF receptor, however, did not affect receptor internalization or kinase activity toward phospholipase C-gamma 1. These data indicate that tyrosine 992 is critical for substrate phosphorylation and internalization only in the context of the truncated receptor, and that minor autophosphorylation sites, such as Y992, may act as compensatory regulatory sties in the absence of the major EGF receptor autophosphorylation sites.

Epidermal growth factor (EGF)-stimulated tyrosine phosphorylation and EGF receptor degradation in cells expressing EGF receptors truncated at residue 973.

Epidermal growth factor (EGF)-stimulated tyrosine phosphorylation of proteins was examined in cells expressing wild-type (WT-EGFR) EGF receptors or EGF receptors truncated at residue 973 (973-EGFR). A much broader spectrum of tyrosine phosphorylated proteins was found following EGF treatment of 973-EGFR expressing cells compared with cells expressing wild-type receptors. Several additional ras GTPase activating protein-associated tyrosine phosphorylated proteins were found in EGF-treated 973-EGFR cells relative to WT-EGFR cells. Additional tyrosine-phosphorylated proteins were also found to co-immunoprecipitate with phospholipase C gamma 1 (PLC gamma 1) following EGF treatment of cells expressing 973-EGFR relative to cells expressing WT-EGFR. EGF-stimulated tyrosine phosphorylation of PLC gamma 1 was found in cells expressing WT-EGFR, but not in cells expressing 973-EGFR. WT-EGF receptor from EGF-treated cells bound well to bacterially expressed src homology (SH) regions of PLC gamma 1 and to a lesser extent to bacterially expressed GTPase activating protein SH regions. No binding of 973-EGF receptor to SH regions of either protein could be detected. EGF treatment greatly reduced the half-life of WT-EGFR, but had relatively little effect on the half-life of 973-EGFR. EGF induced internalization of 973-EGFR at a slower rate than WT-EGFR and caused the appearance of discrete receptor degradation products for both cell types. The data indicate that truncation of the EGF receptor at residue 973 alters receptor substrate specificity, decreases the rate of receptor internalization, and has an inhibitory effect on receptor degradation.

Evidence for intracellular down-regulation of the epidermal growth factor (EGF) receptor during adenovirus infection by an EGF-independent mechanism

We have reported previously that human group C adenoviruses down-regulate the epidermal growth factor (EGF) receptor (EGF-R) (C. R. Carlin, A. E. Tollefson, H. A. Brady, B. L. Hoffman, and W. S. M. Wold, Cell 57:135-144, 1989). Expression of a 13.7-kDa protein encoded by a gene in the E3 transcription unit is necessary and sufficient for this effect (Carlin et al., Cell, 1989; B. L. Hoffman, A. Ullrich, W. S. M. Wold, and C. R. Carlin, Mol. Cell. Biol. 10:5521-5524, 1990). We show here that EGF-R down-regulation is accelerated in cells which overexpress the receptor when these cells are infected with virus mutants that overproduce the 13.7-kDa protein compared with wild-type virus. This is in contrast to EGF stimulation, for which others have shown that high concentrations of ligand are associated with low rates of receptor internalization in EGF-R-overexpressing cells (D. Kuppuswamy and L. J. Pike, J. Biol. Chem. 264:3357-3363, 1989; H. S. Wiley, J. Cell Biol. 107:801-810, 1988). We also show that the E3 protein is not present in media conditioned by infected cells and that it does not induce secretion of an EGF-like autocrine factor. Moreover, while mature membrane-bound EGF-R is down-regulated, the precursor of the membrane-bound form is not. Adenovirus infection also does not affect receptor-related molecules expressed in the secretory pathway. Interestingly, adenovirus-induced down-regulation is not regulated by concentrations of EGF associated with a slow rate of internalization in A431 cells. This suggests that 13.7-kDa protein expression triggers receptor entry by a novel ligand-independent pathway or, alternatively, that it compensates for a cellular factor that may be rate limiting during EGF-mediated endocytosis.

Cation-dependent mannose 6-phosphate receptor contains two internalization signals in its cytoplasmic domain.

The signals required for rapid internalization of the bovine cation-dependent mannose 6-phosphate receptor have been localized to two distinct regions of the 67-amino acid cytoplasmic domain. One signal includes phenylalanine 13 and phenylalanine 18, while the other involves tyrosine 45. The former signal is more potent than the latter, but both must be present for the maximal rate of receptor internalization. Each signal shares similarities with the known internalization signals of other recycling receptors.

The rate of internalization of different receptor–ligand complexes in alveolar macrophages is receptor-specific

To probe the mechanisms of endocytosis in alveolar macrophages, we examined the internalization rates of three different receptors. Initial rates of internalization for mannosylated BSA, diferric transferrin and alpha-macroglobulin-proteinase complexes were all different. Although the absolute rates of internalization varied depending on the cell preparation, transferrin was internalized at 10-20% and alpha-macroglobulin-proteinase complex at 40-60% of the rate of manosylated-BSA. Incubation of cells with transferrin did not affect the rate of internalization of mannosylated BSA or alpha-macroglobulin-proteinase complexes, and the rates of internalization were independent of receptor occupancy. These different internalization rates could not be ascribed to different rates of diacytosis. Altering the distribution of unoccupied surface receptors by either trypsin treatment of cells at 0 degree C or exposure to hyperosmotic solutions resulted in the absolute internalization rates being affected by the experimental condition, but the hierarchy in receptor internalization rates was maintained. The fact that a variety of conditions affect receptor internalization rates to the same degree implies the existence of co-ordinate regulation at a single rate-limiting step. Based on these results, we suggest that differences in internalization rate reflect the ability of ligand-receptor complexes to be captured by coated pits.

Inhibition of the receptor-mediated endocytosis of diferric transferrin is associated with the covalent modification of the transferrin receptor with palmitic acid.

The human transferrin receptor is post-translationally modified by the covalent attachment of palmitic acid to Cys62 and Cys67 via a thio-ester bond. To investigate the role of the acylation of the transferrin receptor, Cys62 and Cys67 were substituted with serine and alanine residues. The properties of the mutant receptors were compared with wild-type receptors after expression in Chinese hamster ovary cells that lack endogenous transferrin receptors. Rapid incorporation of [3H]palmitate into the wild-type transferrin receptor was observed, but the mutant receptors were found to be palmitoylation-defective. The kinetics of endocytosis and recycling of the wild-type and mutant receptors were compared. It was observed that the rate of endocytosis of the palmitoylation-defective transferrin receptors was significantly greater than the rate measured for the wild-type transferrin receptor. In contrast, the mutation of Cys62 and Cys67 was found to have no significant effect on the rate of transferrin receptor recycling. Consistent with these observations, it was found that cells expressing palmitoylation-defective transferrin receptors exhibited an increased rate of accumulation of [59Fe]diferric transferrin. Together, these data indicate that the palmitoylation of the transferrin receptor is associated with an inhibition of the rate of transferrin receptor endocytosis. Addition of insulin to cultured cells causes an increase in the palmitoylation of cell surface transferrin receptors and a decrease in the rate of transferrin receptor internalization. It was observed that the effect of insulin to inhibit the endocytosis of the acylation-defective [Ala62 Ala67]transferrin receptor was attenuated in comparison with the wild-type receptor. The decreased effectiveness of insulin to inhibit the internalization of the acylation-defective transferrin receptor is consistent with the hypothesis that palmitoylation represents a potential mechanism for the regulation of transferrin receptor endocytosis.

Free–Fatty Acid Inhibition of Insulin Binding, Degradation, and Action in Isolated Rat Hepatocytes

The effect of free fatty acids (FFAs) on insulin binding and action was investigated in isolated rat hepatocytes. Oleic acid (0.4 mM) added to the cells rapidly (within 45 min) reduced insulin binding and degradation (each by 45%; P less than 0.001, n = 7) without changing the apparent receptor affinity. The effect was concentration dependent; a half-maximal inhibitory effect occurred at 0.150 +/- 0.050 mM (mean +/- SE). Oleic acid exerted no effect on insulin binding in energy-depleted (KCN-treated) cells. Oleic, palmitic, stearic, palmitoleic, and eicosapentaenoic acids were equally effective in reducing insulin binding. FFA did not change insulin binding to partially purified insulin receptors, thus excluding a direct effect on the insulin receptor. Furthermore, binding to partially purified receptors from solubilized cells pretreated with 0.2 mM oleic acid was not changed, indicating the effect of FFA in intact cells is on the rate of receptor internalization and/or recycling. Concomitant with the effect on insulin binding, oleic acid elicited a concentration-dependent reduction in nonstimulated cellular [14C]aminoisobutyric acid uptake (AIB; 29 +/- 8%, P less than 0.05) and decreased the maximal effect of insulin (39 +/- 7%, P less than 0.05). Thus, in a concentration-dependent manner, different fatty acids can reduce the number of binding sites for insulin and the degradation of insulin by isolated liver cells. Basal and insulin-stimulated AIB transport was reduced, suggesting the presence of postbinding perturbations. These data suggest that FFA exerts an important modulating effect on insulin action in the liver.

Insulin down-regulates insulin receptor number and up-regulates insulin receptor affinity in cells expressing a tyrosine kinase-defective insulin receptor.

We examined the effect of insulin treatment on HTC cells transfected with large numbers of either normal insulin receptors (HTC-IR) or insulin receptors defective in tyrosine kinase (HTC-IR/M-1030). In both HTC-IR and HTC-IR/M-1030 cells, 20 h of insulin treatment (1 microM) at 37 degrees C resulted in a 65% decrease in the number of binding sites with a reciprocal 6-fold increase in affinity. In contrast, treatment with 10 nM insulin (20 h, 37 degrees C) also increased receptor affinity but had a smaller effect on the number of binding sites. 125I-Insulin binding to soluble receptors from HTC-IR and HTC-IR/M-1030 cells pretreated with insulin showed results similar to those obtained in intact cells. In both HTC-IR and HTC-IR/M-1030 cells, insulin enhanced insulin receptor degradation. In HTC-IR/M-1030 cells a 1-h incubation with insulin did not change receptor number and had only a small effect on receptor affinity; also there was no effect of insulin after a 20-h incubation at 15 degrees C. Inhibiting protein synthesis by pretreatment with cycloheximide (100 microM) did not block either the decrease in receptor number or the increase in receptor affinity. Both HTC-IR and HTC-IR/M-1030 cells exhibited a very slow rate of insulin and insulin receptor internalization and no differences were seen in this parameter when HTC-IR cells were compared to HTC-IR/M-1030 cells. These studies indicate, therefore, that in cells expressing kinase-defective insulin receptors, insulin down-regulates insulin receptor number via enhanced receptor degradation, and up-regulates receptor affinity. These effects were time- and temperature-dependent, but not dependent on new protein synthesis, and suggest that activation of tyrosine kinase may not be a prerequisite for certain mechanisms whereby insulin regulates its receptor.

Thyrotropin Modulates Receptors for Atrial Natriuretic Peptide on Intact Human Thyroid Cells

Interest in the mechanism of impaired salt and water metabolism in hypothyroidism has led to growing evidence of an interaction between atrial natriuretic peptide (ANP) and the thyroid, which includes reports of direct effects of thyroid hormone on ANP synthesis and circulating ANP levels, and of the presence of specific ANP receptors in human thyroid tissue, which may act to inhibit thyroglobulin (Tg) secretion. The authors questioned whether or not thyrotropin (TSH) has a role in this interaction. They used 125I-ANP to study the effect of TSH on ANP binding to human thyroid cells in primary culture. Binding competition by increasing concentrations of unlabeled ANP in the presence or absence of TSH was assessed by Scatchard analysis. At lower temperatures of 4 degrees C or 23 degrees C, TSH had no effect either on the ANP receptor equilibrium dissociation constant (Kd) or number of binding sites. However, at 37 degrees C, bovine TSH at 1 mU/ml reduced measurable binding sites by about 50% without affecting receptor affinity (Kd = 0.2 nM). Prolonged (6 days) coincubation of TSH with thyroid cells decreased the assayable ANP receptor. The effects of TSH appear to be specific because human luteinizing hormone, follicle-stimulatory hormone, growth hormone, human chorionic gonadotropin and iodide had no effect on ANP binding. Thus, human thyroid cells possess a single class of high-affinity, specific receptors for ANP with binding activity that is temperature dependent and modulated by TSH at physiologic temperature. TSH-mediated reduction of binding at 37 degrees C but not at 4 degrees C suggests an energy-dependent process that acts possibly by activating an ANP degradative enzyme or by changing the rate of receptor internalization and subsequent degradation.

Insulin Receptor Recycling in Vascular Endothelial Cells: Regulation by insulin and phorbol ester

Endothelial cell insulin receptors mediate the transcytosis of insulin from luminal to abluminal cell surface. We have investigated the kinetics of insulin receptor translocation by immunoprecipitation of radiolabeled receptors at various times before and after trypsin treatment of intact endothelial cells. Insulin receptors were constitutively internalized with t½ = 18 ± 2 min and were recycled to the cell surface. Insulin stimulated receptor internalization and externalization rates 2.6- and 2.4-fold, respectively. Changes in cell-surface binding of 125I-insulin were consistent with the receptor translocation rates observed in surface-labeling experiments. Phorbol myristate acetate (PMA) treatment increased the rate of insulin-stimulated receptor externalization 1.7-fold. PMA treatment increased the constitutive externalization rate 3.5-fold without affecting the constitutive internalization rate, suggesting that recycling might occur via a mobilization of receptors from intracellular sites in a manner independent of internalization rate. Analysis of the intracellular distribution of receptors by 125I-insulin binding and immunogold electron microscopy revealed that less than one-third of the total insulin receptor pool resided on the cell surface. In summary, endothelial cell insulin receptors are constitutively recycled, and internalization and externalization rates are increased by receptor occupancy and PMA treatment.

Internalization and degradation of insulin by a human insulin receptor-v-ros hybrid in Chinese hamster ovary cells

Chinese hamster ovary cell lines expressing either the wild-type human insulin receptor or a hybrid molecule in which the tyrosine kinase domain of the insulin receptor is replaced with that of the oncogene, v-ros were examined for their ability to internalize and degrade insulin. Cells expressing the hybrid receptor were found to internalize and degrade insulin at approximately half the rate of cells expressing the native insulin receptor. Moreover, insulin was incapable of inducing the internalization of the cell-surface hybrid molecule. In contrast, the constitutive rate of receptor internalization was found to be the same for the hybrid and wild-type receptors. These results obtained were similar to those with cells expressing either wild-type or mutant receptors lacking kinase activity. In conclusion, the substitution of the specificity of tyrosine kinase of the insulin receptor with that of the v-ros oncogene product results in defective internalization and degradation of insulin, and loss of ligand-induced receptor internalization.