Light-dependent phosphorylation of rhodopsin by beta-adrenergic receptor kinase

The structural components involved in transduction of extracellular signals as diverse as a photon of light impinging on the retina or a hormone molecule impinging on a cell have been highly conserved. These components include a recognition unit or receptor (for example, the beta-adrenergic receptor (beta AR) for catecholamines or the 'light receptor' rhodopsin), a guanine nucleotide regulatory or transducing protein, and an effector enzyme (for example, adenylate cyclase or cyclic GMP phosphodiesterase). Molecular cloning has revealed that the beta AR shares significant sequence and three-dimensional homology with rhodopsin. The function of the beta AR is diminished by exposure to stimulatory agonists, leading to desensitization. Similarly, 'light adaptation' involves decreased coupling of photoactivated rhodopsin to cGMP phosphodiesterase activation. Both forms of desensitization involve receptor phosphorylation. The latter is mediated by a unique protein kinase, rhodopsin kinase, which phosphorylates only the light-bleached form of rhodopsin. An analogous enzyme (termed beta AR kinase or beta ARK) phosphorylates only the agonist-occupied beta AR. We report here that beta ARK is also capable of phosphorylating rhodopsin in a totally light-dependent fashion. Moreover, rhodopsin kinase can phosphorylate the agonist-occupied beta AR. Thus the mechanisms which regulate the function of these disparate signalling systems also appear to be similar.     

Phosphorylation of the nicotinic acetylcholine receptor regulates its rate of desensitization

Recent studies have provided evidence for a role of protein phosphorylation in the regulation of the function of various potassium and calcium channels (for reviews, see refs 1, 2). As these ion channels have not yet been isolated and characterized, it has not been possible to determine whether phosphorylation of the ion channels themselves alters their properties or whether some indirect mechanism is involved. In contrast, the nicotinic acetylcholine receptor, a neurotransmitter-dependent ion channel, has been extensively characterized biochemically and has been shown to be directly phosphorylated. The phosphorylation of this receptor is catalysed by at least three different protein kinases (cyclic AMP-dependent protein kinase, protein kinase C and a tyrosine-specific protein kinase) on seven different phosphorylation sites. However, the functional significance of phosphorylation of the receptor has been unclear. We have now examined the functional effects of phosphorylation of the nicotinic acetylcholine receptor by cAMP-dependent protein kinase. We investigated the ion transport properties of the purified and reconstituted acetylcholine receptor before and after phosphorylation. We report here that phosphorylation of the nicotinic acetylcholine receptor on the gamma- and delta-subunits by cAMP-dependent protein kinase increases the rate of the rapid desensitization of the receptor, a process by which the receptor is inactivated in the presence of acetylcholine (ACh). These results provide the first direct evidence that phosphorylation of an ion channel protein modulates its function and suggest that phosphorylation of postsynaptic receptors in general may play an important role in synaptic plasticity.     

Phosphorylation and dephosphorylation of the high-affinity receptor for immunoglobulin E immediately after receptor engagement and disengagement.

Triggering of mast cells and basophils by immunoglobulin E (IgE) and antigen induces various biochemical signals, including tyrosine kinase activation, which lead to cell degranulation and the release of mediators of the allergic reaction. The high-affinity receptor for IgE (Fc epsilon RI) responsible for initiating these events is a complex structure composed of an IgE-binding alpha-chain, a beta-chain and a homodimer of gamma-chains. It has been assumed that beta and gamma, which have extensive cytoplasmic domains, play an important but undefined role in coupling Fc epsilon RI to signal transduction mechanisms. Here we show that Fc epsilon RI engagement induces immediate in vivo phosphorylation on beta (tyrosine and serine) and gamma (tyrosine and threonine) by at least two different non-receptor kinases. We take advantage of unique features of this receptor system to demonstrate that the phosphorylation signal is restricted to activated receptors and is immediately reversible upon receptor disengagement by undefined phosphatases. Rapid phosphorylation and dephosphorylation may be a general mechanism to couple and uncouple activated receptors to other effector molecules. This could be particularly relevant to other multimeric receptors containing Fc epsilon RI gamma-chains or the related zeta and eta chains such as the T-cell antigen receptor (TCR) and the low-affinity receptor for immunoglobulin G (Fc gamma RIII, CD16).     

Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence

Morphine is a powerful pain reliever, but also a potent inducer of tolerance and dependence. The development of opiate tolerance occurs on continued use of the drug such that the amount of drug required to elicit pain relief must be increased to compensate for diminished responsiveness. In many systems, decreased responsiveness to agonists has been correlated with the desensitization of G-protein-coupled receptors. In vitro evidence indicates that this process involves phosphorylation of G-protein-coupled receptors and subsequent binding of regulatory proteins called beta-arrestins. Using a knockout mouse lacking beta-arrestin-2 (beta arr2-/-), we have assessed the contribution of desensitization of the mu-opioid receptor to the development of morphine antinociceptive tolerance and the subsequent onset of physical dependence. Here we show that in mice lacking beta-arrestin-2, desensitization of the mu-opioid receptor does not occur after chronic morphine treatment, and that these animals fail to develop antinociceptive tolerance. However, the deletion of beta-arrestin-2 does not prevent the chronic morphine-induced up-regulation of adenylyl cyclase activity, a cellular marker of dependence, and the mutant mice still become physically dependent on the drug.     

Increased phosphorylation of ribosomal protein S6 following microinjection of insulin receptor-kinase into Xenopus oocytes

The protein products of several transforming retroviruses as well as the receptors for several hormones and growth factors, including insulin, have been shown to possess a protein kinase activity in vitro specific for tyrosine residues in protein substrates, including themselves. In the case of pp60src and the insulin receptor, autophosphorylation activates the tyrosine kinase activity towards exogenous substrates. Experiments indicate that, in vivo, many of these viruses or growth factors induce an increase in cellular phosphotyrosine, as well as an increase in the phosphorylation of serine residues on proteins, including ribosomal protein S6. It seems likely that some of the effects of insulin might be mediated by phosphorylation of intracellular substrates by its receptor. As the beta subunit of the receptor is a transmembrane protein, such phosphorylation could occur either while the receptor is still in the membrane or after its internalization. In various cell systems, internalized receptors are degraded, reshuttled back to the plasmalemma or maintained in a separate compartment before reinsertion in the membrane; shuttling of the insulin receptor could provide the opportunity for it to phosphorylate various intracellular components as part of its mechanism of signal transduction. To approach directly the question of whether the receptor can elicit a signal while acting at an intracellular location, we have microinjected Xenopus oocytes with the insulin receptor kinase. The results indicate that an S6 protein-serine kinase is stimulated or an S6 protein-serine phosphatase inhibited by the activity of the insulin receptor, supporting the concept that the insulin receptor acting within the cell can elicit a biological response.     

Functional modulation of the nicotinic acetylcholine receptor by tyrosine phosphorylation

Tyrosine-specific protein phosphorylation has been implicated in the regulation of cell transformation and proliferation. However, recent studies have shown that the expression of protein tyrosine kinases in adult brain is very high, suggesting that tyrosine-specific protein phosphorylation may also have a role in the regulation of neuronal function. Although a number of substrate proteins are phosphorylated on tyrosine residues, the functional alteration of proteins by tyrosine phosphorylation has previously been convincingly demonstrated only for protein tyrosine kinases. The nicotinic acetylcholine receptor, a neurotransmitter-gated ion channel, is phosphorylated by a protein tyrosine kinase in post-synaptic membranes in vitro and in vivo. We demonstrate here that this tyrosine phosphorylation increases the rate of the rapid phase of desensitization of the nicotinic receptor, as measured by single channel recording of purified nicotinic acetylcholine receptor, when reconstituted in lipid vesicles. These data provide direct evidence for the regulation of ion channel properties by tyrosine phosphorylation. The results, which demonstrate a functional role of tyrosine phosphorylation in the nervous system, suggest a widespread role for tyrosine phosphorylation in neuronal signal transduction.     

A kinase-regulated PDZ-domain interaction controls endocytic sorting of the beta2-adrenergic receptor.

A fundamental question in cell biology is how membrane proteins are sorted in the endocytic pathway. The sorting of internalized beta2-adrenergic receptors between recycling endosomes and lysosomes is responsible for opposite effects on signal transduction and is regulated by physiological stimuli. Here we describe a mechanism that controls this sorting operation, which is mediated by a family of conserved protein-interaction modules called PDZ domains. The phosphoprotein EBP50 (for ezrinradixin-moesin(ERM)-binding phosphoprotein-50) binds to the cytoplasmic tail of the beta2-adrenergic receptor through a PDZ domain and to the cortical actin cytoskeleton through an ERM-binding domain. Disrupting the interaction of EBP50 with either domain or depolymerization of the actin cytoskeleton itself causes missorting of endocytosed beta2-adrenergic receptors but does not affect the recycling of transferrin receptors. A serine residue at position 411 in the tail of the beta2-adrenergic receptor is a substrate for phosphorylation by GRK-5 (for G-protein-coupled-receptor kinase-5) and is required for interaction with EBP50 and for proper recycling of the receptor. Our results identify a new role for PDZ-domain-mediated protein interactions and for the actin cytoskeleton in endocytic sorting, and suggest a mechanism by which GRK-mediated phosphorylation could regulate membrane trafficking of G-protein-coupled receptors after endocytosis.     

Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane

The receptor for epidermal growth factor (EGF) is a 170,000-180,000 molecular weight single-chain glycoprotein of 1,186 amino acids. Its sequence suggests that it has an external EGF-binding domain, formed by the NH2-terminal 621 amino acids, linked to a cytoplasmic region by a single membrane-spanning segment. In the cytoplasmic portion, starting 50 residues from the membrane, there is a 250-residue stretch similar to the catalytic domain of the src gene family of retroviral tyrosine protein kinases, and, indeed, a tyrosine-specific protein kinase activity intrinsic to the receptor is stimulated when EGF is bound. Increased tyrosine phosphorylation of cellular proteins, detected in A431 cells following EGF binding, may be important in the mitogenic signal pathway. Tumour promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), counteract this increase, as well as causing loss of a high affinity class of EGF binding sites. The major receptor for TPA has been identified as the serine/threonine-specific Ca2+/phospholipid-dependent diacylglycerol-activated protein kinase, protein kinase C. By substituting for diacylglycerol, TPA stimulates protein kinase C. Protein kinase C phosphorylates purified EGF receptor at specific sites, and this reduces EGF-stimulated tyrosine protein kinase activity. TPA treatment of A431 cells increases serine and threonine phosphorylation of the EGF receptor at the same sites, which suggests that the reduction of EGF receptor kinase activity in TPA-treated cells is a consequence of the receptor's phosphorylation by the kinase. We have attempted to identify these phosphorylation sites and show here that protein kinase C phosphorylates threonine 654 in the human EGF receptor. This threonine is in a very basic sequence nine residues from the cytoplasmic face of the plasma membrane in the region before the protein kinase domain; it is thus in a position to modulate signalling between this internal domain and the external EGF-binding domain.     

MAP2 kinase and 70K S6 kinase lie on distinct signalling pathways.

Activation of protein synthesis is required for quiescent cells to transit the cell cycle, and seems to be mediated in part by phosphorylation of the 40S ribosomal protein, S6. A mitogen-activated S6 kinase of relative molecular mass 70,000 (70K) has been isolated from mouse fibroblasts as well as from avian, rat and rabbit tissues. Comparison of complementary DNA sequences shows that this enzyme is distinct from S6 kinase II (92K) found in Xenopus eggs and fibroblasts. Both kinases are activated by serine/threonine phosphorylation, suggesting that at least one serine/threonine kinase links receptor tyrosine kinases with S6 kinases. A candidate for this link is MAP2 kinase, which is rapidly activated by tyrosine/threonine phosphorylation following mitogenic stimulation. Incubation of MAP2 kinase from insulin-treated 3T3-L1 adipocytes with phosphatase-inactivated S6 kinase II from Xenopus leads to partial reactivation and phosphorylation of the enzyme. These and other findings have led to the suggestion that MAP2 kinase also activates the 70K S6 kinase. Here we refute this idea by showing that the two kinases lie on distinct signalling pathways.     

A chimaeric receptor allows insulin to stimulate tyrosine kinase activity of epidermal growth factor receptor

The cell surface receptors for insulin and epidermal growth factor (EGF) appear to share a common evolutionary origin, as suggested by structural similarity of cysteine-rich regions in their extracellular domains and a highly conserved tyrosine-specific protein kinase domain. Only minor similarity is found outside this catalytic domain, as expected for receptors that have different ligand specificities and generate different biological signals. The EGF receptor is a single polypeptide chain but the insulin receptor consists of distinct alpha and beta subunits that function as an alpha 2 beta 2 heterotetrameric receptor complex. Provoked by this major structural difference in two receptors that carry out parallel functions, we have designed a chimaeric receptor molecule comprising the extracellular portion of the insulin receptor joined to the transmembrane and intracellular domains of the EGF receptor to investigate whether one ligand will activate the tyrosine kinase domain of the receptor for the other ligand. We show here that the EGF receptor kinase domain of the chimaeric protein, expressed transiently in simian cells, is activated by insulin binding. This strongly suggests that insulin and EGF receptors employ closely related or identical mechanisms for signal transduction across the plasma membrane.     

Isoprenylation in regulation of signal transduction by G-protein-coupled receptor kinases.

Rhodopsin kinase and beta-adrenergic receptor kinase (beta ARK) are related members of a serine/threonine kinase family that specifically initiate deactivation of G-protein-coupled receptors. After stimulus-mediated receptor activation, these cytoplasmic kinases translocate to the plasma membrane. Here we show that the molecular basis for this event involves a class of unsaturated lipids called isoprenoids. Covalent modification in vivo of rhodopsin kinase by a 15-C (farnesyl) isoprenoid enables the kinase to anchor to photon-activated rhodopsin. Mutations that alter or eliminate the isoprenoid, fully disable light-specific Rhodopsin kinase translocation. Other receptor kinases (such as beta ARK), which lack an intrinsic lipid, are activated on exposure to brain beta gamma subunits of the signal-transducing G proteins, the gamma subunit of which bears a 20-C (geranylgeranyl) isoprenoid. Using chimaeric beta ARKs that undergo isoprenylation in vitro, we demonstrate that membrane association and activation of these kinases can occur in the absence of beta gamma. These results indicate that rhodopsin kinase (by means of an integral isoprenoid) and beta ARK (through its association with beta gamma) both rely on the function of isoprenyl moieties for their translocation and activity, illustrating distinct, though related, modes of biological regulation of receptor function.     

Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein.

Since the discovery of insulin nearly 70 years ago, there has been no problem more fundamental to diabetes research than understanding how insulin works at the cellular level. Insulin binds to the alpha subunit of the insulin receptor which activates the tyrosine kinase in the beta subunit, but the molecular events linking the receptor kinase to insulin-sensitive enzymes and transport processes are unknown. Our discovery that insulin stimulates tyrosine phosphorylation of a protein of relative molecular mass between 165,000 and 185,000, collectively called pp185, showed that the insulin receptor kinase has specific cellular substrates. The pp185 is a minor cytoplasmic phosphoprotein found in most cells and tissues; its phosphorylation is decreased in cells expressing mutant receptors defective in signalling. We have now cloned IRS-1, which encodes a component of the pp185 band. IRS-1 contains over ten potential tyrosine phosphorylation sites, six of which are in Tyr-Met-X-Met motifs. During insulin stimulation, the IRS-1 protein undergoes tyrosine phosphorylation and binds phosphatidylinositol 3-kinase, suggesting that IRS-1 acts as a multisite 'docking' protein to bind signal-transducing molecules containing Src-homology 2 and Src-homology-3 domains. Thus IRS-1 may link the insulin receptor kinase and enzymes regulating cellular growth and metabolism.     

An unusual feature revealed by the crystal structure at 2.2 A resolution of human transforming growth factor-beta 2.

Transforming growth factor type beta 2 (TGF-beta 2) is a member of an expanding family of growth factors that regulate proliferation and differentiation of many different cell types. TGF-beta 2 binds to various receptors, one of which was shown to be a serine/threonine kinase. TGF-beta 2 is involved in wound healing, bone formation and modulation of immune functions. We report here the crystal structure of TGF-beta 2 at 2.2 A resolution, which reveals a novel monomer fold and dimer association. The monomer consists of two antiparallel pairs of beta-strands forming a flat curved surface and a separate, long alpha-helix. The disulphide-rich core has one disulphide bone pointing through a ring formed by the sequence motifs Cys-Ala-Gly-Ala-Cys and Cys-Lys-Cys, which are themselves connected through the cysteines. Two monomers are connected through a single disulphide bridge and associate such that the helix of one subunit interacts with the concave beta-sheet surface of the other. Four exposed loop regions might determine receptor specificity. The structure provides a suitable model for the TGF-beta s and other members of the super-family and is the basis for the analysis of the TGF-beta 2 interactions with the receptor.     

A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence

Plants sense potential microbial invaders by using pattern-recognition receptors to recognize pathogen-associated molecular patterns (PAMPs). In Arabidopsis thaliana, the leucine-rich repeat receptor kinases flagellin-sensitive 2 (FLS2) (ref. 2) and elongation factor Tu receptor (EFR) (ref. 3) act as pattern-recognition receptors for the bacterial PAMPs flagellin and elongation factor Tu (EF-Tu) (ref. 5) and contribute to resistance against bacterial pathogens. Little is known about the molecular mechanisms that link receptor activation to intracellular signal transduction. Here we show that BAK1 (BRI1-associated receptor kinase 1), a leucine-rich repeat receptor-like kinase that has been reported to regulate the brassinosteroid receptor BRI1 (refs 6,7), is involved in signalling by FLS2 and EFR. Plants carrying bak1 mutations show normal flagellin binding but abnormal early and late flagellin-triggered responses, indicating that BAK1 acts as a positive regulator in signalling. The bak1-mutant plants also show a reduction in early, but not late, EF-Tu-triggered responses. The decrease in responses to PAMPs is not due to reduced sensitivity to brassinosteroids. We provide evidence that FLS2 and BAK1 form a complex in vivo, in a specific ligand-dependent manner, within the first minutes of stimulation with flagellin. Thus, BAK1 is not only associated with developmental regulation through the plant hormone receptor BRI1 (refs 6,7), but also has a functional role in PRR-dependent signalling, which initiates innate immunity.     

Relationship of a putative receptor protein kinase from maize to the S-locus glycoproteins of Brassica

The protein kinase family of enzymes mediates the responses of eukaryotic cells to both inter- and intracellular signals. These enzymes are either serine/threonine-specific or tyrosine-specific. Many of the latter are transmembrane receptors and are important in transduction of extracellular signals across the plasma membrane, whereas few examples of receptor serine kinases have been reported. We have now identified a complementary DNA clone from Zea mays (L.) encoding a putative serine/threonine-specific protein kinase structurally related to the receptor tyrosine kinases. This structural similarity is evidence for a previously undescribed class of transmembrane receptor in higher plants likely to be involved in signal reception and transduction. Furthermore, the catalytic domain of this protein kinase is linked through a transmembrane domain to an extracellular domain similar to that of glycoproteins encoded in the self-incompatibility locus of Brassica which are involved in the self-recognition system between pollen and stigma.     

Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2

Feedback inhibition is a fundamental principle in signal transduction allowing rapid adaptation to different stimuli. In mammalian cells, the major feedback inhibitor for G-protein-coupled receptors (GPCR) is G-protein-coupled receptor kinase 2 (GRK-2), which phosphorylates activated receptors, uncouples them from G proteins and initiates their internalization. The functions of GRK-2 are indispensable and need to be tightly controlled. Dysregulation promotes disorders such as hypertension or heart failure. In our search for a control mechanism for this vital kinase, here we show that the Raf kinase inhibitor protein (RKIP) is a physiological inhibitor of GRK-2. After stimulation of GPCR, RKIP dissociates from its known target, Raf-1 (refs 6-8), to associate with GRK-2 and block its activity. This switch is triggered by protein kinase C (PKC)-dependent phosphorylation of the RKIP on serine 153. The data delineate a new principle in signal transduction: by activating PKC, the incoming receptor signal is enhanced both by removing an inhibitor from Raf-1 and by blocking receptor internalization. A physiological role for this mechanism is shown in cardiomyocytes in which the downregulation of RKIP restrains beta-adrenergic signalling and contractile activity.     

Role of intracellular calcium mobilization in the regulation of protein kinase C-mediated membrane processes

Phorbol esters are potent tumour-promoting agents that exert pleiotropic effects on cells. Among these are the control of growth, stimulation of release of stored bioactive constituents and regulation of growth-factor surface receptors. Phorbol esters bind to and activate protein kinase C, leading to the phosphorylation of specific protein substrates presumed to be necessary for eliciting the full response. Strong evidence exists that specific binding of tumour promoter occurs at the membrane level in intact cells, resulting in activation of protein kinase C. Recent evidence concerning the release of bioactive constituents from platelets and neutrophils has linked agonist-induced protein kinase C activation and Ca2+ mobilization in a synergistic mechanism. Here we present a novel model of synergism between Ca2+ and phorbol esters that leads to transferrin receptor phosphorylation and down-regulation in HL-60 human leukaemic cells. Raising intracellular Ca2+, although ineffective by itself, increases the potency and rate of action of phorbol ester for activating protein kinase C and mediating transferrin receptor phosphorylation and down-regulation. We propose a molecular model in which increased intracellular Ca2+ recruits protein kinase C to the plasma membrane, thus "priming' the system for activation by phorbol ester.     

Regulation of NMDA receptor desensitization in mouse hippocampal neurons by glycine

Responses to the excitatory amino acid N-methyl-D-aspartate (NMDA) are markedly potentiated by nanomolar concentrations of glycine. This is due to the action of glycine at a novel strychnine-resistant binding site with an anatomical distribution identical to that for NMDA receptors, suggesting that the NMDA receptor channel complex contains at least two classes of amino-acid recognition site. Antagonists at the glycine-binding site associated with NMDA receptors act as potent non-competitive antagonists, but do not alter the mean open time or conductance, as estimated by fluctuation analysis. The mechanisms by which glycine acts on NMDA receptors are unknown, but single-channel recording experiments show an increase in opening frequency with no change in mean open time or conductance, suggesting that glycine could regulate transitions to states that are intermediate between binding of NMDA receptor agonists and ion-channel gating. It has been suggested that glycine acts as a co-agonist at the NMDA receptor, and that responses to NMDA cannot be obtained in the complete absence of glycine, but in these experiments the response to NMDA was measured at equilibrium, and it is unlikely that sufficient temporal resolution was achieved to detect rapid alterations in receptor gating. Using a fast perfusion system we find that glycine regulates desensitization at NMDA receptors; this has a major effect on the response to NMDA measured at equilibrium, as would occur with slower applications of agonist. Reduction of NMDA receptor desensitization by glycine provides an example of a novel mechanism for regulation of ion-channel activity.