Phosphorylation-independent desensitization of metabotropic glutamate receptor 5 by G protein-coupled receptor kinase 2 in HEK 293 cells

The metabotropic glutamate receptor 5 (mGluR5) is one of the important excitatory neurotransmitter receptors in the central nervous system, and its desensitization by G protein-coupled receptor kinases (GRKs) plays an important role in neuron protection against receptor overstimulation. It is reported that GRK2 could down-regulate the mGluR5 signaling in both HEK 293 cells and neurons. However, whether GRK2-mediated mGluR5 desensitization is phosphorylation dependent remains controversial. Here, we demonstrated that the signal intensity and kinetics of mGluR5 desensitization was inhibited or changed by GRK2 in HEK 293 cells. By using the catalytically inactive GRK2 mutant K220R, and the receptor mutants that lack potential phosphorylation sites in the C-terminal tail, we demonstrated that the GRK2-mediated mGluR5 desensitization was phosphorylation-independent. Furthermore, overexpression of an N-terminal regulator of G protein signaling (RGS) homology (RH) domain of GRK2 was sufficient to attenuate the mGluR5 signaling, whereas the expression of GRK2 D110A mutant devoid in Gαq binding was unable to inhibit mGluR5 signaling. In summary, this study provides evidence that GRK2 mediates phosphorylationindependent mGluR5 desensitization via the interaction between the RGS domain and Gαq in HEK 293 cells.     

G protein-coupled receptor kinase-mediated desensitization of metabotropic glutamate receptor 1A protects against cell death

Metabotropic glutamate receptors (mGluRs) constitute a unique subclass of G protein-coupled receptors (GPCRs) that bear little sequence homology to other members of the GPCR superfamily. The mGluR subtypes that are coupled to the hydrolysis of phosphoinositide contribute to both synaptic plasticity and glutamate-mediated excitotoxicity in neurons. In the present study, the expression of mGluR1a in HEK 293 cells led to agonist-independent cell death. Since G protein-coupled receptor kinases (GRKs) desensitize a diverse variety of GPCRs, we explored whether GRKs contributed to the regulation of both constitutive and agonist-stimulated mGluR1a activity and thereby may prevent mGluR1a-mediated excitotoxicity associated with mGluR1a overactivation. We find that the co-expression of mGluR1a with GRK2 and GRK5, but not GRK4 and GRK6, reduced both constitutive and agonist-stimulated mGluR1a activity. Agonist-stimulated mGluR1a phosphorylation was enhanced by the co-expression of GRK2 and was blocked by two different GRK2 dominant-negative mutants. Furthermore, GRK2-dependent mGluR1a desensitization protected against mGluR1a-mediated cell death, at least in part by blocking mGluR1a-stimulated apoptosis. Our data indicate that as with other members of the GPCR superfamily, a member of the structurally distinct mGluR family (mGluR1a) serves as a substrate for GRK-mediated phosphorylation and that GRK-dependent "feedback" modulation of mGluR1a responsiveness protects against pathophysiological mGluR1a signaling.     

The metabotropic glutamate G-protein-coupled receptors mGluR3 and mGluR1a are voltage-sensitive

G-protein-coupled receptors play a key role in signal transduction processes. Despite G-protein-coupled receptors being transmembrane proteins, the notion that they exhibit voltage sensitivity is rather novel. Here we examine whether two metabotropic glutamate receptors, mGluR3 and mGluR1a, both involved in fundamental physiological processes, exhibit, by themselves, voltage sensitivity. Measuring mGluR3-induced K(+) currents and mGluR1a-induced Ca(2+)-activated Cl(-) currents in Xenopus oocytes, we show that the apparent affinity toward glutamate decreases (mGluR3) or increases (mGluR1a) upon depolarization. Measurements of binding of [(3)H]glutamate to oocytes expressing either mGluR3 or mGluR1a corroborated the electrophysiological results. Using the chimeric Galpha subunit, we further show that the voltage sensitivity does not reside in the G-protein. To locate sites within the receptors that are involved in the voltage sensitivity, we used chimeric mGluR1a, where the intracellular loops that couple to the G-protein were replaced by those of mGluR3. The voltage sensitivity of the chimeric mGluR1a resembled that of mGluR3 and not that of the parental mGluR1a. The cumulative results indicate that the voltage sensitivity does not reside downstream to the activation of the receptors but rather in the mGluR3 and mGluR1a themselves. Furthermore, the intracellular loops play a crucial role in relaying changes in membrane potential to changes in the affinity of the receptors toward glutamate.     

Phosphorylation-independent desensitization of GABA(B) receptor by GRK4

Agonist-promoted desensitization of the heterodimeric metabotropic GABA(B) receptor was investigated. Whereas no desensitization was observed in HEK293 cells heterologously expressing the receptor, GABA and the synthetic agonist baclofen induced a robust desensitization in cerebellar granule cells endogenously expressing the receptor. Taking advantage of this cell-specific desensitization phenotype, we identified GRK4 as the kinase involved in the neuronal desensitization. Transfection of small interference RNA directed against GRK4 significantly reduced GRK4 levels in cerebellar granule cells and strongly inhibited the agonist-promoted desensitization. Reciprocally, transfection of GRK4 in HEK293 cells restored agonist-promoted desensitization, confirming that this kinase is sufficient to support desensitization. Surprisingly, this desensitization occurred in the absence of ligand-induced receptor phosphorylation and could be promoted by GRK4 mutants deleted of their kinase domain. Taken together, these results suggest that GRK4 plays a central role in the agonist-promoted desensitization of GABA(B) receptor and that it does so through an atypical mechanism that challenges the generally accepted model linking the kinase activity of GRKs to their role in receptor desensitization.     

GRK3 regulation during CRF- and urocortin-induced CRF1 receptor desensitization

The EC(50) values for concentration-dependent stimulation of cAMP accumulation by CRF (1.3nM) and urocortin (1.0nM) were equivalent in human retinoblastoma Y79 cells. The time course and magnitude of CRF- and urocortin-induced CRF(1) receptor desensitization were similar. A significant 3-fold increase in GRK3, but not GRK2, mRNA levels accompanied the emergence of CRF(1) receptor desensitization in Y79 cells exposed to CRF. In preliminary experiments, retinoblastoma GRK3 protein expression became upregulated during a 48-h CRF exposure. Neither GRK3 nor GRK2 expression increased in Y79 cells exposed to urocortin for 10 min to 48 h. We hypothesize that GRK3 upregulation may be a cellular negative feedback process directed at maximizing CRF(1) receptor desensitization by heightening GRK3 phosphorylating capacity during prolonged exposure to high CRF. Regulation of GRK expression associated with urocortin- and CRF-induced CRF(1) receptor desensitization appears to differ, despite a similar level of signaling via the cAMP-protein kinase A pathway.     

Role of G protein-coupled receptor kinase 4 and beta-arrestin 1 in agonist-stimulated metabotropic glutamate receptor 1 internalization and activation of mitogen-activated protein kinases

The metabotropic glutamate 1 (mGlu(1)) receptor in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Here we show that the G protein-coupled receptor kinases (GRK) 2 and 4, which are expressed in these cells, regulate the mGlu(1) receptor by at least in part different mechanisms. Using kinase-dead mutants in HEK293 cells, we found that GRK4, but not GRK2, needs the intact kinase activity to desensitize the mGlu(1) receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated Galpha(q). In cells transfected with GRK4 and exposed to agonist, beta-arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu(1) receptor, and then internalized in vesicles. The receptor was also internalized but in different vesicles. The expression of beta-arrestin V53D dominant negative mutant, which did not affect the mGlu(1) receptor internalization, reduced by 70-80% the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu(1) receptor. The agonist-stimulated differential sorting of the mGlu(1) receptor and beta-arrestin as well as the activation of MAP kinases by mGlu(1) agonist was confirmed in cultured cerebellar Purkinje cells. A major involvement of GRK4 and of beta-arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and expressing beta-arrestin V53D dominant negative mutant by an adenovirus vector. We conclude that GRK2 and GRK4 regulate the mGlu(1) receptor by different mechanisms and that beta-arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule.     

Functional regulation of metabotropic glutamate receptor type 1c: a role for phosphorylation in the desensitization of the receptor

A ratio-fluorescence assay was developed for on-line localization and quantification of lipid oxidation in living cells. The assay explores the oxidative sensitivity of C11-BODIPY(581/591). Upon oxidation, the fluorescence of this fluorophore shifts from red to green. The probe incorporates readily into cellular membranes and is about twice as sensitive to oxidation as arachidonic acid. Using confocal microscopy, the cumene hydroperoxide-induced oxidation of C11-BODIPY(581/591) was visualized at the sub-cellular level in rat-1 fibroblasts. Preloading of the cells with tocopherol retarded this oxidation. The data demonstrate that C11-BODIPY(581/591) is a valuable tool to quantify lipid oxidation and anti-oxidant efficacy in single cells.     

Phosphorylation-independent regulation of metabotropic glutamate receptor signaling by G protein-coupled receptor kinase 2

The accepted paradigm for G protein-coupled receptor kinase (GRK)-mediated desensitization of G protein-coupled receptors involves GRK-mediated receptor phosphorylation followed by the binding of arrestin proteins. Although GRKs contribute to metabotropic glutamate receptor 1 (mGluR1) inactivation, beta-arrestins do not appear to be required for mGluR1 G protein uncoupling. Therefore, we investigated whether the phosphorylation of serine and threonine residues localized within the C terminus of mGluR1a is sufficient to allow GRK2-mediated attenuation of mGluR1a signaling. We find that the truncation of the mGluR1a C-terminal tail prevents mGluR1a phosphorylation and that GRK2 does not contribute to the phosphorylation of an mGluR1 splice variant (mGluR1b). However, mGluR1a-866Delta- and mGluR1b-stimulated inositol phosphate formation is attenuated following GRK2 expression. The expression of the GRK2 C-terminal domain to block membrane translocation of endogenous GRK2 increases mGluR1a-866Delta- and mGluR1b-stimulated inositol phosphate formation, presumably by blocking membrane translocation of GRK2. In contrast, expression of the kinase-deficient GRK2-K220R mutant inhibits inositol phosphate formation by these unphosphorylated receptors. Expression of the GRK2 N-terminal domain (residues 45-185) also attenuates both constitutive and agonist-stimulated mGluR1a, mGluR1a-866Delta, and mGluR1b signaling, and the GRK2 N terminus co-precipitates with mGluR1a. Taken together, our observations indicate that attenuation of mGluR1 signaling by GRK2 is phosphorylation-independent and that the interaction of the N-terminal domain of GRK2 with mGluR1 contributes to the regulation of mGluR1 G protein coupling.     

Mechanisms of metabotropic glutamate receptor desensitization: role in the patterning of effector enzyme activation

Metabotropic glutamate receptors (mGluRs) constitute an unique subclass of G protein-coupled receptors (GPCRs). These receptors are activated by the excitatory amino acid glutamate and play an essential role in regulating neural development and plasticity. In the present review, we overview the current understanding regarding the molecular mechanisms involved in the desensitization and endocytosis of Group 1 mGluRs as well as the relative contribution of desensitization to the spatial-temporal patterning of glutamate receptor signaling. Similar to what has been reported previously for prototypic GPCRs, mGluRs desensitization is mediated by second messenger-dependent protein kinases and GPCR kinases (GRKs). However, it remains to be determined whether mGluRs phosphorylation by GRKs and beta-arrestin binding are absolutely required for desensitization. Group 1 mGluRs endocytosis is both agonist-dependent and -independent. Agonist-dependent mGluRs internalization is mediated by a beta-arrestin- and dynamin-dependent clathrin-coated vesicle dependent endocytic pathway. The activation of Group 1 mGluRs also results in oscillatory Gq protein-coupling leading to the cyclical activation of phospholipase Cbeta thereby stimulating oscillations in both inositol 1,4,5-triphosphate formation and Ca(2+) release from intracellular stores. These glutamate receptor-stimulated Ca(2+) oscillations are translated into the synchronous activation of protein kinase C (PKC), which has led to the hypothesis that oscillatory mGluRs signaling involves the repetitive phosphorylation of mGluRs by PKC. However, recent experimental evidence suggests that oscillatory signaling is an intrinsic glutamate receptor property that is independent of feedback receptor phosphorylation by PKC. The challenge in the future will be to determine the structural determinants underlying mGluRs-mediated spatial-temporal signaling as well as to understand how complex signaling patterns can be interpreted by cells in both the developing and adult nervous systems.     

GRK3 mediates desensitization of CRF1 receptors: a potential mechanism regulating stress adaptation

Potential G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) mediation of homologous desensitization of corticotropin-releasing factor type 1 (CRF1) receptors was investigated in human retinoblastoma Y-79 cells. Inhibition of PKA activity by PKI(5-22) or H-89 failed to attenuate homologous desensitization of CRF1 receptors, and direct activation of PKA by forskolin or dibutyryl cAMP failed to desensitize CRF-induced cAMP accumulation. However, treatment of permeabilized Y-79 cells with heparin, a nonselective GRK inhibitor, reduced homologous desensitization of CRF1 receptors by approximately 35%. Furthermore, Y-79 cell uptake of a GRK3 antisense oligonucleotide (ODN), but not of a random or mismatched ODN, reduced GRK3 mRNA expression by approximately 50% without altering GRK2 mRNA expression and inhibited homologous desensitization of CRF1 receptors by approximately 55%. Finally, Y-79 cells transfected with a GRK3 antisense cDNA construct exhibited an approximately 50% reduction in GRK3 protein expression and an ~65% reduction in homologous desensitization of CRF1 receptors. We conclude that GRK3 contributes importantly to the homologous desensitization of CRF1 receptors in Y-79 cells, a brain-derived cell line.     

The C-terminus of the metabotropic glutamate receptor 1b regulates dimerization of the receptor

The Group C G protein-coupled receptors include the metabotropic glutamate receptors (mGluRs), the GABA_B receptor, the calcium sensor and several taste receptors, most of which are obligate dimers, indeed recent work has shown that dimerization is necessary for the activation of these receptors. Consequently factors that regulate their ability to homo- or heterodimerize are important. The Group 1 mGluRs include mGluR1 and mGluR5 both of which have splice variants with altered C-termini. In this study, we show that mGluR1b is a dimer and that it does not efficiently heterodimerize with mGluR1a, unlike the two splice variants of mGluR5 that can heterodimerize. Mutation of a positively charged motif (RRKK) at the C-terminus of the mGluR1b tail permits mGluR1b to heterodimerize with mGluR1a. Co-expression of mGluR1a and mGluR1b in COS-7 cells results in the accumulation of mGluR1b in intracellular inclusions that do not contain mGluR1a. This behaviour is mimicked by a chimera of the lymphocyte antigen CD2 with the C-terminus of mGluR1b (pCD1b) and depends on the presence of the RRKK motif. These accumulations are immunoreactive for endoplasmic reticulum (ER) markers, but not Golgi and ERGIC markers. This segregation of mGluR1b from other ER proteins may contribute to its failure to dimerize with mGluR1a.     

Structure of the metabotropic glutamate receptor

In the twelve years since the molecular elucidation of the metabotropic glutamate receptor subtype 1, a class III family of G-protein-coupled receptors has emerged; members of this family include the calcium-sensing receptor, the GABA(B) receptor, some odorant receptors and some taste receptors. Atomic structures of the ligand-binding core of the original metabotropic glutamate receptor 1 obtained using X-ray crystallography provide a foundation for determining the initial receptor activation of this important family of G-protein-coupled receptors.     

Pias1 interaction and sumoylation of metabotropic glutamate receptor 8

Group III presynaptic metabotropic glutamate receptors (mGluRs) play a central role in regulating presynaptic activity through G-protein effects on ion channels and signal transducing enzymes. Like all Class C G-protein-coupled receptors, mGluR8 has an extended intracellular C-terminal domain (CTD) presumed to allow for modulation of downstream signaling. In a yeast two-hybrid screen of an adult rat brain cDNA library with the CTDs of mGluR8a and 8b (mGluR8-C) as baits, we identified sumo1 and four different components of the sumoylation cascade (ube2a, Pias1, Piasgamma, Piasxbeta) as interacting proteins. Binding assays using recombinant GST fusion proteins confirmed that Pias1 interacts not only with mGluR8-C but also with all group III mGluR CTDs. Pias1 binding to mGluR8-C required a region N-terminal to a consensus sumoylation motif and was not affected by arginine substitution of the conserved lysine 882 within this motif. Co-transfection of fluorescently tagged mGluR8a-C, sumo1, and enzymes of the sumoylation cascade into HEK293 cells showed that mGluR8a-C can be sumoylated in vivo. Arginine substitution of lysine 882 within the consensus sumoylation motif, but not other conserved lysines within the CTD, abolished in vivo sumoylation. Our results are consistent with post-translational sumoylation providing a novel mechanism of group III mGluR regulation.     

Negative cooperativity of glutamate binding in the dimeric metabotropic glutamate receptor subtype 1

Metabotropic glutamate receptor (mGluR) subtype 1 is a Class III G-protein-coupled receptor that is mainly expressed on the post-synaptic membrane of neuronal cells. The receptor has a large N-terminal extracellular ligand binding domain that forms a homodimer, however, the intersubunit communication of ligand binding in the dimer remains unknown. Here, using the intrinsic tryptophan fluorescence change as a probe for ligand binding events, we examined whether allosteric properties exist in the dimeric ligand binding domain of the receptor. The indole ring of the tryptophan 110, which resides on the upper surface of the ligand binding pocket, sensed the ligand binding events. From saturation binding curves, we have determined the apparent dissociation constants (K(0.5)) of representative agonists and antagonists for this receptor (3.8, 0.46, 40, and 0.89 microm for glutamate, quisqualate, (S)-alpha-methyl-4-carboxyphenylglycine ((S)-MCPG), and (+)-2-methyl-4-carboxyphenylglycine (LY367385), respectively). Calcium ions functioned as a positive modulator for agonist but not for antagonist binding (K(0.5) values were 1.3, 0.21, 59, and 1.2 microm for glutamate, quisqualate, (S)-MCPG, and LY367385, respectively, in the presence of 2.0 mm calcium ion). Moreover, a Hill analysis of the saturation binding curves revealed the strong negative cooperativity of glutamate binding between each subunit in the dimeric ligand binding domain. As far as we know, this is the first direct evidence that the dimeric ligand binding domain of mGluR exhibits intersubunit cooperativity of ligand binding.     

Ligand-induced rearrangement of the dimeric metabotropic glutamate receptor 1alpha

The extracellular domain of the metabotropic glutamate receptor 1alpha (mGluR1alpha) forms a dimer and the ligand, glutamate, induces a structural rearrangement in this domain. However, the conformational change in the cytoplasmic domain, which is critical for mGluR1alpha's interaction with G proteins, remains unclear. Here we investigated the ligand-induced conformational changes in the cytoplasmic domain by fluorescence resonance energy transfer (FRET) analysis of mGluR1alpha labeled with fluorescent protein(s) under total internal reflection field microscopy. Upon ligand binding, the intersubunit FRET efficiency between the second loops increased, whereas that between first loops decreased. In contrast, the intrasubunit FRET did not change clearly. These results show that ligand binding does not change the structure of each subunit, but does change the dimeric allocation of the cytoplasmic regions, which may underlie downstream signaling.     

Expression of G-protein-coupled receptor kinase 6 (GRK6) after acute spinal cord injury in adult rat

Spinal cord injury frequently results in permanent loss of neurological function. It includes many complex molecular and biochemical mechanisms. G-protein-coupled receptor kinase 6 (GRK6) is an intracellular kinase that regulates the sensitivity of certain G-protein-coupled receptors. Some studies reported GRK2 and GRK5 modulate the NFκB pathway in macrophages. Additionally, GRK2 is referred to as regulating activation of spinal cord microglia and GRK6 expression is significantly elevated in most brain regions in the MPTP-lesioned parkinsonian monkeys. However, the expression and function of GRK6 in nervous system lesion and repair are not well understood. In this study, we performed an acute spinal cord injury (SCI) model in adult rats. Western blot analysis showed the expression of GRK6 was upregulated significantly at protein level in spinal cord after SCI. Immunohistochemistry and immunofluorescence revealed wide expression of GRK6 in the normal spinal cord. After injury, GRK6 expression was increased predominantly in microglia, which expressed F4/80 (marker of macrophages and activated microglia) strongly. To understand whether GRK6 played a role in microglia activation, we applied lipopolysaccharide (LPS) to induce microglia activation in vitro. Western blot analysis demonstrated up-regulation in GRK6 protein expression after LPS stimulation was time- and dose-dependent and that up-regulation in F4/80 expression was concomitant with GRK6. These data suggested that GRK6 might be involved in the pathophysiology of SCI.     

Phosphorylation-independent regulation of metabotropic glutamate receptor 1 signaling requires g protein-coupled receptor kinase 2 binding to the second intracellular loop

Metabotropic glutamate receptors (mGluRs) are members of a unique class of G protein-coupled receptors (class III) that include the calcium-sensing and gamma-aminobutyric acid type B receptors. The activity of mGluRs is regulated by second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). The attenuation of both mGluR1a and mGluR1b signaling by GRK2 is phosphorylation- and beta-arrestin-independent and requires the concomitant association of GRK2 with both the receptor and Galpha(q/11). G protein interactions are mediated, in part, by the mGluR1 intracellular second loop, but the domains required for GRK2 binding are unknown. In the present study, we showed that GRK2 binds to the second intracellular loop of mGluR1a and mGluR1b and also to the mGluR1a carboxyl-terminal tail. Alanine scanning mutagenesis revealed a discrete domain within loop 2 that contributes to GRK2 binding, and the mutation of either lysine 691 or 692 to an alanine within this domain resulted in a loss of GRK2 binding to both mGluR1a and mGluR1b. Mutation of either Lys(691) or Lys(692) prevented GRK2-mediated attenuation of mGluR1b signaling, whereas the mutation of only Lys(692) prevented GRK2-mediated inhibition of mGluR1a signaling. Thus, the mGluR1a carboxyl-terminal tail may also be involved in regulating the signaling of the mGluR1a splice variant. Taken together, our findings indicated that kinase binding to an mGluR1 domain involved in G protein-coupling is essential for the phosphorylation-independent attenuation of signaling by GRK2.     

Protein kinase A directly phosphorylates metabotropic glutamate receptor 5 to modulate its function

Metabotropic glutamate receptor 5 (mGluR5) regulates excitatory post‐synaptic signaling in the central nervous system (CNS) and is implicated in various CNS disorders. Protein kinase A (PKA) signaling is known to play a critical role in neuropsychiatric disorders such as Parkinson's disease, schizophrenia, and addiction. Dopamine signaling is known to modulate the properties of mGluR5 in a cAMP‐ and PKA‐dependent manner, suggesting that mGluR5 may be a direct target for PKA. Our study identifies mGluR5 at Ser870 as a direct substrate for PKA phosphorylation and demonstrates that this phosphorylation plays a critical role in the PKA‐mediated modulation of mGluR5 functions such as extracellular signal‐regulated kinase phosphorylation and intracellular Ca²⁺ oscillations. The identification of the molecular mechanism by which PKA signaling modulates mGluR5‐mediated cellular responses contributes to the understanding of the interaction between dopaminergic and glutamatergic neuronal signaling.

     

Membrane topology of a metabotropic glutamate receptor

The metabotropic glutamate receptors (mGluRs) have been predicted to have a classical seven transmembrane domain structure similar to that seen for members of the G-protein-coupled receptor (GPCR) superfamily. However, the mGluRs (and other members of the family C GPCRs) show no sequence homology to the rhodopsin-like GPCRs, for which this seven transmembrane domain structure has been experimentally confirmed. Furthermore, several transmembrane domain prediction algorithms suggest that the mGluRs have a topology that is distinct from these receptors. In the present study, we set out to test whether mGluR5 has seven true transmembrane domains. Using a variety of approaches in both prokaryotic and eukaryotic systems, our data provide strong support for the proposed seven transmembrane domain model of mGluR5. We propose that this membrane topology can be extended to all members of the family C GPCRs.