PDZ7 of glutamate receptor interacting protein binds to its target via a novel hydrophobic surface area

Glutamate receptor interacting protein 1 (GRIP1) is a scaffold protein composed of seven PDZ (Postsynaptic synaptic density-95/Discs large/Zona occludens-1) domains. The protein plays important roles in the synaptic targeting of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The interaction between GRIP1 PDZ7 and a Ras guanine nucleotide exchange factor, GRASP-1, regulates synaptic distribution of AMPA receptors. Here, we describe the three-dimensional structure of GRIP1 PDZ7 determined by NMR spectroscopy. GRIP1 PDZ7 contains a closed carboxyl group-binding pocket and a narrow alphaB/betaB-groove that is not likely to bind to classical PDZ ligands. Unexpectedly, GRIP1 PDZ7 contains a large solvent-exposed hydrophobic surface at a site distinct from the conventional ligand-binding alphaB/betaB-groove. NMR titration experiments show that GRIP1 PDZ7 binds to GRASP-1 via this hydrophobic surface. Our data uncover a novel PDZ domain-mediated protein interaction mode that may be responsible for multimerization of other PDZ domain-containing scaffold proteins.     

Ligand--protein interactions in the glutamate receptor

Fourier transform infrared spectroscopy was used to investigate ligand-protein interactions in the ligand-binding domain of the GluR4 glutamate receptor subunit. Glutamate binding induces more extensive secondary structural changes in the ligand-binding domain than does kainate binding. Glutamate also alters the hydrogen bonding strength of the single free cysteine side chain in the domain, while kainate does not. On the other hand, the interaction of a binding site arginine residue with kainate appears to be stronger than that with glutamate. These results identify chemical and structural differences that may explain the different functional characteristics of the two agonists acting on ionotropic glutamate receptors. In doing so, they complement and extend recent crystallographic structures of the ligand-binding domain.     

Cell-cell interactions in regulating lung function

Tight junction barrier formation and gap junctional communication are two functions directly attributable to cell-cell contact sites. Epithelial and endothelial tight junctions are critical elements of the permeability barrier required to maintain discrete compartments in the lung. On the other hand, gap junctions enable a tissue to act as a cohesive unit by permitting metabolic coupling and enabling the direct transmission of small cytosolic signaling molecules from one cell to another. These components do not act in isolation since other junctional elements, such as adherens junctions, help regulate barrier function and gap junctional communication. Some fundamental elements related to regulation of pulmonary barrier function and gap junctional communication were presented in a Featured Topic session at the 2004 Experimental Biology Conference in Washington, DC, and are reviewed in this summary.     

Evolution of glutamate interactions during binding to a glutamate receptor

Glutamate receptors are the predominant mediators of excitatory synaptic signals in the central nervous system and are important in learning and memory as well as in diverse neuropathologies including epilepsy and ischemia. Their primary function is to receive the chemical signal glutamate (1), which binds to an extracellular domain in the receptor, and convert it into an electrical signal through the formation of cation-permeable transmembrane channels. Recently described end-state apo and ligated structures of the ligand-binding domain of a rat glutamate receptor provide a first view of specific molecular interactions between the ligand and the receptor that are central to the allosteric regulation of function in this protein. Yet there is little information on the mechanism and the structures of intermediates (if any) formed during the ligand-binding process. Here we have used time-resolved vibrational spectroscopy to show that the process involves a sequence of interleaved ligand and protein changes that starts with the docking of glutamate at the alpha-carboxylate moiety and ends with the establishment of the interactions between the gamma-carboxylate of glutamate and the protein.     

Synaptic PDZ domain-mediated protein interactions are disrupted by inhalational anesthetics

Anesthetics exert multiple effects on the central nervous system through altering synaptic transmission, but the mechanisms for this process are poorly understood. PDZ domain-mediated protein interactions play a central role in organizing signaling complexes around synaptic receptors for efficient signal transduction. We report here that clinically relevant concentrations of inhalational anesthetics dose-dependently and specifically inhibit the PDZ domain-mediated protein interaction between PSD-95 or PSD-93 and the N-methyl-d-aspartate receptor or neuronal nitric-oxide synthase. These inhibitory effects are immediate, potent, and reversible and occur at a hydrophobic peptide-binding groove on the surface of the second PDZ domain of PSD-95 in a manner relevant to anesthetic action. These findings reveal the PDZ domain as a new molecular target for inhalational anesthetics.     

Cell-cell interactions in regulating osteogenesis and osteoblast function

Endochondral bone formation requires an elaborate interplay among autocrine, paracrine, and endocrine signals, positional cues, and cell-cell contacts to mediate the complex three-dimensional architecture and function of the skeleton. Embryonic bone development occurs by migration, aggregation, and condensation of immature mesenchymal progenitor cells to form the cartilaginous anlage. Upon vascular invasion, the cartilaginous scaffold is colonized and subsequently mineralized by osteoblasts. Likewise, bone remodeling in the adult skeleton is a dynamic process that requires coordinated cellular activities among osteoblasts, osteocytes, and osteoclasts to maintain bone homeostasis. This review examines the role of cell-cell interactions mediated by adherens junctions formed by cadherins and communicative gap junctions formed by connexins in regulating bone development and osteogenic function.     

The PDZ proteins PICK1, GRIP, and syntenin bind multiple glutamate receptor subtypes. Analysis of PDZ binding motifs

Using sequence homology searches, yeast two-hybrid assays and glutathione S-transferase (GST)-pull-down approaches we have identified a series of glutamate receptor subunits that interact differentially with the PDZ proteins GRIP, PICK1, and syntenin. GST-pull-down experiments identified more interactions than detected by yeast two-hybrid assays. We report several receptor-protein interactions, strong ones include: (i) GRIP and syntenin with mGluR7a, mGluR4a, and mGluR6; (ii) PICK1 and GRIP with mGluR3; and (iii) syntenin with all forms of GluR1-4 and mGluR7b. We further characterized the novel mGluR7a-GRIP interaction found both in yeast two-hybrid and GST-pull-down assays and observed that mGluR7a localization overlapped with GRIP with in hippocampal neurons. The wide range of targets for PICK1, GRIP, and syntenin suggests they may represent a molecular mechanism that can concentrate and/or regulate a number of different receptors at a common site on a synapse. These data also suggest that the structural determinants involved in PDZ interactions are more complex than originally envisaged.     

GABAB receptor association with the PDZ scaffold Mupp1 alters receptor stability and function

gamma-Aminobutyric acid, type B (GABA(B)) receptors are heterodimeric G protein-coupled receptors that mediate slow inhibitory synaptic transmission in the central nervous system. To identify novel interacting partners that might regulate GABA(B) receptor (GABA(B)R) functionality, we screened the GABA(B)R2 carboxyl terminus against a recently created proteomic array of 96 distinct PDZ (PSD-95/Dlg/ZO-1 homology) domains. The screen identified three specific PDZ domains that exhibit interactions with GABA(B)R2: Mupp1 PDZ13, PAPIN PDZ1, and Erbin PDZ. Biochemical analysis confirmed that full-length Mupp1 and PAPIN interact with GABA(B)R2 in cells. Disruption of the GABA(B)R2 interaction with PDZ scaffolds by a point mutation to the carboxyl terminus of the receptor dramatically decreased receptor stability and attenuated the duration of GABA(B) receptor signaling. The effects of mutating the GABA(B)R2 carboxyl terminus on receptor stability and signaling were mimicked by small interference RNA knockdown of endogenous Mupp1. These findings reveal that GABA(B) receptor stability and signaling can be modulated via GABA(B)R2 interactions with the PDZ scaffold protein Mupp1, which may contribute to cell-specific regulation of GABA(B) receptors in the central nervous system.     

Proteomic analysis of beta1-adrenergic receptor interactions with PDZ scaffold proteins

Many G protein-coupled receptors possess carboxyl-terminal motifs ideal for interaction with PDZ scaffold proteins, which can control receptor trafficking and signaling in a cell-specific manner. To gain a panoramic view of beta1-adrenergic receptor (beta AR) interactions with PDZ scaffolds, the beta1AR carboxyl terminus was screened against a newly developed proteomic array of PDZ domains. These screens confirmed beta1AR associations with several previously identified PDZ partners, such as PSD-95, MAGI-2, GIPC, and CAL. Moreover, two novel beta1AR-interacting proteins, SAP97 and MAGI-3, were also identified. The beta1AR carboxyl terminus was found to bind specifically to the first PDZ domain of MAGI-3, with the last four amino acids (E-S-K-V) of beta1AR being the key determinants of the interaction. Full-length beta1AR robustly associated with full-length MAGI-3 in cells, and this association was abolished by mutation of the beta1AR terminal valine residue to alanine (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. MAGI-3 co-expression with beta1AR profoundly impaired beta1AR-mediated ERK1/2 activation but had no apparent effect on beta1AR-mediated cyclic AMP generation or agonist-promoted beta1AR internalization. These findings revealed that the interaction of MAGI-3 with beta1AR can selectively regulate specific aspects of receptor signaling. Moreover, the screens of the PDZ domain proteomic array provide a comprehensive view of beta1AR interactions with PDZ scaffolds, thereby shedding light on the molecular mechanisms by which beta1 AR signaling and trafficking can be regulated in a cell-specific manner.     

PDZ domains: the building blocks regulating tumorigenesis

Over 250 PDZ (PSD95/Dlg/ZO-1) domain-containing proteins have been described in the human proteome. As many of these possess multiple PDZ domains, the potential combinations of associations with proteins that possess PBMs (PDZ-binding motifs) are vast. However, PDZ domain recognition is a highly specific process, and much less promiscuous than originally thought. Furthermore, a large number of PDZ domain-containing proteins have been linked directly to the control of processes whose loss, or inappropriate activation, contribute to the development of human malignancies. These regulate processes as diverse as cytoskeletal organization, cell polarity, cell proliferation and many signal transduction pathways. In the present review, we discuss how PBM-PDZ recognition and imbalances therein can perturb cellular homoeostasis and ultimately contribute to malignant progression.     

The structure and function of glutamate receptor ion channels

As in the case of many ligand-gated ion channels, the biochemical and electrophysiological properties of the ionotropic glutamate receptors have been studied extensively. Nevertheless, we still do not understand the molecular mechanisms that harness the free energy of agonist binding, first to drive channel opening, and then to allow the channel to close (desensitize) even though agonist remains bound. Recent crystallographic analyses of the ligand-binding domains of these receptors have identified conformational changes associated with agonist binding, yielding a working hypothesis of channel function. This opens the way to determining how the domains and subunits are assembled into an oligomeric channel, how the domains are connected, how the channel is formed, and where it is located relative to the ligand-binding domains, all of which govern the processes of channel activation and desensitization.     

Osteoblastic glutamate receptor function regulates bone formation and resorption

Previous studies showed that a variety of bone cells express protein components necessary for neuronal-like glutamatergic signaling and implicated glutamate as having a role in mechanically induced bone remodeling. Initial functional studies concentrated on the role of glutamate signaling in bone resorption and provided compelling evidence to suggest that glutamate signaling through functional NMDA type ionotropic glutamate receptors (iGluRs) is a prerequisite for in vitro osteoclastogenesis. Originally, effects of iGluR antagonists seen in co-cultures were attributed to antagonists acting directly on osteoclast precursors. However, in the light of recent osteoblast studies it now seems likely that the observed effects on osteoclastogenesis are an indirect effect of modulating the function of pre-osteoblast present within these cultures. The presence of iGluRs in osteoblasts suggests a role for them in bone formation and this paper reviews and discusses the emerging data relating to the role of glutamate signaling in osteoblasts. A number of recently published studies have shown that osteoblasts not only express a wide number of 'pre-synaptic' glutamatergic proteins but also possess the ability to both regulate glutamate release and actively recycle extracellular glutamate. The functionality of osteoblastic 'post-synaptic' glutamatergic components has also been shown as both primary and clonal osteoblasts express electrophysiologically active iGluRs, metabotropic type glutamate receptors (mGluRs) along with a variety of glutamate receptor associated signaling proteins. There is, however, little published data regarding the actual role of glutamatergic signaling in osteoblastic bone formation. In vivo and in vitro studies performed provide evidence that glutamatergic signaling is a necessity for normal osteoblast function. In a number of different models of in vitro bone formation, the addition of non-competitive antagonists of iGluRs prevents the formation of mineralized bone, moreover antagonizing some sub-types of iGluR mediates the differentiation of pre-osteoblasts. iGluR antagonists modulate osteoblast function in a manner that correlates with the previously reported data regarding in vitro osteoclastogenesis. Interestingly iGluR mediated glutamate signaling appears to function differently in osteoblasts derived from flat and long bones. This implies the components of osteoblastic glutamatergic signaling may be adapted in vivo possibly to reflect the differential function of osteoblasts in those regions of the skeleton.     

Importance of Shank3 protein in regulating metabotropic glutamate receptor 5 (mGluR5) expression and signaling at synapses

Shank3/PROSAP2 gene mutations are associated with cognitive impairment ranging from mental retardation to autism. Shank3 is a large scaffold postsynaptic density protein implicated in dendritic spines and synapse formation; however, its specific functions have not been clearly demonstrated. We have used RNAi to knockdown Shank3 expression in neuronal cultures and showed that this treatment specifically reduced the synaptic expression of the metabotropic glutamate receptor 5 (mGluR5), but did not affect the expression of other major synaptic proteins. The functional consequence of Shank3 RNAi knockdown was impaired signaling via mGluR5, as shown by reduction in ERK1/2 and CREB phosphorylation induced by stimulation with (S)-3,5-dihydroxyphenylglycine (DHPG) as the agonist of mGluR5 receptors, impaired mGluR5-dependent synaptic plasticity (DHPG-induced long-term depression), and impaired mGluR5-dependent modulation of neural network activity. We also found morphological abnormalities in the structure of synapses (spine number, width, and length) and impaired glutamatergic synaptic transmission, as shown by reduction in the frequency of miniature excitatory postsynaptic currents (mEPSC). Notably, pharmacological augmentation of mGluR5 activity using 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide as the positive allosteric modulator of these receptors restored mGluR5-dependent signaling (DHPG-induced phosphorylation of ERK1/2) and normalized the frequency of mEPSCs in Shank3-knocked down neurons. These data demonstrate that a deficit in mGluR5-mediated intracellular signaling in Shank3 knockdown neurons can be compensated by 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide; this raises the possibility that pharmacological augmentation of mGluR5 activity represents a possible new therapeutic approach for patients with Shank3 mutations.     

PDZ interactions regulate rapid turnover of the scaffolding protein EBP50 in microvilli

Scaffolding proteins containing PDZ (postsynaptic density 95/discs large/zonula occludens-1) domains are believed to provide relatively stable linkages between components of macromolecular complexes and in some cases to bridge to the actin cytoskeleton. The microvillar scaffolding protein EBP50 (ERM-binding phosphoprotein of 50 kD), consisting of two PDZ domains and an ezrin-binding site, retains specific proteins in microvilli and is necessary for microvillar biogenesis. Our analysis of the dynamics of microvillar proteins in vivo indicated that ezrin and microvillar membrane proteins had dynamics consistent with actin treadmilling and microvillar lifetimes. However, EBP50 was highly dynamic, turning over within seconds. EBP50 turnover was reduced by mutations that inactivate its PDZ domains and was enhanced by protein kinase C phosphorylation. Using a novel in vitro photoactivation fluorescence assay, the EBP50-ezrin interaction was shown to have a slow off-rate that was dramatically enhanced in a PDZ-regulated manner by addition of cell extract to near in vivo levels. Thus, the linking of relatively stable microvillar components can be mediated by surprisingly dynamic EBP50, a finding that may have important ramifications for other scaffolding proteins.     

Crystal structures of autoinhibitory PDZ domain of Tamalin: implications for metabotropic glutamate receptor trafficking regulation

Metabotropic glutamate receptors (mGluRs) function as neuronal G-protein-coupled receptors and this requires efficient membrane targeting through associations with cytoplasmic proteins. However, the molecular mechanism regulating mGluR cell-surface trafficking remains unknown. We report here that mGluR trafficking is controlled by the autoregulatory assembly of a scaffold protein Tamalin. In the absence of mGluR, Tamalin self-assembles into autoinhibited conformations, through its PDZ domain and C-terminal intrinsic ligand motif. X-ray crystallographic analyses visualized integral parts of the oligomeric self-assemblies of Tamalin, which require not only the novel hydrophobic dimerization interface but also canonical and noncanonical PDZ/ligand autoinhibitory interactions. The mGluR cytoplasmic region can competitively bind to Tamalin at a higher concentration, disrupting weak inhibitory interactions. The atomic view of mGluR association suggests that this rearrangement is dominated by electrostatic attraction and repulsion. We also observed in mammalian cells that the association liberates the intrinsic ligand toward a motor protein receptor, thereby facilitating mGluR cell-surface trafficking. Our study suggests a novel regulatory mechanism of the PDZ domain, by which Tamalin switches between the trafficking-inhibited and -active forms depending on mGluR association.     

NMDA receptor subunit composition controls synaptic plasticity by regulating binding to CaMKII

Calcium entry through postsynaptic NMDA-Rs and subsequent activation of CaMKII trigger synaptic plasticity in many brain regions. Active CaMKII can bind to NMDA-Rs, but the physiological role of this interaction is not well understood. Here, we test if association between active CaMKII and synaptic NMDA-Rs is required for synaptic plasticity. Switching synaptic NR2B-containing NMDA-Rs that bind CaMKII with high affinity with those containing NR2A, a subunit with low affinity for CaMKII, dramatically reduces LTP. Expression of NR2A with mutations that increase association to active CaMKII recovers LTP. Finally, driving into synapses NR2B with mutations that reduce association to active CaMKII prevents LTP. Spontaneous activity-driven potentiation shows similar results. We conclude that association between active CaMKII and NR2B is required for different forms of synaptic enhancement. The switch from NR2B to NR2A content in synaptic NMDA-Rs normally observed in many brain regions may contribute to reduced plasticity by controlling the binding of active CaMKII.     

The prevalence and significance of PDZ domain-phosphoinositide interactions

PDZ domains predominate in multi-cellular organisms. They are ubiquitous protein-interaction modules recognizing short peptide sequences generally situated at the C-terminal end of plasma membrane proteins. They contribute to the formation and spatial confinement of protein complexes and thereby play an essential role in the control of cell signaling. Recent studies indicate that PDZ domains can also interact with phosphoinositides (PIPs), signaling lipids with key-roles in receptor signal transduction, membrane trafficking, cytoskeleton remodeling and nuclear processes. In particular the PDZ domains of syntenin-1 and syntenin-2 bind to phosphatidylinositol 4, 5-bisphosphate (PIP2) with high-affinity. Syntenin-1/PIP2 interaction is important for receptor cargo recycling and syntenin-2 plays a role in the organization of nuclear PIP2. In addition, other lower-affinity PDZ domain/PIPs interactions are documented. Here, we summarize and discuss the present knowledge about the occurrence, the biochemistry and the biology of PDZ domain-lipid interactions.     

The role of receptor interactions in regulating ethylene signal transduction

The phytohormone ethylene is perceived in Arabidopsis by a five-member receptor family. Earlier work has demonstrated that the basic functional unit for an ethylene receptor is a disulfide-linked homodimer. We recently reported in The Journal of Biological Chemistry that the ethylene-receptor ETR1 physically associates with other ethylene receptors through higher order interactions, suggesting the existence of receptor clusters. Here we consider the implications of such clusters upon the mechanism of ethylene signal transduction. In particular, we consider how such clustering provides a cooperative mechanism, akin to what has been found for the prokaryotic chemoreceptors, by which plant sensitivity to ethylene may be increased. In addition, we consider how the dominant ethylene insensitivity conferred by some receptor mutations, such as etr1-1, may also be propagated by interactions among members of the ethylene receptor family.Key words: ethylene, receptor, ETR1, cooperativity, ArabidopsisThe plant hormone ethylene regulates growth and development, and is perceived by a five-member family of receptors (ETR1, ERS1, ETR2, ERS2 and EIN4) in Arabidopsis.¹ Genetic analysis indicates that ethylene receptors are functionally redundant and negatively regulate ethylene responses through interactions with the Raf-like kinase CTR1.^2–5 The functional unit of an ethylene receptor in a disulfide-linked homodimer, with each homodimer capable of binding one ethylene molecule.^6,7 However, several observations suggest that propagation of the ethylene signal through the receptors is likely to involve more than just ethylene-induced changes within individual receptor homodimers. First, Arabidopsis is amazingly sensitive to ethylene and can respond to ethylene concentrations as low as 0.2 nl/L,⁸ 300-fold lower than the K_d of the receptors for ethylene, which suggests that some mechanism exists for amplifying the input signal.^7,9 Second, ethylene-insensitive mutations in the binding sites of the receptors exhibit greater dominance than would be predicted solely from a lesion within one member of the receptor family.¹⁰In our paper published in The Journal of Biological Chemistry,¹¹ we demonstrate that the Arabidopsis ethylene receptor ETR1 physically associates with other ethylene receptors through higher order interactions. Such physical interactions suggest that the receptors exist in plants as clusters, and that models for cooperative signaling previously applied to the histidine-kinaselinked chemoreceptors of bacteria may also be applicable to the evolutionarily related ethylene receptors of plants. In bacteria, the highly packed chemoreceptors are found in clusters at one or both poles of the cell.^12,13 Structural studies indicate that chemoreceptors can associate to form a ‘trimer of dimers’^14,15 and also support the possibility that domain swapping may occur to produce a large interconnected array of receptors. 16 Our studies indicate that ethylene receptors can interact through their cytosolic GAF domains, identifying one possible interface through which conformational changes could be propagated in an ethylene receptor cluster.A higher-order cooperative mechanism among the ethylene receptors may explain the high sensitivity of plants to ethylene. In this model, the ethylene receptors amplify ethylene signaling by lateral signal output. Binding of ethylene to one receptor induces the conformation change of the receptor from a tense state (T) to a relaxed state (R). This conformational change is then propagated to other empty receptors in the cluster due to their physical associations with the receptor in the R state. As a result empty receptors also adopt the relaxed state (R′), resulting in amplification of the initial signal. It should be noted here that mutational evidence supports the unbound state of the receptors (T state) as being the lower energy conformation of the receptors.¹⁷ Thus, according to this model, part of the energy from ligand binding would be used to transmit conformational changes to the neighboring receptors.An alternative model that may also explain the high sensitivity of ethylene responsiveness in plants, and one that is not necessarily incompatible with the previous model, is a conjugation model.¹⁸ Here it is hypothesized that, due to the physical proximity of the ethylene receptors, that ethylene released from one receptor then binds to another receptor rather than diffusing away. Through this conjugation mechanism, one ethylene molecule could amplify its signal by converting the conformations of multiple ethylene receptors from the ethylene-unbound state (T) to the ethylene-bound state (R). This model is based on several assumptions. One assumption is that a single ethylene molecule can bind ethylene receptors in the same cluster multiple times due to the dynamic binding of ethylene and ethylene receptor. A second assumption is that, after ethylene is released from one ethylene receptor, the recovery time for that receptor to resume the T state is longer than the time required for the released ethylene to bind to and convert another receptor from the T to the R state.Models for cooperativity need to also explain the dominant ethylene insensitivity of various mutant receptors such as etr1-1, in which a missense mutation results in a receptor incapable of binding ethylene. Several studies indicate that the etr1-1 mutant receptor acts cooperatively to affect the signal output from other wild-type receptors (i.e., the presence of the etr1-1 receptor in its T state increases the likelihood of other receptors adopting the T state).^10,11 This observation can be most readily explained if the dominant ethylene-insensitive mutations result in a receptor that requires more energy to undergo the T to R transition than do the wild-type receptors. For example, the etr1-1 mutation may increase the stability of the T form (a T′ state). There is evidence to support this possibility. The etr1-1 missense mutation results in a receptor unable to chelate a copper cofactor necessary for ethylene binding,¹⁹ but the effects of this mutation on signaling are different from wild-type receptors that lack their copper cofactor. The etr1-1 mutant receptor appears locked in its T state, whereas wild-type receptors lacking the copper cofactor appear to be in the R state.²⁰ Thus etr1-1 is truly a gain-of-function mutation that alters the conformation of the receptor in ways not necessarily predicted from just the loss of the copper cofactor.In conclusion, we have attempted here to provide models that can resolve an apparent contradiction in the cooperative signaling behavior exhibited by ethylene receptors. The high sensitivity of plants to ethylene suggest cooperative changes in which an R state can be propagated within a receptor cluster, but the dominance of the ethylene ethylene-insensitive mutant etr1-1 suggests that the T state can also be propagated within a receptor cluster. It should be born in mind, however, that ethylene signaling is mediated by multiple signaling components. The ethylene receptors regulate ethylene responses through interaction with and modulation of CTR1 kinase activity. Thus, the total kinase activity of CTR1 represents the signal output from the receptors. This situation is very similar to that of the bacterial chemoreceptors, which regulate the activity of an associated histidine kinase, and, as with the chemoreceptors, the stoichiometry of CTR1 interactions with the ethylene receptors and the means by which its kinase activity is regulated are important for the elucidation of the mechanism of ethylene signal transduction.