Compartmentalized IgE Receptor–mediated Signal Transduction in Living Cells

Several receptor-mediated signal transduction pathways, including EGF and IgE receptor pathways, have been proposed to be spatially restricted to plasma membrane microdomains. However, the experimental evidence for signaling events in these microdomains is largely based on biochemical fractionation and immunocytochemical studies and only little is known about their spatial dynamics in living cells. Here we constructed green fluorescent protein–tagged SH2 domains to investigate where and when IgE receptor (FcεRI)–mediated tyrosine phosphorylation occurs in living tumor mast cells. Strikingly, within minutes after antigen addition, tandem SH2 domains from Syk or PLC-γ1 translocated from a uniform cytosolic distribution to punctuate plasma membrane microdomains. Colocalization experiments showed that the microdomains where tyrosine phosphorylation occurred were indistinguishable from those stained by cholera toxin B, a marker for glycosphingolipids. Competitive binding studies with coelectroporated unlabeled Syk, PLC-γ1, and other SH2 domains selectively suppressed the induction of IgE receptor–mediated calcium signals as well as the binding of the fluorescent SH2 domains. This supports the hypothesis that PLC-γ1 and Syk SH2 domains selectively bind to Syk and IgE receptors, respectively. Unlike the predicted prelocalization of EGF receptors to caveolae microdomains, fluorescently labeled IgE receptors were found to be uniformly distributed in the plasma membrane of unstimulated cells and only transiently translocated to glycosphingolipid rich microdomains after antigen addition. Thus, these in vivo studies support a plasma membrane signaling mechanism by which IgE receptors transiently associate with microdomains and induce the spatially restricted activation of Syk and PLC-γ1.     

Membrane order and molecular dynamics associated with IgE receptor cross-linking in mast cells

Cholesterol-rich microdomains (or "lipid rafts") within the plasma membrane have been hypothesized to exist in a liquid-ordered phase and play functionally important roles in cell signaling; however, these microdomains defy detection using conventional imaging. To visualize domains and relate their nanostructure and dynamics to mast cell signaling, we use two-photon (760 nm and 960 nm) fluorescence lifetime imaging microscopy and fluorescence polarization anisotropy imaging, with comparative one-photon anisotropy imaging and single-point lifetime and anisotropy decay measurements. The inherent sensitivity of ultrafast excited-state dynamics and rotational diffusion to the immediate surroundings of a fluorophore allows for real-time monitoring of membrane structure and organization. When the high affinity receptor for IgE (FcepsilonRI) is extensively cross-linked with anti-IgE, molecules associated with cholesterol-rich microdomains (e.g., saturated lipids (the lipid analog diI-C(18) or glycosphingolipids)) and lipid-anchored proteins coredistribute with cross-linked IgE-FcepsilonRI. We find an enhancement in fluorescence lifetime and anisotropy of diI-C(18) and Alexa 488-labeled IgE-FcepsilonRI in the domains where these molecules colocalize. Our results suggest that fluorescence lifetime and, particularly, anisotropy permit us to correlate the recruitment of lipid molecules into more ordered domains that serve as platforms for IgE-mediated signaling.     

Engagement of Gab1 and Gab2 in erythropoietin signaling.

Several signaling cascades are activated during engagement of the erythropoietin receptor to mediate the biological effects of erythropoietin. The members of the insulin receptor substrate (IRS) family of proteins play a central role in signaling for various growth factor receptors and cytokines by acting as docking proteins for the SH2 domains of signaling elements, linking cytokine receptors to diverse downstream pathways. In the present study we provide evidence that the recently cloned IRS-related proteins, Gab1 and Gab2, of the Gab family of proteins, are rapidly phosphorylated on tyrosine during erythropoietin treatment of erythropoietin-responsive cells and provide docking sites for the engagement of the SHP2 phosphatase and the p85 subunit of the phosphatidylinositol 3'-kinase. Furthermore, our data show that Gab1 is the primary IRS-related protein activated by erythropoietin in primary erythroid progenitor cells. In studies to identify the erythropoietin receptor domains required for activation of Gab proteins, we found that tyrosines 425 and 367 in the cytoplasmic domain of the erythropoietin receptor are required for the phosphorylation of Gab2. Taken together, our data demonstrate that Gab proteins are engaged in erythropoietin signaling to mediate downstream activation of the SHP2 and phosphatidylinositol 3'-kinase pathways and possibly participate in the generation of the erythropoietin-induced mitogenic responses.     

The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways.

Bone morphogenetic proteins (BMPs) are multifunctional proteins regulating cell growth, differentiation, and apoptosis. BMP-2 signals via two types of receptors (BRI and BRII) that are expressed at the cell surface as homomeric as well as heteromeric complexes. Prior to ligand binding, a low but measurable level of BMP-receptors is found in preformed hetero-oligomeric complexes. The major fraction of the receptors is recruited into hetero-oligomeric complexes only after ligand addition. For this, BMP-2 binds first to the high affinity receptor BRI and then recruits BRII into the signaling complex. However, ligand binding to the preformed complex composed of BRII and BRI is still required for signaling, suggesting that it may mediate activating conformational changes. Using several approaches we have addressed the following questions: (i) Are preformed complexes incompetent of signaling in the absence of BMP-2? (ii) Which domains of the BRII receptors are essential for this complex formation? (iii) Are there differences in signals sent from BMP-induced versus preformed receptor complexes? By measuring the activation of Smads, of p38 MAPK and of alkaline phosphatase, we show that the ability of kinase-deficient BRII receptor mutants to inhibit BMP signaling depends on their ability to form heteromeric complexes with BRI. Importantly, a BRII mutant that is incapable in forming preassembled receptor complexes but recruits into a BMP-induced receptor complex does not interfere with the Smad pathway but does inhibit the induction of alkaline phosphatase as well as p38 phosphorylation. These results indicate that signals induced by binding of BMP-2 to preformed receptor complexes activate the Smad pathway, whereas BMP-2-induced recruitment of receptors activates a different, Smad-independent pathway resulting in the induction of alkaline phosphatase activity via p38 MAPK.     

Microdomain-dependent regulation of Lck and Fyn protein-tyrosine kinases in T lymphocyte plasma membranes

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.     

Insulin signaling in retinal neurons is regulated within cholesterol-enriched membrane microdomains

Neuronal cell death is an early pathological feature of diabetic retinopathy. We showed previously that insulin receptor signaling is diminished in retinas of animal models of diabetes and that downstream Akt signaling is involved in insulin-mediated retinal neuronal survival. Therefore, further understanding of the mechanisms by which retinal insulin receptor signaling is regulated could have therapeutic implications for neuronal cell death in diabetes. Here, we investigate the role of cholesterol-enriched membrane microdomains to regulate PKC-mediated inhibition of Akt-dependent insulin signaling in R28 retinal neurons. We demonstrate that PKC activation with either a phorbol ester or exogenous application of diacylglycerides impairs insulin-induced Akt activation, whereas PKC inhibition augments insulin-induced Akt activation. To investigate the mechanism by which PKC impairs insulin-stimulated Akt activity, we assessed various upstream mediators of Akt signaling. PKC activation did not alter the tyrosine phosphorylation of the insulin receptor or IRS-2. Additionally, PKC activation did not impair phosphatidylinositol 3-kinase activity, phosphoinositide-dependent kinase phosphorylation, lipid phosphatase (PTEN), or protein phosphatase 2A activities. Thus, we next investigated a biophysical mechanism by which insulin signaling could be disrupted and found that disruption of lipid microdomains via cholesterol depletion blocks insulin-induced Akt activation and reduces insulin receptor tyrosine phosphorylation. We also demonstrated that insulin localizes phosphorylated Akt to lipid microdomains and that PMA reduces phosphorylated Akt. In addition, PMA localizes and recruits PKC isotypes to these cholesterol-enriched microdomains. Taken together, these results demonstrate that both insulin-stimulated Akt signaling and PKC-induced inhibition of Akt signaling depend on cholesterol-enriched membrane microdomains, thus suggesting a putative biophysical mechanism underlying insulin resistance in diabetic retinopathy.     

Signaling through sphingolipid microdomains of the plasma membrane: The concept of signaling platform

Transmembrane signaling requires modular interactions between signaling proteins, phosphorylation or dephosphorylation of the interacting protein partners [1] and temporary elaboration of supramolecular structures [2], to convey the molecular information from the cell surface to the nucleus. Such signaling complexes at the plasma membrane are instrumental in translating the extracellular cues into intracellular signals for gene activation. In the most straightforward case, ligand binding promotes homodimerization of the transmembrane receptor which facilitates modular interactions between the receptor's cytoplasmic domains and intracellular signaling and adaptor proteins [3]. For example, most growth factor receptors contain a cytoplasmic protein tyrosine kinase (PTK) domain and ligand-mediated receptor dimerization leads to cross phosphorylation of tyrosines in the receptor's cytoplasmic domains, an event that initiates the signaling cascade [4]. In other signaling pathways where the receptors have no intrinsic kinase activity, intracellular non-receptor PTKs (i.e. Src family PTKs, JAKs) are recruited to the cytoplasmic domain of the engaged receptor. Execution of these initial phosphorylations and their translation into efficient cellular stimulation requires concomitant activation of diverse signaling pathways. Availability of stable, preassembled matrices at the plasma membrane would facilitate scaffolding of a large array of receptors, coreceptors, tyrosine kinases and other signaling and adapter proteins, as it is the case in signaling via the T cell antigen receptor [5]. The concept of the signaling platform [6] has gained usage to characterize the membrane structure where many different membrane-bound components need to be assembled in a coordinated manner to carry out signaling.The structural basis of the signaling platform lies in preferential assembly of certain classes of lipids into distinct physical and functional compartments within the plasma membrane [7,8]. These membrane microdomains or rafts (Figure 1) serve as privileged sites where receptors and proximal signaling molecules optimally interact [9]. In this review, we shall discuss first how signaling platforms are assembled and how receptors and their signaling machinery could be functionally linked in such structures. The second part of our review will deal with selected examples of raft-based signaling pathways in T lymphocytes and NK cells to illustrate the ways in which rafts may facilitate signaling.     

Signaling through sphingolipid microdomains of the plasma membrane: the concept of signaling platform

Transmembrane signaling requires modular interactions between signaling proteins, phosphorylation or dephosphorylation of the interacting protein partners and temporary elaboration of supramolecular structures, to convey the molecular information from the cell surface to the nucleus. Such signaling complexes at the plasma membrane are instrumental in translating the extracellular cues into intracellular signals for gene activation. In the most straightforward case, ligand binding promotes homodimerization of the transmembrane receptor which facilitates modular interactions between the receptor's cytoplasmic domains and intracellular signaling and adaptor proteins. For example, most growth factor receptors contain a cytoplasmic protein tyrosine kinase (PTK) domain and ligand-mediated receptor dimerization leads to cross phosphorylation of tyrosines in the receptor's cytoplasmic domains, an event that initiates the signaling cascade. In other signaling pathways where the receptors have no intrinsic kinase activity, intracellular nonreceptor PTKs (i.e. Src family PTKs, JAKs) are recruited to the cytoplasmic domain of the engaged receptor. Execution of these initial phosphorylations and their translation into efficient cellular stimulation requires concomitant activation of diverse signaling pathways. Availability of stable, preassembled matrices at the plasma membrane would facilitate scaffolding of a large array of receptors, coreceptors, tyrosine kinases and other signaling and adapter proteins, as it is the case in signaling via the T cell antigen receptor. The concept of the signaling platform has gained usage to characterize the membrane structure where many different membrane-bound components need to be assembled in a coordinated manner to carry out signaling. The structural basis of the signaling platform lies in preferential assembly of certain classes of lipids into distinct physical and functional compartments within the plasma membrane. These membrane microdomains or rafts (Figure 1) serve as privileged sites where receptors and proximal signaling molecules optimally interact. In this review, we shall discuss first how signaling platforms are assembled and how receptors and their signaling machinery could be functionally linked in such structures. The second part of our review will deal with selected examples of raft-based signaling pathways in T lymphocytes and NK cells to illustrate the ways in which rafts may facilitate signaling.     

On the cross-regulation of protein tyrosine phosphatases and receptor tyrosine kinases in intracellular signaling

Intracellular signaling proteins are very often regulated by site-specific phosphorylation. For example, growth factor receptors in eukaryotic cells contain intrinsic tyrosine kinase activity and use inter- and intra-molecular interactions to recruit and orient potential protein substrates for phosphorylation. Equally important in determining the magnitude and kinetics of such a response is protein dephosphorylation, catalysed by phosphatase enzymes. A growing body of evidence indicates that certain protein tyrosine phosphatases (PTPs), like tyrosine kinases, are affected by intermolecular interactions that alter the specific activity or localization of their catalytic domains. Using a detailed kinetic modeling framework, we theoretically explore the regulation of PTPs through their association with receptor tyrosine kinases, as noted for the Src homology 2-domain-containing PTPs, SHP-1 and -2. Receptor-PTP binding, in turn, is expected to influence the phosphorylation pattern of those receptors and proteins they associate with, and we show how PTPs might serve to co- or counter-regulate parallel pathways in a signaling network.     

Cytoplasmic cAMP concentrations in intact cardiac myocytes

In cardiac myocytes there is evidence that activation of some receptors can regulate protein kinase A (PKA)-dependent responses by stimulating cAMP production that is limited to discrete intracellular domains. We previously developed a computational model of compartmentalized cAMP signaling to investigate the feasibility of this idea. The model was able to reproduce experimental results demonstrating that both beta(1)-adrenergic and M(2) muscarinic receptor-mediated cAMP changes occur in microdomains associated with PKA signaling. However, the model also suggested that the cAMP concentration throughout most of the cell could be significantly higher than that found in PKA-signaling domains. In the present study we tested this counterintuitive hypothesis using a freely diffusible fluorescence resonance energy transfer-based biosensor constructed from the type 2 exchange protein activated by cAMP (Epac2-camps). It was determined that in adult ventricular myocytes the basal cAMP concentration detected by the probe is approximately 1.2 muM, which is high enough to maximally activate PKA. Furthermore, the probe detected responses produced by both beta(1) and M(2) receptor activation. Modeling suggests that responses detected by Epac2-camps mainly reflect what is happening in a bulk cytosolic compartment with little contribution from microdomains where PKA signaling occurs. These results support the conclusion that even though beta(1) and M(2) receptor activation can produce global changes in cAMP, compartmentation plays an important role by maintaining microdomains where cAMP levels are significantly below that found throughout most of the cell. This allows receptor stimulation to regulate cAMP activity over concentration ranges appropriate for modulating both higher (e.g., PKA) and lower affinity (e.g., Epac) effectors.     

Functional role of glycosphingolipids and gangliosides in control of cell adhesion, motility, and growth, through glycosynaptic microdomains

At cell surface microdomains, glycosyl epitopes, carried either by glycosphingolipids, N- or O-linked oligosaccharides, are recognized by carbohydrate-binding proteins or complementary carbohydrates. In both cases, the carbohydrate epitopes may be clustered with specific signal transducers, tetraspanins, adhesion receptors or growth factor receptors. Through this framework, carbohydrates can mediate cell signaling leading to changes in cellular phenotype. Microdomains involved in carbohydrate-dependent cell adhesion inducing cell activation, motility, and growth are termed "glycosynapse". In this review a historical synopsis of glycosphingolipids-enriched microdomains study leading to the concept of glycosynapse is presented. Examples of glycosynapse as signaling unit controlling the tumor cell phenotype are discussed in three contexts: (i) Cell-to-cell adhesion mediated by glycosphingolipids-to-glycosphingolipids interaction between interfacing glycosynaptic domains, through head-to-head (trans) carbohydrate-to-carbohydrate interaction. (ii) Functional role of GM3 complexed with tetraspanin CD9, and interaction of such complex with integrins, or with fibroblast growth factor receptor, to control tumor cell phenotype and its reversion to normal cell phenotype. (iii) Inhibition of integrin-dependent Met kinase activity by GM2/tetraspanin CD82 complex in glycosynaptic microdomain. Data present here suggest that the organizational status of glycosynapse strongly affects cellular phenotype influencing tumor cell malignancy.     

Human oxytocin receptors in cholesterol-rich vs. cholesterol-poor microdomains of the plasma membrane.

We analyzed the properties of a G protein-coupled receptor localized in cholesterol-poor vs. cholesterol-rich microdomains of the plasma membrane. For this purpose, the human oxytocin receptor, which is very sensitive against alterations of the membrane cholesterol level, was stably expressed in HEK293 cells. To calculate the total number of receptors independent of ligand binding studies, the oxytocin receptor was tagged with an enhanced green fluorescent protein (EGFP) which did not change the functional properties of the receptor. Only 1% of the oxytocin receptors were present in cholesterol-rich detergent-insoluble domains. In contrast, employing a detergent-free fractionation scheme that preserves the functional activity of the receptor, we detected 10-15% of the receptors in cholesterol-rich low-density membranes and therein the high-affinity state receptors were twofold enriched. In cholesterol-poor vs. cholesterol-rich domains, high-affinity oxytocin receptors behaved similar with respect to their agonist binding kinetics and GTP sensitivity. However, high-affinity oxytocin receptors localized in cholesterol-rich low-density membranes showed a markedly enhanced (t (1/2) approximately threefold) stability at 37 degrees C as compared with the oxytocin receptors localized in the cholesterol-poor high-density membranes. Addition of cholesterol to the high-density membranes fully protected the oxytocin receptors against loss of function. The importance of cholesterol to stabilize the oxytocin receptor was supported in experiments with solubilized receptors. Cholesterol markedly delayed the inactivation of oxytocin receptors solubilized with Chapso. In conclusion, the data of this report suggest that functional properties of heptahelical receptor proteins could differ in dependence of their localization in different membrane microdomains.     

Selective inhibition of T cell activation via CD147 through novel modulation of lipid rafts

The plasma membrane is compartmentalized into microdomains and the association/dissociation of receptors and signaling molecules with/from these membrane domains is a major principle for regulation of signal transduction. By following the reorganization of microdomains on living cells and performing biochemical studies, we show that Ab targeting of the T cell activation-associated Ag CD147 prevents TCR stimulation-dependent reorganization and clustering of microdomains. Triggering CD147 induces a displacement of the GPI-anchored coreceptors CD48 and CD59 from microdomains in human T lymphocytes. This perturbation of microdomains is accompanied by a selective inhibition of TCR-mediated T cell proliferation. The CD147-inhibited cells secret normal levels of IL-2 but acquire reduced amounts of the IL-2 receptor alpha-chain CD25. These results indicate that negative regulating signals can modulate microdomains and suggest a general mechanism for inhibition of receptor signaling.     

Real-time cross-correlation image analysis of early events in IgE receptor signaling

Signaling in mast cells and basophils is mediated through IgE and its high affinity cell surface receptor, FcepsilonRI. Crosslinking of the receptors by a cognate multivalent antigen leads to degranulation and release of mediators of the allergic immune response. Using multicolor fluorescence confocal microscopy, we probed the spatio-temporal dynamics of early events in the IgE receptor signal cascade. We monitored the recruitment of GFP-/CFP-labeled signaling proteins by acquiring sequential images with time resolution of 3 s during stimulation of RBL-2H3 mast cells with multivalent antigen. A fluorescent tag on the antigen allowed us to visualize the plasma membrane localization of crosslinked receptors, and fluorescent cholera toxin B served as a plasma membrane marker. We developed an automated image analysis scheme to quantify the recruitment of fluorescent intracellular proteins to the plasma membrane and to assess the time-dependent colocalization of these and other membrane-associated proteins with crosslinked receptors as measured by cross-correlation between the plasma membrane distributions of the two fluorophores. This automated method permits analysis of thousands of individual images from multiple experiments for each cross-correlation pair. We systematically applied this analysis to characterize stimulated interactions of IgE receptors with several signaling proteins, including the tyrosine kinases Lyn and Syk, and the adaptor protein LAT. Notably, for Syk-CFP we observed a rapid stimulated translocation to the plasma membrane but very little colocalization with aggregated receptors. Our results demonstrate the utility of this simple, automated method to monitor protein interactions quantitatively during cell signaling.     

Sequence-dependent sorting of recycling proteins by actin-stabilized endosomal microdomains

The functional consequences of signaling receptor endocytosis are determined by the endosomal sorting of receptors between degradation and recycling pathways. How receptors recycle efficiently, in a sequence-dependent manner that is distinct from bulk membrane recycling, is not known. Here, in live cells, we visualize the sorting of a prototypical sequence-dependent recycling receptor, the beta-2 adrenergic receptor, from bulk recycling proteins and the degrading delta-opioid receptor. Our results reveal a remarkable diversity in recycling routes at the level of individual endosomes, and indicate that sequence-dependent recycling is an active process mediated by distinct endosomal subdomains distinct from those mediating bulk recycling. We identify a specialized subset of tubular microdomains on endosomes, stabilized by a highly localized but dynamic actin machinery, that mediate this sorting, and provide evidence that these actin-stabilized domains provide the physical basis for a two-step kinetic and affinity-based model for protein sorting into the sequence-dependent recycling pathway.     

Floating the raft hypothesis for immune receptors: access to rafts controls receptor signaling and trafficking

The B cell antigen receptor (BCR) is a member of an important family of multichain immune recognition receptors, which are complexes composed of ligand-binding domains associated with signal-transduction complexes. The signaling components of these receptors have no inherent kinase activity but become tyrosine phosphorylated in their cytoplasmic domains by Src-family kinases upon oligomerization, thus initiating signaling cascades. The BCR is unique in this family in that, in addition to its signaling function, it also serves to deliver antigen to intracellular compartments where the antigen is processed and presented bound to major histocompatibility complex (MHC) class II molecules. Recent evidence indicates that both the signaling and antigen-trafficking functions of the BCR are regulated by cholesterol- and sphingolipid-rich plasma membrane microdomains termed rafts. Indeed, upon oligomerization, the BCR translocates into rafts that concentrate the Src-family kinase Lyn and is subsequently internalized directly from the rafts. Thus, translocation into rafts allows the association of the oligomerized BCR with Lyn and the initiation of both signaling and trafficking. Significantly, the access of the BCR to rafts appears to be controlled by a variety of B lymphocyte co-receptors, as well as factors including the developmental state of the B cell and viral infection. Thus, the translocation of the immune receptors into signaling-competent microdomains may represent a novel mechanism to initiate and regulate immune-cell activation.     

Src Regulates Sequence‐Dependent Beta‐2 Adrenergic Receptor Recycling via Cortactin Phosphorylation

The recycling of internalized signaling receptors, which has direct functional consequences, is subject to multiple sequence and biochemical requirements. Why signaling receptors recycle via a specialized pathway, unlike many other proteins that recycle by bulk, is a fundamental unanswered question. Here, we show that these specialized pathways allow selective control of signaling receptor recycling by heterologous signaling. Using assays to visualize receptor recycling in living cells, we show that the recycling of the beta‐2 adrenergic receptor (B2AR), a prototypic signaling receptor, is regulated by Src family kinases. The target of Src is cortactin, an essential factor for B2AR sorting into specialized recycling microdomains on the endosome. Phosphorylation of a single cortactin residue, Y466, regulates the rate of fission of B2AR recycling vesicles from these microdomains and, therefore, the rate of delivery of B2AR to the cell surface. Together, our results indicate that actin‐stabilized microdomains that mediate signaling receptor recycling can serve as a functional point of convergence for crosstalk between signaling pathways.      

Fc Rgamma -independent signaling by the platelet collagen receptor glycoprotein VI

The platelet collagen receptor glycoprotein VI (GPVI) is structurally homologous to multisubunit immune receptors and signals through the immune receptor adaptor Fc Rgamma. Multisubunit receptors are composed of specialized subunits thought to be dedicated exclusively to ligand binding or signal transduction. However, recent studies of the intracellular region of GPVI, a ligand-binding subunit, have suggested the existence of protein-protein interactions that could regulate receptor signaling. In the present study we have investigated the signaling role of the GPVI intracellular domain by stably expressing GPVI mutants in RBL-2H3 cells, a model system that accurately reproduces the GPVI signaling events observed in platelets. Studies of mutant GPVI receptor protein-protein interaction and calcium signaling reveal the existence of discrete domains within the receptor's intracellular tail that mediate interaction with Fc Rgamma, calmodulin, and Src family tyrosine kinases. These receptor interactions are modular and mediated by non-overlapping regions of the receptor transmembrane and intracellular domains. GPVI signaling requires all three of these domains as receptor mutants able to couple to only two interacting proteins exhibited severe signaling defects despite normal surface expression. Our results demonstrate that the ligand-binding subunit of the GPVI-Fc Rgamma receptor participates directly in receptor signaling by interacting with downstream signaling molecules other than Fc Rgamma through an adaptor-like mechanism.     

G protein-dependent Ca2+ signaling complexes in polarized cells

Polarized cells signal in a polarized manner. This is exemplified in the patterns of [Ca2+]i waves and [Ca2+]i oscillations evoked by stimulation of G protein-coupled receptors in these cells. Organization of Ca(2+)-signaling complexes in cellular microdomains, with the aid of scaffolding proteins, is likely to have a major role in shaping G protein-coupled [Ca2+]i signal pathways. In epithelial cells, these domains coincide with sites of [Ca2+]i-wave initiation and local [Ca2+]i oscillations. Cellular microdomains enriched with Ca(2+)-signaling proteins have been found in several cell types. Microdomains organize communication between Ca(2+)-signaling proteins in the plasma membrane and internal Ca2+ stores in the endoplasmic reticulum through the interaction between the IP3 receptors in the endoplasmic reticulum and Ca(2+)-influx channels in the plasma membrane. Ca2+ signaling appears to be controlled within the receptor complex by the regulators of G protein-signaling (RGS) proteins. Three domains in RGS4 and related RGS proteins contribute important regulatory features. The RGS domain accelerates GTP hydrolysis on the G alpha subunit to uncouple receptor stimulation from IP3 production; the C-terminus may mediate interaction with accessory proteins in the complex; and the N-terminus acts in a receptor-selective manner to confer regulatory specificity. Hence, RGS proteins have both catalytic and scaffolding function in Ca2+ signaling. Organization of Ca(2+)-signaling proteins into complexes within microdomains is likely to play a prominent role in the localized control of [Ca2+]i and in [Ca2+]i oscillations.     

Membrane microdomain may be a platform for immune signaling

Arabidopsis RPS2 is a typical disease resistance (R) protein with nucleotide-binding leucine-rich repeats (NB-LRR). Previously, we reported that RPS2 is physically associated with some Arabidopsis hypersensitive induced reaction (AtHIR) proteins, which are enriched in membrane microdomains. Biochemical and genetic analyses suggested that members of the AtHIR gene family have a function in RPS2-mediated immune signaling. Here, we provide evidence that the pattern recognition receptor (PRR) FLS2 is also physically associated with AtHIR2 in a N. benthamiana transient expression system. We thus speculate that PM microdomains provide a platform for both types of immune receptors, R proteins and PRRs, and that the activation of the receptors is facilitated by AtHIR proteins.