Compartmentalized signal transduction by receptor tyrosine kinases

Signal transduction through receptor tyrosine kinases is believed to occur mainly at the plasma membrane. Ligands bind to their cognate receptors and trigger autophosphorylation events, which are detected by intracellular signalling molecules. However, ligands, such as epidermal growth factor and insulin, induce the rapid internalization of their receptors into endosomes. Although this event is traditionally thought to attenuate the ligand-induced response, in this article the authors discuss an alternative scenario in which selective and regulated signal transduction from receptor tyrosine kinases occurs within the endosome.     

The trick of the tail: protein-protein interactions of metabotropic glutamate receptors

It was initially believed that G-protein-coupled receptors, such as metabotropic glutamate receptors, could simply be described as individual proteins that are associated with intracellular signal cascades via G-proteins. This view is no longer tenable. Today we know that metabotropic glutamate receptors (mGluRs) can dimerize and bind to a variety of proteins in addition to trimeric G-proteins. These newly identified protein interactions led to the discovery of new regulatory mechanisms that are independent of and sometimes synergistic with the classical G-protein-coupled second messenger pathways. Notably, several of these mechanisms connect mGluR-mediated signaling to other receptor classes, thereby creating a network of different receptor types and associated signal cascades. The intracellular C-termini of mGluRs play a key role in the regulation of these networks, and various new protein interactions of these domains were described recently. Because mGluRs are involved in a variety of physiological and pathophysiological processes, some of the proteins interacting with this receptor class have potential as valuable pharmaceutical targets. This review will give a comprehensive overview of proteins interacting with mGluR C-termini, highlight new evolving regulatory mechanisms for glutamatergic signal transduction and discuss possibilities for future drug development.     

The TNF-TNF receptor system

Different forms of tumor necrosis factor (TNF) interact with two specific receptors for TNF (TNFR) on the cell membrane to induce a variety of effects. While sharing structural similarities in their extracellular domains, the two TNFRs differ in their intracellular domain, their signal transduction, and consequently their function. In addition, one of the two TNFRs can be expressed in two differently located isoforms. This makes the TNF-TNFR system very complex. The dual TNF function for either cell death or survival upon interaction of members of the TNF ligand family with members of the TNF receptor family will be discussed.     

Heterogeneity of signal requirements in T cell activation within a panel of human proliferative T cell clones

Activation of T lymphocytes is initiated by receptor ligand interactions at the cell surface leading to the transduction of intracellular signals followed by the de novo synthesis and expression of T cell activation markers (including receptors for interleukin 2 (IL 2) and transferrin), production of lymphokines, and T cell proliferation. This requisite first step for activation of T lymphocytes can be mimicked in certain situations with a variety of stimuli. These include antibodies to certain integral membrane proteins, phorbol esters, and plant lectins that act as mitogens. In this paper, we report that at least two classes of human T cell clones can be distinguished based upon signal requirements necessary to induce proliferation. Although all clones analyzed expressed IL 2 receptors and secreted IL 2 after non-antigenic activation, one subset of clones did not proliferate in response to the same non-antigenic signals. In that subset, complete activation leading to proliferation required interaction of the T cell with specific antigen. The ability to subset these T cell clones into two groups did not correlate with phenotypic differences, source of the clone, nor with magnitude of intracellular calcium mobilization. By studying the stimulation requirements of these two subsets of human T cell clones through the use of specific antigen or antigen-independent stimuli, it was possible to demonstrate that different stimuli varied in their ability to induce steps of T cell activation. Analysis of reactivity of these clones to suboptimal stimulation allowed the definition of intermediate stages of T cell activation. Such intermediate stages might reflect a diversity of intracellular signaling pathways or a complexity of regulatory mechanisms distal to the events that allow intracellular calcium mobilization. Thus for the first time, it has been possible to study ordered events of T cell activation in non-transformed, antigen-dependent human T lymphocytes. The data presented in this paper suggest that T cell activation is not an all or nothing phenomenon, and there is an ordered sequence of events that can be differentiated based upon signal requirements at the T cell membrane.     

Membrane lipid rafts: new targets for immunoregulation

Engagement of immune receptors by antigen may lead to activation, cell proliferation, differentiation and effector functions. It has recently been proposed that the initiation and propagation of the signaling events taking place in immune cells occur in specialized membrane regions called lipid rafts. These detergent-insoluble glycolipid domains are specialized membrane compartments enriched in cholesterol and glycolipids. They also contain many lipid-modified signaling proteins such as tyrosine kinases of the Src family, GPI (glycosylphosphatidylinositol)-linked proteins as well as adaptor proteins. The confinement of signaling molecules in membrane subdomains suggests that lipid rafts function as platforms for the formation of multicomponent transduction complexes. Indeed, upon receptor binding, immune receptors become raft-associated and additional components of the signaling pathways are recruited to rafts in order to form signaling complexes. It has been speculated that the entry of immune receptors into rafts can regulate cell activation. Accordingly, numerous experiments have provided substantial evidence that raft integrity is crucial for the initiation and maintenance of intracellular signals. Recent studies have also shown that the access and translocation of immune receptors to lipid rafts are developmentally regulated (immature versus mature cells, Th1 versus Th2 lymphocytes) and sensitive to pharmacological agents. The aim of the present review is to summarize the current knowledge of immune receptor signal transduction with particular emphasis on the role of membrane compartments in immune activation. Finally, experimental evidences indicating that these membrane structures may represent clinically relevant potential targets for immune regulation, will be discussed.     

Signal transduction in monocytes: the role of zinc ions

The availability of zinc has a regulatory role in the immune system. It can have either pro- or anti-inflammatory effects, which both seem to be a consequence of a direct interaction of zinc with the cytokine secretion by monocytes. In this review, the molecular basis for this effect, the interaction of zinc with the signal transduction of monocytes, is discussed. In particular, zinc seems to activate or inhibit several signaling pathways that interact with the signal transduction of pathogen sensing receptors, the so-called Toll-like receptors (TLR), which sense pathogen-derived molecular structures and, upon activation, lead to secretion of pro-inflammatory cytokines. The interaction of zinc with protein tyrosine phosphatases and protein kinase C, and a direct modulation of lipopolysaccharide binding to its receptor (TLR-4) all result in enhanced cytokine production. On the other hand, a complex interaction between zinc, NO and cyclic nucleotide signaling, and inhibition of interleukin-1 receptor associated kinase-1, and inhibitor of kappa B kinase all counteract the production of pro-inflammatory cytokines. A role for the zinc binding protein metallothionein as a regulator for intracellular zinc signaling is discussed. By acting on all these signaling molecules, the zinc status of monocytes can have a direct effect on inflammation.     

Regulation of receptors and transporters by ubiquitination: new insights into surprisingly similar mechanisms

The function of many receptors and transport proteins that reside at the surface of the cell is regulated by endocytosis and postendocytic trafficking. Modification of receptors and transporters by ubiquitin conjugation has recently emerged as the major regulatory mechanism of internalization and intracellular sorting of these membrane proteins. This review will describe recent advances in elucidating the mechanisms of ubiquitination of mammalian receptors and transporters using two examples: the receptor for epidermal growth factor and the dopamine transporter. How ubiquitination controls the endocytosis and turnover of these proteins will be also discussed.     

Involvement of JAK-family tyrosine kinases in hematopoietin receptor signal transduction

A variety of cytokines, hormones and hematopoietic growth factors signal through the hematopoietin family of membrane receptors, which share several structural features, including a Trp-Ser-X-Trp-Ser motif and four paired cysteine residues. The signal transduction mechanisms utilized by these receptors have remained elusive, although tyrosine kinase activation has been one common element. Recently, a role for the cytoplasmic tyrosine kinases of the Janus kinase (JAK) family has been implicated in signalling by these receptors. There are currently three known JAK family kinases, including JAK1, JAK2 and TYK2. This review will focus on the role of such tyrosine kinases in hematopoietin receptor signal transduction, and address the possibility of the involvement also of unidentified Janus kinases.     

A 20 amino acid synthetic peptide of a region from the 55 kDa human TNF receptor inhibits cytolytic and binding activities of recombinant human tumour necrosis factor in vitro.

Tumour Necrosis Factor (TNF) and Lymphotoxin (LT) can exert a wide range of effects on cells and tissues and they are important effector molecules in cell mediated immunity. All these effects are induced subsequent to the binding of these cytokines to specific membrane receptors. Recently, two of these membrane receptors of 55 and 75 kDa, have been identified which share some amino acid (AA) homology in their N-terminal extracellular domains but differ in their intracellular domains. We synthesized two synthetic 20 AA peptides from hydrophilic regions of the N-terminal extracellular domains of the 55 kDa receptor; peptide A shares homology with both 55 and 75 kDa receptors, peptide B is unique. We found peptide B inhibits both the binding and cytolytic activity of recombinant human TNF when tested on murine L929 cells in vitro. Polyclonal antiserum generated against peptide B will block binding of 125I-labelled TNF to these cells in vitro. However, peptide A and antiserum prepared against peptide A are without effect in these same assay systems. These data suggest that the 20 AA sequences from AA 175 to 194 in the N-terminal extracellular domain of the 55 kDa TNF receptor are expressed on the cell surface and are involved in the binding of TNF.     

Lipid raft-independent B cell receptor-mediated antigen internalization and intracellular trafficking

The Ag-specific B cell receptor (BCR) expressed by B lymphocytes has two distinct functions upon interaction with cognate Ag: signal transduction (generation of intracellular second messenger molecules) and Ag internalization for subsequent processing and presentation. While it is known that plasma membrane domains, termed lipid rafts, are involved in BCR-mediated signal transduction, the precise role of plasma membrane lipid rafts in BCR-mediated Ag internalization and intracellular trafficking is presently unclear. Using a highly characterized model system, it was determined that while plasma membrane lipid rafts can be internalized by B lymphocytes, lipid rafts do not represent a major pathway for the rapid and efficient internalization of cell surface Ag-BCR complexes. Moreover, internalized plasma membrane lipid rafts are delivered to intracellular compartments distinct from those to which the bulk of internalized Ag-BCR complexes are delivered. These results demonstrate that B lymphocytes, like other cell types, possess at least two distinct endocytic pathways (i.e., clathrin-coated pits and plasma membrane lipid rafts) that deliver internalized ligands to distinct intracellular compartments. Furthermore, Ag-BCR complexes differentially access these two distinct internalization pathways.     

JAK/STAT: Why choose a classical or an alternative pathway when you can have both?

A subset of cytokines triggers the JAK‐STAT pathway to exert various functions such as the induction of inflammation and immune responses. The receptors for these cytokines are dimers/trimers of transmembrane proteins devoid of intracellular kinase activity. Instead, they rely on Janus kinases (JAKs) for signal transduction. Classical JAK‐STAT signalling involves phosphorylation of cytokine receptors'' intracellular tyrosines, which subsequently serve as docking sites for the recruitment and activation of STATs. However, there is evidence to show that several cytokine receptors also use a noncanonical, receptor tyrosine‐independent path to induce activation of STAT proteins. We identified two main alternative modes of STAT activation. The first involves an association between a tyrosine‐free region of the cytokine receptor and STATs, while the second seems to depend on a direct interaction between JAK and STAT proteins. We were able to identify the use of noncanonical mechanisms by almost a dozen cytokine receptors, suggesting they have some importance. These alternative pathways and the receptors that employ them are discussed in this review.     

Mechanisms of polarized targeting of the FSH receptor]

Gonadotropin and TSH receptors belong to a subgroup of G protein-coupled receptors. TSH and FSH receptor present a particular intracellular traffic: they present a polarized basolateral expression in thyroid follicular cells and in Sertoli cells respectively. By contrast, the LH receptor is expressed circumferentially in target gonadic cells. We expressed these receptors in MDCK cells (a well characterized model of polarized epithelial cells) to understand this difference of properties. We show that the three receptors have a polarized basolateral expression in these cells. All contain a basolateral targeting signal. Furthermore, gonadotropin receptors undergo a partial transcytosis which is not observed for the TSH receptor. We show that heterotrimeric G proteins play a role in this mechanism of transcytosis. This effect is not mediated by adenylate cyclase activation and involves a population of G proteins different from that involved in signal transduction. We thus used in vitro mutagenesis to delineate the basolateral localization signal of the FSH receptor. Surprisingly, the signal is localized in the C-terminal tail of the intracellular domain which is not conserved between the three receptors. It contains 14 amino-acids and its activity is mainly dependent on a tyrosine and a leucine residue. The basolateral localization signal of the FSHR is not colinear with its internalization signal. This signal is autonomous and dominant because, when transferred to an apically targeted membrane protein, the neurotrophin receptor, it redirects the chimeric construct to the basolateral domain of MDCK cells. The basolateral localization signal of the FSH receptor is thus the first signal identified for a G protein-coupled receptor and more generally for a hormone receptor.     

Ceramide-enriched membrane domains—Structure and function

Membrane lipids seem to be organized and not randomly distributed in the cell membrane. In particular, sphingolipids seem to interact with cholesterol in the outer leaflet of the cell membrane resulting in the formation of distinct membrane domains, i.e. rafts. The generation of ceramide within rafts alters their biophysical properties and results in the formation of large ceramide-enriched membrane platforms. These platforms serve to cluster receptor molecules and to organize intracellular signalling molecules to facilitate signal transduction via a receptor upon stimulation. Thus, ceramide-enriched membrane domains amplify not only receptor-, but also stress-mediated signalling events. Although many receptors cluster, the molecular mechanisms mediating this important and general event in signal transduction need to be identified.     

Ceramide-enriched membrane domains--structure and function

Membrane lipids seem to be organized and not randomly distributed in the cell membrane. In particular, sphingolipids seem to interact with cholesterol in the outer leaflet of the cell membrane resulting in the formation of distinct membrane domains, i.e. rafts. The generation of ceramide within rafts alters their biophysical properties and results in the formation of large ceramide-enriched membrane platforms. These platforms serve to cluster receptor molecules and to organize intracellular signalling molecules to facilitate signal transduction via a receptor upon stimulation. Thus, ceramide-enriched membrane domains amplify not only receptor-, but also stress-mediated signalling events. Although many receptors cluster, the molecular mechanisms mediating this important and general event in signal transduction need to be identified.     

Astrocyte beta1-adrenergic receptor immunoreactivity and agonist induced increases in [Ca2+]i: differential results indicative of a modified membrane receptor

Antibodies against the C-terminus of the beta1-adrenergic receptor were used for staining cultured astrocytes from the rat cerebral cortex. Immunoreactivity was found to be localized exclusively to an intracellular organelle structure similar to the Golgi complex, with no staining of the plasma membrane. The astrocytes stained positive with BODIPY CGP 12177, a FITC-conjugated beta-adrenergic receptor agonist, and this staining was blocked by the beta1-adrenergic antagonist atenolol, indicating that these receptors are expressed on the surface of the astrocytes. The presence of functional plasma membrane beta1-adrenergic receptors was further verified using microspectrofluorometry for measurements of intracellular calcium changes upon beta-adrenergic agonist stimulation. Intracellular immunoreactivity confined to the organelles was also found in astrocytes from mixed astroglial-neuronal cultures. In contrast, the neurons in these cultures showed a strong labeling of the cell bodies by the beta1-adrenergic receptor antibodies. Thus, the beta1-adrenergic receptor antibody, which stains the cell bodies of the neurons, recognizes the astroglial receptors only intracellularly, although functional beta1-adrenergic receptors are present on the astroglial surface. Taken together, these data suggest that the beta1-adrenergic receptors observed intracellularly might be processed on their passage to the surface to a modified form of the final plasma membrane receptor, which is not recognized by the antibodies.     

Dissociation of beta-arrestin from internalized bradykinin B2 receptor is necessary for receptor recycling and resensitization

Beta-arrestins are multifunctional adaptors that bind agonist-activated G protein-coupled receptors (GPCRs), mediate their desensitization and internalization, and control the rate at which receptors recycle back at the plasma membrane ready for subsequent stimulation. The activation of the bradykinin (BK) type 2 receptor (B2R) results in the rapid desensitization and internalization of the receptor. Little is known, however, about the role of beta-arrestin in regulating the intracellular trafficking and the resensitization of the B2R. Using confocal microscopy, we show that BK stimulation of COS-7 cells expressing B2R induces the colocalization of the agonist-activated receptor with beta-arrestin into endosomes. Fluorescent imaging and ligand binding experiments also reveal that upon agonist removal, beta-arrestin rapidly dissociates from B2R into endosomes, and that receptors return back to the plasma membrane, fully competent for reactivating B2R signaling as measured by NO production upon a second BK challenge. However, when the receptor is mutated in its C-terminal domain to increase its avidity for beta-arrestin, B2R remains associated with beta-arrestin into endosomes, and receptors fail to recycle to the plasma membrane postagonist wash. Similarly, the recycling of receptors is prevented when a beta-arrestin mutant exhibiting increased avidity for agonist-bound GPCRs is expressed with B2R. Stabilizing receptor/beta-arrestin complexes into endosomes results in the dampening of the BK-mediated NO production. These results provide evidence for the involvement of beta-arrestin in the intracellular trafficking of B2R, and highlight the importance of receptor recycling in reestablishing B2R signaling.