(Biol Res 35: 117-125, 2002 )

Introduction to supramolecular complex formation in cell
signaling and disease

LISETTE LEYTON AND ANDREW F. G. QUEST*

Program of Morphology and *Program of Cellular and Molecular Biology, Laboratory of Cellular Communication, ICBM (Instituto de Ciencias Biomédicas)-Faculty of Medicine
University of Chile, Independencia 1027, Santiago, Chile.
lleyton@machi.med.uchile.cl, aquest@machi.med.uchile.cl

Cells are constantly exposed to a plethora of signals from their environment. Incoming information, which often impinges on cells as membrane-impermeable molecules, must be converted into a "language" that allows the cell to respond in an appropriate fashion. This process, called signal transduction, culminates in cellular responses as diverse as secretion, cell division, differentiation, cell movement or cell death, to name a few. Signaling pathways, the intracellular link between incoming information and outgoing response, are becoming increasingly convoluted and intertwined and the question concerning specificity ever more demanding to answer. One important aspect to keep in mind when thinking about such processes is that the molecules involved, frequently proteins, are not randomly distributed within the cell. Even those that constitute what is referred to as "cytosol" are associated with other molecules. However, interactions may be weak and occur in such a transient manner that detection is not possible by available methods of analysis. Thus, the real challenge becomes not signaling per se, but rather understanding the principles governing cellular organization particularly in those domains where structure is less apparent. As the following summary reveals, progress has been made but much more remains yet to be accomplished.

Early on, signaling events linked to membrane-bound lipid second messengers, like diacylglycerol (DAG), were associated with the concept of compartmentalization. The identification of Protein Kinase C (PKC) as a molecular target for DAG and tumor promoting agents (phorbol esters) highlighted the possible relevance of lipid-mediated signaling events to disease. Quite remarkably, such thinking was developed at least 10 years before compartmentalization, localization and complex formation became central themes in signal transduction (revised in Quest, 1996; Quest et al., 1996). In the early nineties, phosphorylation on tyrosine residues of receptor tyrosine kinases was shown to generate docking sites for effector proteins with appropriate binding motifs (e.g. SH2 domains) that were essential for signal propagation (Reedijk et al., 1992). From there, in the wake of many more important discoveries, emerged our current view of proteins as modular entities, comprised to a considerable extent of elements that define location (proximity to other molecules) rather than function (activity), and hence also our current appreciation of the importance of complex formation and localization in the process of signal transduction.

Since lipids have played a crucial role in developing concepts like "compartmentalization" and in appreciating the importance of "positional information" in cellular signaling, the fact that this symposium will start with a discussion of recent insights concerning membrane microdomains and their role in signaling is probably not surprising.

In contrast to earlier held common wisdom, recent evidence shows that depending on the nature of the lipid itself, lateral mobility can be more or less restrained. Particularly, phospholipids with saturated fatty acid side chains, sphingolipids and cholesterol have a tendency to form "microdomains". Thanks to their "ordered" nature, these membrane regions are more resistant to non-ionic detergents. Thus, terms like detergent-resistant membranes (DRMs), detergent-insoluble glycolipid enriched domains (DIGs) or rafts, to indicate their insular nature, have been coined. The ability to biochemically isolate such structures has been instrumental in their functional characterization and in providing a link to cellular signaling events. Indeed, such preparations are highly enriched in a variety of molecules involved in signal transduction. Thus the term "signalosome" has also been developed in reference to these sites where multiple signaling pathways appear to converge (Bender et al., 2002; Magee et al., 2002).

Protein presence in such domains may vary depending on the activation state and is often, but not exclusively, linked to the presence of lipid modifications (Bender et al., 2002; Magee et al., 2002; Patterson, 2002). Unfortunately, tremendous variations are also observed depending on the methods of preparation. For instance, Quest and co-workers compare here two methods typically employed to isolate such membrane domains and show that different proteins may be found associated with such preparations starting from the same biological material (Bender et al., 2002). These limitations underscore the necessity to refine our approaches in the future and hopefully develop the tools to characterize the different sub-populations of microdomains that undoubtedly exist. Yet, as crude as our current experimental approaches may appear, they have been instrumental in developing the idea that supramolecular structures dedicated to signal transduction (signalosomes) exist in the plasma membrane. The importance of such structures for cellular signaling is perhaps best illustrated by experiments showing how cholesterol depletion/sequestration modulates microdomain structure and signaling functions alike (Bender et al., 2002; Magee et al., 2002).

Caveolins, a protein family comprised of at least three mammalian isoforms, are important components of some microdomains. Particularly in the presence of caveolin-1 or -3, microdomains are morphologically distinguishable flask-shaped membrane invaginations of 50-100 nm, referred to as caveolae, that can be easily detected by electron microscopy. However, it is important to emphasize that membrane microdomains exist and are functionally relevant in cells in the absence of caveolins. T cells are a nice example in this respect, since they lack caveolin expression but do possess microdomains enriched in cholesterol and sphingolipids, glycosyl phosphatidylinositol (GPI)_anchored proteins, Src-family kinases and a number of other proteins implicated in T cell signaling. Magee and coworkers discuss the dynamics of lipid rafts upon T cell receptor (TCR) stimulation, indicating that aggregation of such domains is a crucial event in T cell activation. Molecules like the Src-family kinase Lck, the linker for activation of T cells (LAT) and the TCR colocalize in these domains once the TCR is activated, thereby promoting tyrosine kinase activation and hence tyrosine phosphorylation of proteins like LAT and the z chain of the TCR. Enhanced tyrosine phosphorylation appears to be favored by the formation of a supramolecular complex from which the tyrosine phosphatase CD45 is excluded (Magee et al., 2002).

T cell activation is an essential part of the cellular immune response that is mounted in response to infection with the objective of eliminating infected cells and the infectious agent alike. Initiation of an antigen-specific immune response in T cells occurs upon recognition by the TCR of antigenic peptides presented on the surface of an antigen-presenting cell (APC) in the context of a major histocompatibility complex molecule. In this instance, a specialized structure, termed the immunological synapse, comprised of both adhesion and signaling molecules, is formed at the cell-cell interface. Formation of this structure was considered a pre-requisite to T cell signaling (reviewed in Bromley et al., 2001). More recently, however, it has been proposed that TCR signaling occurs prior to formation of the immunological synapse, implying that this structure may play a different role to that once contemplated (Lee et al., 2002). Using different imaging-based approaches, Valitutti and co-workers show that, although not essential for productive TCR triggering per se, the immunological synapse does support the signaling process and promotes events leading to full-scale T cell activation (Faroudi et al., 2002).

Imaging techniques are currently very much "en vogue" to study compartmentalization in signal transduction and a number of researchers will discuss experiments of this type. For instance, conventional immunofluorescence experiments and analysis by confocal microscopy are employed to follow formation of focal contacts, focal adhesions and stress fibers (Arthur et al., 2002; Avalos et al., 2002). Expression of signaling proteins tagged with green fluorescence protein (GFP) is also utilized here to study protein localization and translocation events in signaling, as well as to investigate domain organization of different molecules of a particular signaling cascade by fluorescence energy transfer (Magee et al., 2002). Finally, high-resolution 3D confocal microscopy is used to visualize formation of the immmunological synapse between APCs and T cells (Faroudi et al., 2002). The image chosen for the cover page of this special edition and the symposium poster was obtained from immunofluoresence studies. Upon stimulating astrocytes by allowing them to grow on coverslips coated with the molecule Thy-1, tyrosine phosphorylation is observed in select locations throughout the cell (Leyton et al., 2001). Such images illustrate, in a very appealing manner, how highly localized cellular signaling events can be.

Studies of cell signaling in lymphocytes indicate that efficient activation may also be achieved in vitro by cross-linking the TCR with antibodies attached to a solid phase. In contrast, soluble anti-TCR antibodies only stimulate T cells to modest levels unless added together with antibodies against accessory molecules, such as the GPI-anchored protein Thy-1 (Leyton et al., 1999). The src family protein tyrosine kinase (PTK) Lck is essential for TCR signaling and colocalizes with GPI-anchored proteins in rafts. Targeting to such membrane regions occurs in both cases via post-translational lipid modifications (Magee et al., 2002; Patterson, 2002) and is considered essential for efficient signaling.

Similar importance is also attributed to lipid modifications in the development of neuronal growth cones. There, alterations in such modifications, observed as a function of growth cone maturation, may determine the nature of signaling events triggered in response to a given stimulus (Patterson, 2002).

Caveolin-1 is also covalently modified by lipids, but these modifications are neither considered essential for membrane localization nor presence in detergent resistant membrane microdomains. However, they do appear to be important for cholesterol transport and also caveolin phosphorylation by src kinase. Cytoplasmic N- and C-terminal domains flank a central 33 amino acid hydrophobic domain that is presumed to form a tight alpha-helical loop and anchor caveolin-1 to the membrane. A juxta-membrane motif of caveolin-1, called the scaffolding domain, binds directly to a number of signaling proteins implicated in cell proliferation and inhibits their function. Accordingly, reduced caveolin-1 levels were proposed to enhance cell proliferation and anchorage-independent growth, thereby favoring cell transformation. Consistent with this notion caveolin-1 levels are reduced in a number of tumors and cancer cell lines (Bender et al., 2002). Results will be discussed showing that caveolin-1 levels are significantly lower in colon tumor mucosa when compared with normal mucosa and only low levels of caveolin-1 expression are detected in colon carcinoma cell lines. In addition, ectopic expression of caveolin-1 in HT-29 and DLD-1 reduces the tumor forming ability of these cells (Bender et al., 2000). Targets of caveolin-1 that might explain this ability were sought. Caveolin-1 was found to inhibit inducible nitric oxide synthase (iNOS) by an unexpected mechanism, namely by promoting degradation via the proteasome pathway (Felley-Bosco et al., 2000; 2002). More recently, microarray experiments have identified a number of genes that are regulated at the transcriptional level by the presence of caveolin-1 in colon carcinoma cells (Bender et al., 2002). Thus caveolin-1-dependent control of cellular function is rapidly becoming far more complex than once anticipated.

Compartmentalization is not only important for lipids and proteins, but also for second messengers like calcium. In this case, liberation at distinct locations in conjunction with variations in kinetics and magnitude are employed to explain the ability of Ca²⁺ to regulate such a large variety of different processes. Increases in intracellular calcium levels are achieved by opening of channels located either at the plasma membrane or intracellular membranes, such as the endoplasmic reticulum. On the other hand, powerful Ca²⁺ pumps are responsible for restoring basal Ca²⁺ levels in the cytosol after stimulation (Petersen, 2002). Given the central role calcium plays in cellular signaling, it is easy to appreciate that all these activities are subject to stringent regulation by a wide variety of mechanisms. For example, Hidalgo and coworkers discuss how redox modification of the ryanodyne receptor affects Ca²⁺ release from the endoplasmic reticulum in mammalian cardiac and skeletal muscle cells (Hidalgo et al., 2002). On the other hand, Jaimovich and coworkers have found that Ca²⁺ levels increase in both the cytosol and nuclear compartment of muscle cells by independent inositol -3,4,5-trisphosphate linked mechanisms. Clearly, calcium transients in the nucleus are important for regulation of gene transcription while those in the cytosol trigger muscle contraction. How these signaling events are controlled in an independent fashion is currently under investigation (Jaimovich and Carrasco, 2002). Perturbations of any of these mechanisms involved in calcium homeostasis can lead to disease. For instance the ability of bile acids to induce acute pancreatitis is probably linked to excessive and prolonged cytosolic Ca²⁺ signals (Petersen, 2002). Alternatively, mutations in some genes encoding calcium channel subunits are associated with muscle disorders or malfunction of neuromuscular junctions (Hidalgo et al., 2002; Jaimovich and Carrasco, 2002).

Deregulation of calcium signaling may also lead to cell death. For instance, treatment of pancreatic cells with the oxidant menadione triggers apoptosis by modulating calcium signals and permeability of the mitochondrial transition pores (Gerasimenko et al., 2002; Petersen, 2002). Interestingly, however, alterations in calcium homeostasis are also implicated in an alternative form of cell death called necrosis (Barros et al., 2002; Simon et al., 2002).

Apoptosis, or programmed cell death, refers to a mode of cell death frequently employed during development or as part of normal tissue homeostasis to eliminate cells without triggering an immune response. Given this physiological role, it is not surprising that apoptotic dysfunction is intimately associated with the etiology of various disorders including cancer, AIDS and neurodegenerative diseases. Thus, understanding the reasons why apoptotic checkpoints fail is critical to achieve appropriate treatments (Razik and Cidlowski, 2002).

During apoptosis decreases in cell volume and chromatin condensation are observed prior to cell fragmentation into apoptotic bodies, which are then phagocytosed by specialized cells. In recent years, this essentially morphological definition has become better understood at the molecular level. Two distinct pathways are distinguished depending on whether the stimulus that leads to cell death is intrinsic (mitochondrial pathway) or extrinsic (surface receptor dependent pathway). In both cases, multiprotein complexes are formed that activate a series of proteases, called caspases, which then orchestrate the demise of the cell. The decline in cell volume associated with apoptosis has generally been considered an interesting but non-essential part of the process. However, more recent evidence suggests that decreases in K⁺ concentration associated with cell shrinkage favor the activation of proteases and nucleases implicated in the execution of apoptotic cell death (Razik and Cidlowski, 2002).

Necrosis, in contrast to apoptosis, is not considered a regulated event and is often referred to as accidental cell death. Cells that die by necrosis swell and ultimately are lysed, thereby provoking an inflammatory response. Interestingly, different ions and ion fluxes are associated with the two types of cell death as discussed by Barros and coworkers. While apoptosis is characterized by an intracellular increase in protons and a decrease in K⁺, large increases in Ca²⁺, Na⁺ and Mg²⁺ are associated with cells death by necrosis (Barros et al., 2002). In response to hydrogen peroxide mediated oxidative stress, liver cells die by necrosis. Swelling, in this case, is due mainly to influx of Na⁺ through nonselective cation channels (NSCC) and treatment of cells with flufenamic acid blocks both cell swelling and necrosis (Simon et al., 2002). Interestingly, apoptosis and necrosis need not be mutually exclusive events. Both were recently shown to occur simultaneously in response to FasL in Jurkat T and A20 B-lymphoma cells. FasL-dependent necrosis required caspase 8 activation and was linked to lipid scrambling at the plasma membrane followed by delayed increases in intracellular levels of the lipid second messenger ceramide (Hetz et al., 2000; 2002). Thus, contrary to general thinking, necrosis also appears to be initiated as a regulated and controllable process, at least under certain circumstances.

DAG was the first membrane-bound lipid second messenger to be identified. Meanwhile, many more have been described and lipid second messengers are easily becoming the largest and most diverse group of cellular messenger molecules characterized to date. As such they are implicated in the control of all aspects of cell growth, including proliferation, differentiation and apoptosis. One important intracellular target is PKC, a family of serine/threonine kinases with meanwhile at least eleven members. For DAG, in particular, showing that it served as a specific activator of PKCs was a key observation in establishing its role as a second messenger. Also, it was argued that separate pools of DAG must be utilized in cells for lipid biosynthesis and signaling. Both of these concepts are now subject to scrutiny. First, DAG may no longer be considered a specific activator of only PKCs, since cysteine-rich motifs responsible for the interaction with DAG are now known to be present in many other proteins. Second, biosynthetic DAG may also play a role in signaling events leading to apoptosis (Quest, 2000; Quest et al., 1996; Wright and McMaster, 2002).

Apoptosis is also regulated by cell adhesion and is triggered in non-transformed cells by preventing the interaction with extracellular matrix (ECM) proteins. This form of apoptosis that is induced by anchorage-independent growth has been termed "Anoikis". Cell characteristics like shape and cytoskeletal organization are also important in this context. Since integrins are important ECM receptors in vivo, integrin signaling has been implicated as a critical element in preventing activation of the apoptotic machinery.

Integrins not only mediate cell-matrix interactions but also cell-cell interactions as discussed by Leyton and coworkers. Recently, a novel interaction between the immunoglobulin superfamily member Thy-1, present in neurons, and the integrin avß3, found in astrocytes, was described. This interaction promotes astrocyte adhesion and spreading by stimulating focal adhesion and stress fiber formation (Leyton et al., 2001). Integrins and cadherins, another important group of receptors that mediates cell-cell interactions, are both known modulators of the Rho-family GTPases. Leyton and coworkers present evidence suggesting that the morphological changes observed in astrocytes are indeed dependent upon signaling events triggered by the small G protein RhoA (Avalos et al., 2002). On the other hand, Burridge and coworkers discuss how these small G proteins are regulated by the two aforementioned groups of receptors and propose a model in which feedback mechanisms between the two potentiate cell-matrix and cell-cell interactions (Arthur et al., 2002).

Small GTP-binding proteins, like the larger heterotrimeric G-proteins, cycle between an OFF and ON state by exchanging GDP for GTP. Activation and inhibition are regulated by different sets of proteins know as "Guanine nucleotide Exchanger Factors" (GEFs) and GTPase-Activating Proteins (GAPs). Both families of proteins and their respective regulation are discussed here (Arthur et al., 2002; Hawadle et al., 2002). Another group of small GTP binding proteins that will be discussed is the ADP-ribosylation factor (ARF) family. ARFs are generally implicated in vesicular transport and control of cytoskeletal and cell adhesion events. The means by which extracellular signals regulate specific ARF signaling pathways and the part played by macromolecular complexes in controlling such events is addressed in studies by Jackson and coworkers (Hawadle et al., 2002; Jackson et al., 2000).

Cell proliferation, cell movement, cell adhesion, cell-cell interaction, cell differentiation and apoptosis are all important events in the development of multicellular organisms with different tissues and organs. The formation of regions with completely distinct functions starting from a common ancestor relies on differential gene expression. Given that the genome is essentially identical in all cells of an organism, the question becomes how such differential expression is coordinated in a temporally meaningful way. During embryo gastrulation three major regions of cells are defined, termed endoderm (precursors for gut and internal organs), mesoderm (precursor cells for muscle, connective tissue and vascular system) and ectoderm (precursor cells for epidermis and the nervous system). The way such specification is currently thought to occur is by differential exposure to gradients of "morphogens". Depending on their local concentration, these molecules induce different behaviors in the exposed groups of cells. In this context, Mayor and coworkers discuss how a series of sequentially defined signals are responsible for induction of the neural crest. Initially, a gradient of bone morphogenetic proteins is established in the ectoderm that leads to segregation of the neural plate, epidermis and neural folds. This initial neural fold is of anterior character and exposure to additional posteriorizing signals, such as Wnt ligands, retinoic acid and fibroblast growth factor, is utilized to define the prospective neural crest. Finally, Notch/Delta signaling is required for induced cells to complete neural crest formation (Aybar et al., 2002).

As outlined above, Wnts play a critical role in determining cell fate during embryogenesis (Aybar et al., 2002). In adult individuals, however, excessive signaling through this same pathway is strongly implicated in tumorigenesis, since mutations in downstream elements are associated in a causative manner with the development of different types of cancers. In the canonical Wnt signaling pathway (b-catenin pathway), multiprotein complexes play a crucial role by regulating b-catenin degradation via the proteasome pathway. For instance, the tumor suppressor protein adenomatous polyposis coli (APC), which is frequently mutated in human colon cancer, is a scaffolding protein that associates with Axin/Conductin and b-catenin, promotes phosphorylation of the latter by GSK3b and subsequent ubiquitination. Wnt signaling via its receptor Frizzled blocks this sequence. Thereby stabilized b-catenin accumulates in the cytoplasm where it interacts with members of the TCF/LEF family of transcription factors and activates gene expression (Hendriks and Reichmann, 2002).

Cancer is a multistep process involving genetic alterations that favor proliferation and/or decreased susceptibility to apoptosis. Several oncogenes and tumor suppressors are implicated with varying frequencies. For example, oncogenic mutations of Ras are detected in roughly 30% of all human cancers. Ras-dependent signaling involves amongst many other possibilities, activation of the mitogen-activated-protein kinase (MAPK) cascade.

This evolutionarily highly conserved signaling cascade is another excellent example in which complex formation between individual pathway elements and scaffolding proteins are essential for efficient signal transduction. Interestingly, oncogenic ras induces a senescence-like permanent growth arrest phenotype in primary cells, suggesting that cells may avoid oncogene-mediated transformation by inducing senescence (Lin and Lowe, 2001). Sierra and coworkers have identified genes that are specifically over-expressed in rats as they approach the end of their lifespan. Serum levels of T-kininogen (T-KG) were found to increase dramatically in these animals. Upon overexpression of T-KG in both fibroblasts and endothelial cells, proliferation was dramatically reduced. This was linked to reduced signaling via the Ras/ERK pathway, since T-KG inhibited proteases that control the half-life of MAPK phosphatases (Leiva-Salcedo et al., 2002). Thus, senescence may be viewed as a potential safeguard against neoplasia in aging individuals.

As apparent so far, generation of intracellular signals often occurs at or near the plasma membrane. Multi-protein complex formation in receptor proximity represents an initial crucial step that is reasonably well understood for many signaling pathways. A big part of the challenge ahead resides in understanding how these signals traverse the cytosol and ultimately lead to alterations in gene expression. Nuclear receptors are a family of transcription factors that essentially bypass this sequence by directly regulating transcription in a ligand-dependent manner. Despite the apparent differences, activation or repression of gene expression by such receptors is intimately linked to the formation of complexes with molecules that serve as co-activators or co-repressors by modifying local DNA structure. Alterations in signaling events linked to this receptor family are associated with various human cancers (Hart, 2002).

Long-term cellular responses involve not only changes in gene transcription but also protein synthesis. What remains largely unrecognized is that most of what is generated by the translational machinery is more of precisely the same components. For instance, in an exponentially growing carcinoma cell 7500 ribosomes or 600000 ribosomal proteins are synthesized per minute. Thus, synthesis of ribosomal components is easily the most energy-consuming anabolic process in a growing cell and precise control is mandatory to avoid unnecessary depletion of energy reserves. S6Kinase (S6K) phosphorylates the 40S ribosomal protein S6 and thereby increases translation of mRNAs coding for components of the protein synthesis machinery. Here, G. Thomas describes how lack of S6K in flies and mice leads to the generation of smaller individuals. This probably is due to decreased protein synthesis that results in smaller cells. Surprisingly, in the mouse, such S6K deficiency led to glucose intolerance associated with hypoinsulinemia that could be accounted for by a reduction in size of the b cells in Langerhans islets (Thomas, 2002).

In summary, formation of multi-protein complexes is an essential if not defining feature of eukaryotic signaling. Clearly, we now appreciate this fact and are beginning to grasp some of the underlying guiding principles. However, much remains yet to be accomplished. The hope is that insights to these mechanisms will not only further our understanding of fundamental processes in biology like signal transduction, but will also help in the development of new strategies to treat human disease.

ACKNOWLEDGEMENTS

The following organizations and enterprises are gratefully acknowledged for sponsoring the Intenational Symposium "Supramolecular complex formation in signaling and disease" : International Centre for Genetic Engineering and Biotechnology (ICGEB); International Cell Research Organization (ICRO)-European Molecular Biology Organization (EMBO)- The United Nations Educational, Scientific and Cultural Organization (UNESCO); Centro FONDAP de Estudios Moleculares de la Célula (CEMC, ICBM, Facultad de Medicina, Universidad de Chile); International Union for Biochemistry and Molecular Biology (IUBMB); Latin American Network of Biological Sciences (RELAB); Proyecto Mecesup ICBM, Facultad de Medicina, Universidad de Chile; Fundación para Estudios Biomédicos Avanzados (FEBA); Ursula Biggemann & Co. Ltda., Analítica Weisser S.A. and Arquimed. In addition we wish to acknowledge the support we received for the International Training Course entitled "Supramolecular complex formation in cellular signaling" from the same sources and the following additional enterprises: MERCK, Equilab, Gene X-Press (HyClone), Biosonda (Pierce), Andes Import and Fermelo.

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Received: July 10, 2002. In revised form: July 30, 2002. Accepted: August 07, 2002