Phorbol diesters and transferrin modulate lymphoblastoid cell transferrin receptor expression by two different mechanisms

Expression of transferrin receptors (TfR) by activated lymphocytes is necessary for lymphocyte DNA synthesis and proliferation. Regulation of TfR expression, therefore, is a mechanism by which the lymphocyte's proliferative potential may be directed and controlled. We studied mechanisms by which lymphoblastoid cells modulate TfR expression during treatment with phorbol diesters or iron transferrin (FeTf), agents which cause downregulation of cell surface TfR. Phorbol diester-induced TfR downregulation occurred rapidly, being detectable at 2 min and reaching maximal decreases of 50% by 15 min. It was inhibited by cold but not by agents that destabilize cytoskeletal elements. Furthermore, this downregulation was reversed rapidly by washing or by treatment with the membrane interactive agent, chlorpromazine. In contrast, FeTf-induced TfR downregulation occurred slowly. Decreased expression of TfR was detectable only after 15 min and maximal downregulation was achieved after 60 min. Although FeTf-induced downregulation also was inhibited by cold, it was inhibited in addition by a group of microtubule destabilizing agents (colchicine, vinblastine, podophyllotoxin) or cytochalasin B, a microfilament inhibitor. Furthermore, FeTf-induced downregulation was not reversed readily by washing or by treatment with chlorpromazine. The inactive colchicine analogues, beta- and gamma-lumicolchicine, did not inhibit FeTf-induced TfR downregulation. Similarly, when cells were pretreated with taxol to stabilize microtubules, colchicine no longer inhibited FeTf-induced downregulation. Therefore, FeTf causes TfR downregulation in lymphoblastoid cells by a cytoskeleton-dependent mechanism. Phorbol diesters cause TfR downregulation by a cytoskeleton-independent mechanism. In other experiments, treatment of cells with both a phorbol diester and FeTf, either simultaneously or sequentially, produced additive effects on TfR expression. These data indicate that TfR expression is regulated by two independent mechanisms in lymphoblastoid cells, and they provide the possibility that downregulation of TfR by different mechanisms may result in different effects in these cells.     

The achondroplasia mutation does not alter the dimerization energetics of the fibroblast growth factor receptor 3 transmembrane domain

The Gly380 --> Arg mutation in the TM domain of fibroblast growth factor receptor 3 (FGFR3) of the RTK family is linked to achondroplasia, the most common form of human dwarfism. The molecular mechanism of pathology induction is under debate, and two different mechanisms have been proposed to contribute to pathogenesis: (1) Arg380-mediated FGFR3 dimer stabilization and (2) slow downregulation of the activated mutant receptors. Here we show that the Gly380 --> Arg mutation does not alter the dimerization energetics of the FGFR3 transmembrane domain in detergent micelles or in lipid bilayers. This result indicates that pathogenesis in achondroplasia cannot be explained simply by a higher dimerization propensity of the mutant FGFR3 TM domain, thus highlighting the importance of the observed slow downregulation in phenotype induction.     

Bruce/apollon promotes hippocampal neuron survival and is downregulated by kainic acid

Prolonged or excess stimulation of excitatory amino acid receptors leads to seizures and the induction of excitotoxic nerve cell injury. Kainic acid acting on glutamate receptors produces degeneration of vulnerable neurons in parts of the hippocampus and amygdala, but the exact mechanisms are not fully understood. We have here investigated whether the anti-apoptotic protein Bruce is involved in kainic acid-induced neurodegeneration. In the rat hippocampus and cortex, Bruce was exclusively expressed by neurons. The levels of Bruce were rapidly downregulated by kainic acid in hippocampal neurons as shown both in vivo and in cell culture. Caspase-3 was activated in neurons exhibiting low levels of Bruce causing cell death. Likewise, downregulation of Bruce using antisense oligonucleotides decreased viability and enhanced the effect of kainic acid in the hippocampal neurons. The results show that Bruce is involved in neurodegeneration caused by kainic acid and the downregulation of the protein promotes neuronal death.     

Integrins and EGFR coordinately regulate the pro-apoptotic protein Bim to prevent anoikis

Epithelial cells must adhere to the extracellular matrix (ECM) for survival, as detachment from matrix triggers apoptosis or anoikis. Integrins are major mediators of adhesion between cells and ECM proteins, and transduce signals required for cell survival. Recent evidence suggests that integrin receptors are coupled to growth factor receptors in the regulation of multiple biological functions; however, mechanisms involved in coordinate regulation of cell survival are poorly understood and mediators responsible for anoikis have not been well characterized. Here, we identify the pro-apoptotic protein Bim as a critical mediator of anoikis in epithelial cells. Bim is strongly induced after cell detachment and downregulation of Bim expression by RNA interference (RNAi) inhibits anoikis. Detachment-induced expression of Bim requires a lack of beta(1)-integrin engagement, downregulation of EGF receptor (EGFR) expression and inhibition of Erk signalling. Overexpressed EGFR was uncoupled from integrin regulation, resulting in the maintenance of Erk activation in suspension, and a block in Bim expression and anoikis. Thus, Bim functions as a key sensor of integrin and growth factor signals to the Erk pathway, and loss of such coordinate regulation may contribute to tumour progression.     

Complexity of agonist- and cyclic AMP-mediated downregulation of the human beta 1-adrenergic receptor: role of internalization,degradation, and mRNA destabilization

Prolonged agonist exposure often induces downregulation of G protein-coupled receptors (GPCRs). Although downregulation of the prototypical beta(2)-adrenergic receptor (beta(2)AR) has been extensively studied, the underlying mechanisms have yet to be resolved. As even less is known about the beta(1)-subtype, we investigated the downregulation of human beta(1)AR stably expressed in Chinese hamster fibroblasts in response to the agonist isoproterenol or the cell-permeable, chlorophenylthio-cAMP (CPT-cAMP). While either effector mediated decreases in both beta(1)AR binding activity and steady-state beta(1)AR mRNA levels, there were significant differences in their actions. Whereas agonist-mediated downregulation of beta(1)AR followed first-order kinetics, that induced by CPT-cAMP was delayed for several hours and approximately 50% of the former. Furthermore, agonist but not CPT-cAMP induced beta(1)AR internalization, and inhibiting internalization also suppressed agonist-mediated downregulation. The latter, however, was more sensitive than the former to agonist concentration (EC(50) of 0.3 vs 48 nM). Thus, at < or =1 nM agonist, downregulation occurred without internalization and with a pattern similar to that mediated by CPT-cAMP. The amounts of beta(1)AR downregulated or internalized were proportional to initial receptor levels but reached saturation at approximately 2 and 3 pmol/mg of protein, respectively. The fate of beta(1)AR protein during downregulation was determined by immunoblotting with anti-C-terminal antibodies. In agonist-treated cells, beta(1)AR protein disappeared with time and without any immunoreactive degradation products. Agonist-mediated downregulation of the human beta(1)AR appears to be a complex process that consists of both agonist- and cAMP-specific components. The former involves both receptor internalization and degradation whereas the latter involves a reduction in receptor mRNA.     

Endocytic downregulation of ErbB receptors: mechanisms and relevance in cancer

ErbB receptors (EGFR (ErbB1), ErbB2, ErbB3, and ErbB4) are important regulators of normal growth and differentiation, and they are involved in the pathogenesis of cancer. Following ligand binding and receptor activation, EGFR is endocytosed and transported to lysosomes where the receptor is degraded. This downregulation of EGFR is a complex and tightly regulated process. The functions of ErbB2, ErbB3, and ErbB4 are also regulated by endocytosis to some extent, although the current knowledge of these processes is sparse. Impaired endocytic downregulation of signaling receptors is frequently associated with cancer, since it can lead to increased and uncontrolled receptor signaling. In this review we describe the current knowledge of ErbB receptor endocytic downregulation. In addition, we outline how ErbB receptors can escape endocytic downregulation in cancer, and we discuss how targeted anti-cancer therapy may induce endocytic downregulation of ErbB receptors.     

Ligand stimulation induces clathrin- and Rab5-dependent downregulation of the kinase-dead EphB6 receptor preceded by the disruption of EphB6-Hsp90 interaction

Ligand-induced internalisation and subsequent downregulation of receptor tyrosine kinases (RTKs) serve to determine biological outputs of their signalling. Intrinsically kinase-deficient RTKs control a variety of biological responses, however, the mechanism of their downregulation is not well understood and its analysis is focused exclusively on the ErbB3 receptor.The Eph group of RTKs is represented by the EphA and EphB subclasses. Each bears one kinase-inactive member, EphA10 and EphB6, respectively, suggesting an important role for these molecules in the Eph signalling network. While EphB6 effects on cell behaviour have been assessed, the mechanism of its downregulation remains elusive.Our work reveals that EphB6 and its kinase-active relative, and signalling partner, EphB4, are downregulated in a similar manner in response to their common ligand, ephrin-B2. Following stimulation, both receptors are internalised through clathrin-coated pits and are degraded in lysosomes. Their targeting for lysosomal degradation relies on the activity of an early endosome regulator, the Rab5 GTPase, as this process is inhibited in the presence of a Rab5 dominant-negative mutant. EphB6 also interacts with the Hsp90 chaperone and EphB6 downregulation is preceded by their rapid dissociation. Moreover, the inhibition of Hsp90 results in EphB6 degradation, mimicking its ligand-induced downregulation. These processes appear to rely on overlapping mechanisms, since Hsp90 inhibition does not significantly enhance ligand-induced EphB6 elimination.Taken together, our observations define a novel mechanism for intrinsically kinase-deficient RTK downregulation and support an intriguing model, where Hsp90 dissociation acts as a trigger for ligand-induced receptor removal.     

The switch from latent to productive infection in epstein-barr virus-infected B cells is associated with sensitization to NK cell killing

Following activation of Epstein-Barr virus (EBV)-infected B cells from latent to productive (lytic) infection, there is a concomitant reduction in the level of cell surface major histocompatibility complex (MHC) class I molecules and an impaired antigen-presenting function that may facilitate evasion from EBV-specific CD8+ cytotoxic T cells. In some other herpesviruses studied, most notably human cytomegalovirus (HCMV), evasion of virus-specific CD8+ effector responses via downregulation of surface MHC class I molecules is supplemented with specific mechanisms for evading NK cells. We now report that EBV differs from HCMV in this respect. While latently infected EBV-positive B cells were resistant to lysis by two NK lines and by primary polyclonal NK cells from peripheral blood, these effectors efficiently killed cells activated into the lytic cycle. Susceptibility to NK lysis coincided not only with downregulation of HLA-A, -B, and -C molecules that bind to the KIR family of inhibitory receptors on NK cells but also with downregulation of HLA-E molecules binding the CD94/NKG2A inhibitory receptors. Conversely, ULBP-1 and CD112, ligands for the NK cell-activating receptors NKG2D and DNAM-1, respectively, were elevated. Susceptibility of the virus-producing target cells to NK cell lysis was partially reversed by blocking ULBP-1 or CD112 with specific antibodies. These results highlight a fundamental difference between EBV and HCMV with regards to evasion of innate immunity.     

ErbB2 and ErbB4 Cbl binding sites can functionally replace the ErbB1 Cbl binding site

Poor downregulation of ErbB receptors is associated with enhanced downstream signaling and tumorigenesis. It has been suggested that poor downregulation of ErbB-2, -3 and -4 receptors when compared to ErbB1 is due to decreased recruitment of Cbl E3 ligase proteins. However, a highly conserved Cbl binding site is not only present in ErbB1/EGFR (FLQRpY¹⁰⁴⁵SSDP), but also in ErbB2 (PLQRpY¹⁰⁹¹SEDP) and ErbB4 (STQRpY¹¹⁰³SADP). We therefore replaced the ErbB1 Cbl binding site by that of ErbB2 and ErbB4. Whereas retrovirally infected NIH3T3 cells containing the EGFR Y1045F mutation showed dramatically impaired Cbl recruitment, EGFR ubiquitination and delayed EGFR degradation, replacement of the EGFR Cbl binding site by that of ErbB2 or ErbB4 did not affect Cbl recruitment, receptor-ubiquitination, -degradation, -downregulation or ligand degradation. We conclude that poor downregulation of ErbB2 and ErbB4 receptors is not due to sequence variations in the Cbl binding site of these receptors.     

The critical role of adenosine A2A receptors in downregulation of inflammation and immunity in the pathogenesis of infectious diseases

Adenosine can be described as a retaliatory metabolite, the production and release of which is usually enhanced under adverse environmental conditions. Binding via specific receptors, adenosine activates endogenous protective mechanisms aiming at the restoration of tissue homeostasis. While adenosinergic downregulation of tissue damage is beneficial in acute inflammation, chronic suppression of the immune system by adenosine may account for immunoparalysis in long-term septic patients.     

The mechanisms of nucleotide actions in insulin resistance

Insulin resistance contributes to metabolic disorders in obesity and type 2 diabetes. In mechanisms of insulin resistance, the roles of glucose, fatty acids, and amino acids have been extensively documented in literature. However, the activities of nucleotides remain to be reviewed comprehensively in the regulation of insulin sensitivity. Nucleotides are well known for their activities in biosynthesis of DNA and RNA as well as their signaling activities in the form of cAMP and cGAMP. Their activities in insulin resistance are dependent on the derivatives and corresponding receptors. ATP and NADH, derivatives of adenosine, inhibit insulin signaling inside cells by downregulation of activities of AMPK and SIRT1, respectively. ATP, ADP and AMP, the well-known energy carriers, regulate cellular responses to insulin outside cells through the purinergic receptors in cell surface. Current evidence suggests that ATP, NADH, cGAMP, and uridine are potential biomarkers of insulin resistance. However, GTP and cGMP are likely the markers of insulin sensitization. Here, studies crossing the biomedical fields are reviewed to characterize nucleotide activities in the regulation of insulin sensitivity. The knowledge brings new insights into the mechanisms of insulin resistance.     

Downregulation of tumor necrosis factor receptors of macrophages by interferons and interleukin-1. Role of protein kinase C activation

Freshly harvested murine peritoneal macrophages and a line of transformed murine macrophages (RAW) were used in experiments designed to investigate the effect of different interferons (IFN) and interleukin-1 (IL-1) on tumor necrosis factor (TNF) receptors. Low concentrations of IFN-gamma or somewhat higher concentrations of IFN-alpha drastically downregulated the TNF receptors of RAW cells. A similar, but less pronounced, downregulation of TNF receptors was observed in peritoneal macrophages treated with these IFNs. This downregulation could not be accounted for by an induction of TNF secretion. Furthermore, IFN-alpha and gamma interacted synergistically in downregulating TNF receptors of RAW cells. IL-1 also downregulated TNF receptors. When RAW cells were treated with inhibitors of protein kinase C, the downregulation of TNF receptors by IFNs or IL-1 was reversed, and TNF binding increased up to 2-fold over that of untreated cells. Such increase was also observed in RAW cells treated only with the inhibitor of protein kinase C, staurosporine. However, TNF receptors decreased in peritoneal macrophages treated with staurosporine. This finding was explained by activation of macrophages by staurosporine, which induced secretion of TNF. These findings indicate that protein kinase C activity regulates TNF receptors in macrophages.     

α1-Adrenergic Receptor-Mediated Downregulation of Angiotensin II Receptors in Neuronal Cultures

Previous evidence has suggested that brain catecholamine levels are important in the regulation of central angiotensin II receptors. In the present study, the effects of norepinephrine and 3,4-dihydroxyphenylethylamine (dopamine) on angiotensin II receptor regulation in neuronal cultures from rat hypothalamus and brainstem have been examined. Both catecholamines elicit significant decreases in [125I]angiotensin II-specific binding to neuronal cultures prepared from normotensive rats, effects that are dose dependent and that are maximal within 4-8 h of preincubation. Saturation and Scatchard analyses revealed that the norepinephrine-induced decrease in the binding is due to a decrease in the number of angiotensin II receptors in neuronal cultures, with little effect on the receptor affinity. Norepinephrine has no significant actions on [125I]angiotensin II binding in cultures prepared from spontaneously hypertensive rats. The downregulation of angiotensin II receptors by norepinephrine or dopamine is blocked by alpha 1-adrenergic and not by other adrenergic antagonists, a result suggesting that this effect is initiated at the cell surface involving alpha 1-adrenergic receptors. This is further supported by our data indicating a parallel downregulation of specific alpha 1-adrenergic receptors elicited by norepinephrine. In summary, these results show that norepinephrine and dopamine are able to alter the regulation of neuronal angiotensin II receptors by acting at alpha 1-adrenergic receptors, which is a novel finding.     

Internalization and desensitization of adenosine receptors

Until now, more than 800 distinct G protein-coupled receptors (GPCRs) have been identified in the human genome. The four subtypes of the adenosine receptor (A(1), A(2A), A(2B) and A(3) receptor) belong to this large family of GPCRs that represent the most widely targeted pharmacological protein class. Since adenosine receptors are widespread throughout the body and involved in a variety of physiological processes and diseases, there is great interest in understanding how the different subtypes are regulated, as a basis for designing therapeutic drugs that either avoid or make use of this regulation. The major GPCR regulatory pathway involves phosphorylation of activated receptors by G protein-coupled receptor kinases (GRKs), a process that is followed by binding of arrestin proteins. This prevents receptors from activating downstream heterotrimeric G protein pathways, but at the same time allows activation of arrestin-dependent signalling pathways. Upon agonist treatment, adenosine receptor subtypes are differently regulated. For instance, the A(1)Rs are not (readily) phosphorylated and internalize slowly, showing a typical half-life of several hours, whereas the A(2A)R and A(2B)R undergo much faster downregulation, usually shorter than 1 h. The A(3)R is subject to even faster downregulation, often a matter of minutes. The fast desensitization of the A(3)R after agonist exposure may be therapeutically equivalent to antagonist occupancy of the receptor. This review describes the process of desensitization and internalization of the different adenosine subtypes in cell systems, tissues and in vivo studies. In addition, molecular mechanisms involved in adenosine receptor desensitization are discussed.     

Cell survival through Trk neurotrophin receptors is differentially regulated by ubiquitination

Specificity of neurotrophin factor signaling is dictated through the action of Trk receptor tyrosine kinases. Once activated, Trk receptors are internalized and targeted for degradation. However, the mechanisms implicated in this process are incompletely understood. Here we report that the Trk receptors are multimonoubiquitinated in response to neurotrophins. We have identified an E3 ubiquitin ligase, Nedd4-2, that associates with the TrkA receptor and is phosphorylated upon NGF binding. The binding of Nedd4-2 to TrkA through a PPXY motif leads to the ubiquitination and downregulation of TrkA. Activated TrkA receptor levels and the survival of NGF-dependent sensory neurons, but not BDNF-dependent sensory neurons, are directly influenced by Nedd4-2 expression. Unexpectedly, Nedd4-2 does not bind or ubiquitinate related TrkB receptors, due to the lack of a consensus PPXY motif. Our results indicate that Trk neurotrophin receptors are differentially regulated by ubiquitination to modulate the survival of neurons.     

ANG II type 1 receptor downregulation does not require receptor endocytosis or G protein coupling

ANG II type 1 (AT₁) receptors respond to sustained exposure to ANG II byundergoing downregulation of absolute receptor numbers. It has beenassumed previously that downregulation involves endocytosis. Thepresent study hypothesized that AT₁ receptor downregulation occurs independently of receptor endocytosis or G protein coupling. Mutant AT₁ receptors with carboxy-terminal deletionsinternalized <5% of radioligand compared with 65% for wild-typeAT₁ receptors. The truncated AT₁ receptorsretained the ability to undergo downregulation. These data suggest theexistence of an alternative pathway to AT₁ receptordegradation that does not require endocytosis, per se. Point mutationsin either the second transmembrane region or second intracellular loopimpaired G protein (G_q) coupling. These receptors exhibiteda biphasic pattern of downregulation. The earliest phase ofdownregulation (0-2 h) was independent of coupling toG_q, but no additional downregulation was observed after2 h of ANG II exposure in the receptors with impaired coupling toG_q. These data suggest that coupling to G_q isrequired for the later phase (2-24 h) of AT₁ receptor downregulation.

     

Analysis of expression of growth factor receptors in replicatively and oxidatively senescent human fibroblasts

Replicatively and oxidatively senescent human fibroblasts demonstrate an impaired response to mitogens. To investigate whether this is due to downregulation of growth factor receptors we examined their expression in these two types of senescence. mRNA and protein levels of the insulin receptor and platelet-derived growth factor (PDGF) alpha-receptor decreased in replicatively senescent cells. The PDGF beta-receptor and insulin-like growth factor 1 receptor at the protein level also decreased but remained readily detectable. However, these major growth factor receptors remained unchanged in oxidatively premature senescent cells. This suggests that mechanisms underlying diminished responsiveness to mitogens might be different in replicative senescence and oxidatively premature senescence.     

Role of Ubiquitination in Endocytic Trafficking of G‐Protein‐Coupled Receptors

Lysyl ubiquitination has long been known to target cytoplasmic proteins for proteasomal degradation, and there is now extensive evidence that ubiquitination functions in vacuolar/lysosomal targeting of membrane proteins from both the biosynthetic and endocytic pathways. G‐protein‐coupled receptors (GPCRs) represent the largest and most diverse family of membrane proteins, whose function is of fundamental importance both physiologically and therapeutically. In this review, we discuss the role of ubiquitination in the vacuolar/lysosomal downregulation of GPCRs through the endocytic pathway, with a primary focus on lysosomal trafficking in mammalian cells. We will summarize evidence indicating that mammalian GPCRs are regulated by ubiquitin‐dependent mechanisms conserved in budding yeast, and then consider evidence for additional ubiquitin‐dependent and ‐independent regulation that may be specific to animal cells.     

Human immunodeficiency virus-1 infection requires pertussis toxin sensitive G-protein-coupled signalling and mediates cAMP downregulation

The human immunodeficiency virus-1 (HIV-1) utilises CD4 and certain beta-chemokine receptors, mainly CCR-5 and CXCR4, for attachment and virus entry into T-lymphocytes and monocytes/macrophages. CD4 and beta-chemokine receptors participate in intracellular signalling via protein tyrosine kinases and G-protein-coupled signalling. The factors which influence HIV-1 replication and the intracellular signalling mechanisms elicited by the virus are not well understood. In this study, it was demonstrated that exposure of peripheral blood lymphocytes (PBLs) to a T-cell tropic strain of HIV-1 evokes signal(s) which results in downregulation of intracellular cAMP. In addition, pre-incubation of PBLs with the Gi-protein inhibitor Pertussis toxin mediated a significant inhibition of HIV-1 replication. These data strongly suggest that HIV-1 employs CD4 receptors and Gi-coupled proteins for entry into target cells and that productive HIV-1 infection is dependent on an active signalling event.