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.     

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.     

The second intracellular loop of metabotropic glutamate receptors recognizes C termini of G-protein alpha-subunits

Heptahelical receptor coupling selectivity to G-proteins is controlled by a large contact area that involves several portions of the receptor and each subunit of the G-protein. In the G-protein alpha subunit, the C-terminal 5 residues, the N terminus, and the alpha N-beta 1 and alpha 4-alpha 5 loops play important roles. On the receptor side, both the second and third (i2 and i3) intracellular loops as well as the C-terminal tail probably contact these different regions of the G-protein. It is now accepted that the C terminus of the alpha subunit binds in a cavity formed by the i2 and i3 loops. Among the various G-protein-coupled receptors (GPCRs), class III receptors that include metabotropic glutamate (mGlu) receptors greatly differ from the rhodopsin-like GPCRs, but the contact zone between these receptors and the G-protein is less understood. The C terminus of the alpha subunit has been shown to play a pivotal role in the selective recognition of class III GPCRs. Indeed, the mGlu2 and mGlu4 and -8 receptors can discriminate between alpha subunits that differ at the level of their C-terminal end only (such as Gqo and Gqz). Here, we examine the role of the i2 loop of mGluRs in the selective recognition of this region of the alpha subunit. To that aim, we analyzed the coupling properties of mGlu2 and mGlu4 or -8 receptors and chimeras containing the i2 loop of the converse receptor to G-protein alpha subunits that only differ by their C termini (Gqo,Gqz, and their point mutants). Our data demonstrate that the central portion of the i2 loop is responsible for the selective recognition of the C-terminal end of the alpha subunit, especially the residue on position -4. These data are consistent with the proposal that the C-terminal end of the G-protein alpha subunit interacts with residues in a cavity formed by the i2 and i3 loops in class III GPCRs, as reported for class I GPCRs.     

Protein kinase C isoform-specific differences in the spatial-temporal regulation and decoding of metabotropic glutamate receptor1a-stimulated second messenger responses

Metabotropic glutamate receptors (mGluRs) coupled via Gq to the hydrolysis of phosphoinositides stimulate Ca(2+) and PKCbetaII oscillations in both excitable and non-excitable cells. In the present study, we show that mGluR1a activation stimulates the repetitive plasma membrane translocation of each of the conventional and novel, but not atypical, PKC isozymes. However, despite similarities in sequence and cofactor regulation by diacyglycerol and Ca(2+), conventional PKCs exhibit isoform-specific oscillation patterns. PKCalpha and PKCbetaI display three distinct patterns of activity: (1) agonist-independent oscillations, (2) agonist-stimulated oscillations, and (3) persistent plasma membrane localization in response to mGluR1a activation. In contrast, only agonist-stimulated PKCbetaII translocation responses are observed in mGluR1a-expressing cells. PKCbetaI expression also promotes persistent increases in intracellular diacyglycerol concentrations in response to mGluR1a stimulation without affecting PKCbetaII oscillation patterns in the same cell. PKCbetaII isoform-specific translocation patterns are regulated by specific amino acid residues localized within the C-terminal PKC V5 domain. Specifically, Asn-625 and Lys-668 localized within the V5 domain of PKCbetaII cooperatively suppress PKCbetaI-like response patterns for PKCbetaII. Thus, redundancy in PKC isoform expression and differential decoding of second messenger response provides a novel mechanism for generating cell type-specific responses to the same signal.     

The second cytoplasmic loop of metabotropic glutamate receptor functions at the third loop position of rhodopsin.

G protein-coupled receptors identified so far are classified into at least three major families based on their amino acid sequences. For the family of receptors homologous to rhodopsin (family 1), the G protein activation mechanism has been investigated in detail, but much less for the receptors of other families. To functionally compare the G protein activation mechanism between rhodopsin and metabotropic glutamate receptor (mGluR), which belong to distinct families, we prepared a set of bovine rhodopsin mutants whose second or third cytoplasmic loop was replaced with either the second or third loop of Gi/Go- or Gq-coupled mGluR (mGluR6 or mGluR1). Among these mutants, the mutants in which the second or third loop was replaced with the corresponding loop of mGluR exhibited no G protein activation ability. In contrast, the mutant whose third loop was replaced with the second loop of Gi/Go-coupled mGluR6 efficiently activated Gi but not Gt: this activation profile is almost identical with those of the mutant rhodopsins whose third loop was replaced with those of the Gi/Go-coupled receptors in family 1 [Yamashita et al. (2000) J. Biol. Chem. 275, 34272-34279]. The mutant whose third loop was replaced with the second loop of Gq-coupled mGluR1 partially retained the Gi coupling ability of rhodopsin, which is in contrast to the fact that all the rhodopsin mutants having the third loops of Gq-coupled receptors in family 1 exhibit no detectable Gi activation. These results strongly suggest that the molecular architectures of rhodopsin and mGluR are different, although the G protein activation mechanism involving the cytoplasmic loops is common.     

A novel constitutively active mutation in the second cytoplasmic loop of metabotropic glutamate receptor

G protein-coupled receptors have a common structural motif of seven transmembrane alpha-helices and are classified into different families showing no sequence similarity. Extensive studies have been conducted on the structure-function relationship in family 1 receptors, but those in other families have not been well studied. In this study, to investigate the molecular basis leading to the G protein activation by metabotropic glutamate receptor (mGluR), the member of family 3, we searched for the amino acid residues responsible for the G protein activation in the second cytoplasmic loop, which was thought to be the main G protein binding region. Analyses of the systematical mutations of Gi/Go-coupled mGluR8 revealed the presence of a constitutively active mutation in the C-terminal region of the second loop. The corresponding mutation in the second loop of Gq-coupled mGluR1 also exhibited high agonist-independent activity. These results indicate that there is a common constitutive active mutation site regardless of mGluR subtypes, suggesting that the structural change of the junction between the second cytoplasmic loop and helix IV is strongly linked to the formation of the active state.     

G Protein-coupled receptor kinase 2 regulator of G protein signaling homology domain binds to both metabotropic glutamate receptor 1a and Galphaq to attenuate signaling

Heterotrimeric guanine nucleotide-binding (G) protein-coupled receptor kinases (GRKs) are cytosolic proteins that contribute to the adaptation of G protein-coupled receptor signaling. The canonical model for GRK-dependent receptor desensitization involves GRK-mediated receptor phosphorylation to promote the binding of arrestin proteins that sterically block receptor coupling to G proteins. However, GRK-mediated desensitization, in the absence of phosphorylation and arrestin binding, has been reported for metabotropic glutamate receptor 1 (mGluR1) and gamma-aminobutyric acid B receptors. Here we show that GRK2 mutants impaired in Galphaq/11 binding (R106A, D110A, and M114A), bind effectively to mGluR1a, but do not mediate mGluR1a adaptation. Galphaq/11 is immunoprecipitated as a complex with mGluR1a in the absence of agonist, and either agonist treatment or GRK2 overexpression promotes the dissociation of the receptor/Galphaq/11 complex. However, these mGluR1a/Galphaq/11 interactions are not antagonized by the overexpression of either GRK2 mutants defective in Galphaq/11 binding or RGS4. We have also identified a GRK2-D527A mutant that binds Galphaq/11 in an AlF4(-)-dependent manner but is unable to either bind mGluR1a or attenuate mGluR1a signaling. We conclude that the mechanism underlying GRK2 phosphorylation-independent attenuation of mGluR1a signaling is RH domain-dependent, requiring the binding of GRK2 to both Galphaq/11 and mGluR1a. This serves to coordinate GRK2 interactions with Galphaq/11 and to disrupt receptor/Galphaq/11 complexes. Our findings indicate that GRK2 regulates receptor/G protein interactions, in addition to its traditional role as a receptor kinase.     

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.     

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.     

Structural determinants of calmodulin binding to the intracellular C-terminal domain of the metabotropic glutamate receptor 7A

Calmodulin (CaM) binds in a Ca2+-dependent manner to the intracellular C-terminal domains of most group III metabotropic glutamate receptors (mGluRs). Here we combined mutational and biophysical approaches to define the structural basis of CaM binding to mGluR 7A. Ca2+/CaM was found to interact with mGluR 7A primarily via its C-lobe at a 1:1 CaM:C-tail stoichiometry. Pulldown experiments with mutant CaM and mGluR 7A C-tail constructs and high resolution NMR with peptides corresponding to the CaM binding region of mGluR 7A allowed us to define hydrophobic and ionic interactions required for Ca2+/CaM binding and identified a 1-8-14 CaM-binding motif. The Ca2+/CaM.mGluR 7A peptide complex displays a classical wraparound structure that closely resembles that formed by Ca2+/CaM upon binding to smooth muscle myosin light chain kinase. Our data provide insight into how Ca2+/CaM regulates group III mGluR signaling via competition with intracellular proteins for receptor-binding sites.     

Selective regulation of H1 histamine receptor signaling by G protein-coupled receptor kinase 2 in uterine smooth muscle cells

Histamine stimulates uterine contraction; however, little is known regarding the mechanism or regulation of uterine histamine receptor signaling. Here we investigated the regulation of Galpha(q/11)-coupled histamine receptor signaling in human myometrial smooth muscle cells using the inositol 1,4,5-trisphosphate biosensor pleckstrin homology domain of phospholipase-delta1 tagged to enhanced green fluorescent protein and the Ca(2+)-sensitive dye Fluo-4. Histamine addition caused concentration-dependent increases in inositol 1,4,5-trisphosphate and [Ca(2+)](i) in the ULTR human uterine smooth muscle cell line and primary human myometrial cells. These effects were completely inhibited by the H(1) histamine receptor antagonist, diphenhydramine, and were unaffected by the H(2) histamine receptor antagonist, cimetidine. ULTR and primary myometrial cells were transfected with either dominant-negative G protein-coupled receptor kinases (GRKs) or small interfering RNAs targeting specific GRKs to assess the roles of this protein kinase family in H(1) histamine receptor desensitization. Dominant-negative GRK2, but not GRK5 or GRK6, prevented H(1) histamine receptor desensitization. Similarly, transfection with short interfering RNAs (that each caused >70% depletion of the targeted GRK) for GRK2, but not GRK3 or GRK6, also prevented H(1) histamine receptor desensitization. Our data suggest that histamine stimulates phospholipase C-signaling in myometrial smooth muscle cells through H(1) histamine receptors and that GRK2 recruitment is a key mechanism in the regulation of H(1) histamine receptor signaling in human uterine smooth muscle. These data provide insights into the in situ regulation of this receptor subtype and may inform pathophysiological functioning in preterm labor and other conditions involving uterine smooth muscle dysregulation.     

Agonist-induced internalization of metabotropic glutamate receptor 1A: structural determinants for protein kinase C- and G protein-coupled receptor kinase-mediated internalization

To investigate the role of the intracellular C-terminal tail of the rat metabotropic glutamate receptor 1a (mGlu1a) in receptor regulation, we constructed three C-terminal tail deletion mutants (Arg847stop, DM-I; Arg868stop, DM-II; Val893stop, DM-III). Quantification of glutamate-induced internalization provided by ELISA indicated that DM-III, like the wild-type mGlu1a, underwent rapid internalization whilst internalization of DM-I and DM-II was impaired. The selective inhibitor of protein kinase C (PKC), GF109203X, which significantly reduced glutamate-induced mGlu1a internalization, had no effect on the internalization of DM-I, DM-II, or DM-III. In addition activation by carbachol of endogenously expressed M1 muscarinic acetylcholine receptors, which induces PKC- and Ca2+-calmodulin-dependent protein kinase II-dependent internalization of mGlu1a, produced negligible internalization of the deletion mutants. Co-expression of a dominant negative mutant form of G protein-coupled receptor kinase 2 (DNM-GRK2; Lys220Arg) significantly attenuated glutamate-induced internalization of mGlu1a and DM-III, whilst internalization of DM-I and DM-II was not significantly affected. The glutamate-induced internalization of mGlu1a and DM-III, but not of DM-I or DM-II, was inhibited by expression of DNM-arrestin [arrestin-2(319-418)]. In addition glutamate-induced rapid translocation of arrestin-2-Green Fluorescent Protein (arr-2-GFP) from cytosol to membrane was only observed in cells expressing mGlu1a or DM-III. Functionally, in cells expressing mGlu1a, glutamate-stimulated inositol phosphate accumulation was increased in the presence of PKC inhibition, but so too was that in cells expressing DM-II and DM-III. Together these results indicate that different PKC mechanisms regulate the desensitization and internalization of mGlu1a. Furthermore, PKC regulation of mGlu1a internalization requires the distal C terminus of the receptor (Ser894-Leu1199), whilst in contrast glutamate-stimulated GRK- and arrestin-dependent regulation of this receptor depends on a region of 25 amino acids (Ser869-Val893) in the proximal C-terminal tail.     

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.     

Bimodal regulation of the human H1 histamine receptor by G protein-coupled receptor kinase 2

The H1 histamine receptor (H1HR) is a member of the G protein-coupled receptor superfamily and regulates numerous cellular functions through its activation of the G(q/11) subfamily of heterotrimeric G proteins. Although the H1HR has been shown to undergo desensitization in multiple cell types, the mechanisms underlying the regulation of H1HR signaling are poorly defined. To address this issue, we examined the effects of wild type and mutant G protein-coupled receptor kinases (GRKs) on the phosphorylation and signaling of human H1HR in HEK293 cells. Overexpression of GRK2 promoted H1HR phosphorylation in intact HEK293 cells and completely inhibited inositol phosphate production stimulated by H1HR, whereas GRK5 and GRK6 had lesser effects on H1HR phosphorylation and signaling. Interestingly, catalytically inactive GRK2 (GRK2-K220R) also significantly attenuated H1HR-mediated inositol phosphate production, as did an N-terminal fragment of GRK2 previously characterized as a regulator of G protein signaling (RGS) protein for Galpha(q/11). Disruption of this RGS function in holo-GRK2 by mutation (GRK2-D110A) partially reversed the quenching effect of GRK2, whereas deletion of both the kinase activity and RGS function (GRK2-D110A/K220R) effectively relieved the inhibition of inositol phosphate generation. To evaluate the role of endogenous GRKs on H1HR regulation, we used small interfering RNAs to selectively target GRK2 and GRK5, two of the primary GRKs expressed in HEK293 cells. A GRK2-specific small interfering RNA effectively reduced GRK2 expression and resulted in a significant increase in histamine-promoted calcium flux. In contrast, knockdown of GRK5 expression was without effect on H1HR signaling. These findings demonstrate that GRK2 is the principal kinase mediating H1 histamine receptor desensitization in HEK293 cells and suggest that rapid termination of H1HR signaling is mediated by both the kinase activity and RGS function of GRK2.     

Proteomic analysis reveals novel binding partners of metabotropic glutamate receptor 1

Regulated trafficking of neurotransmitter receptors is critical to normal neurodevelopment and neuronal signaling. Group I mGluRs (mGluR1/5 and their splice variants) are G protein-coupled receptors enriched at excitatory synapses, where they serve to modulate glutamatergic transmission. The mGluR1 splice variants mGluR1a and mGluR1b are broadly expressed in the central nervous system and differ in their signaling and trafficking properties. Several proteins have been identified that selectively interact with mGluR1a and participate in receptor trafficking but no proteins interacting with mGluR1b have thus far been reported. We have used a proteomic strategy to isolate and identify proteins that co-purify with mGluR1b in Madin-Darby Canine Kidney (MDCK) cells, an established model system for trafficking studies. Here, we report the identification of 10 novel candidate mGluR1b-interacting proteins. Several of the identified proteins are structural components of the cell cytoskeleton, while others serve as cytoskeleton-associated adaptors and motors or endoplasmic reticulum-associated chaperones. Findings from this work will help unravel the complex cellular mechanisms underlying mGluR trafficking under physiological and pathological conditions.     

A relationship between protein kinase C phosphorylation and calmodulin binding to the metabotropic glutamate receptor subtype 7.

Metabotropic glutamate receptor subtype 7 (mGluR7) is coupled to the inhibitory cyclic AMP cascade and is selectively activated by a glutamate analogue, L-2-amino-4-phosphonobutyrate. Among L-2-amino-4-phosphonobutyrate-sensitive mGluR subtypes, mGluR7 is highly concentrated at the presynaptic terminals and is thought to play an important role in modulation of glutamatergic synaptic transmission by presynaptic inhibition of glutamate release. To gain further insight into the intracellular signaling mechanisms of mGluR7, with the aid of glutathione S-transferase fusion affinity chromatography, we attempted to identify proteins that interact with the intracellular carboxyl terminus of mGluR7. Here, we report that calmodulin (CaM) directly binds to the carboxyl terminus of mGluR7 in a Ca(2+)-dependent manner. The CaM-binding domain is located immediately following the 7th transmembrane segment. We also show that the CaM-binding domain of mGluR7 is phosphorylated by protein kinase C (PKC). This phosphorylation is inhibited by the binding of Ca(2+)/CaM to the receptor. Conversely, the Ca(2+)/CaM binding is prevented by PKC phosphorylation. Collectively, these results suggest that mGluR7 serves to cross-link the cyclic AMP, Ca(2+), and PKC phosphorylation signal transduction cascades.     

Regulation of g protein-coupled receptor signaling by a-kinase anchoring proteins

Specificity of transduction events is controlled at the molecular level by scaffold, anchoring, and adaptor proteins, which position signaling enzymes at proper subcellular localization. This allows their efficient catalytic activation and accurate substrate selection. A-kinase anchoring proteins (AKAPs) are group of functionally related proteins that compartmentalize the cAMP-dependent protein kinase (PKA) and other signaling enyzmes at precise subcellular sites in close proximity to their physiological substrate(s) and favor specific phosphorylation events. Recent evidence suggests that AKAP transduction complexes play a key role in regulating G protein-coupled receptor (GPCR) signaling. Regulation can occur at multiple levels because AKAPs have been shown both to directly modulate GPCR function and to act as downstream effectors of GPCR signaling. In this minireview, we focus on the molecular mechanisms through which AKAP-signaling complexes modulate GPCR transduction cascades.     

The G-protein-coupled receptor kinase GRK4 mediates homologous desensitization of metabotropic glutamate receptor 1.

G-protein-coupled receptor kinases (GRKs) are involved in the regulation of many G-protein-coupled receptors. As opposed to the other GRKs, such as rhodopsin kinase (GRK1) or beta-adrenergic receptor kinase (beta ARK, GRK2), no receptor substrate for GRK4 has been so far identified. Here we show that GRK4 is expressed in cerebellar Purkinje cells, where it regulates mGlu(1) metabotropic glutamate receptors, as indicated by the following: 1) When coexpressed in heterologous cells (HEK293), mGlu(1) receptor signaling was desensitized by GRK4 in an agonist-dependent manner (homologous desensitization). 2) In transfected HEK293 and in cultured Purkinje cells, the exposure to glutamate agonists induced internalization of the receptor and redistribution of GRK4. There was a substantial colocalization of the receptor and kinase both under basal condition and after internalization. 3) Kinase activity was necessary for desensitizing mGlu(1a) receptor and agonist-dependent phosphorylation of this receptor was also documented. 4) Antisense treatment of cultured Purkinje cells, which significantly reduced the levels of GRK4 expression, induced a marked modification of the mGlu(1)-mediated functional response, consistent with an impaired receptor desensitization. The critical role for GRK4 in regulating mGlu(1) receptors implicates a major involvement of this kinase in the physiology of Purkinje cell and in motor learning.     

Inhibition of metabotropic glutamate receptor signaling by the huntingtin-binding protein optineurin

Huntington disease is caused by a polyglutamine expansion in the huntingtin protein (Htt) and is associated with excitotoxic death of striatal neurons. Group I metabotropic glutamate receptors (mGluRs) that are coupled to inositol 1,4,5-triphosphate formation and the release of intracellular Ca(2+) stores play an important role in regulating neuronal function. We show here that mGluRs interact with the Htt-binding protein optineurin that is also linked to normal pressure open angled glaucoma and, when expressed in HEK 293 cells, optineurin functions to antagonize agonist-stimulated mGluR1a signaling. We find that Htt is co-precipitated with mGluR1a and that mutant Htt functions to facilitate optineurin-mediated attenuation of mGluR1a signaling. In striatal cell lines derived from Htt(Q111/Q111) mutant knock-in mice mGluR5-stimulated inositol phosphate formation is also severely impaired when compared with striatal cells derived from Htt(Q7/Q7) knock-in mice. In addition, we show that a missense single nucleotide polymorphism optineurin H486R variant previously identified to be associated with glaucoma is selectively impaired in mutant Htt binding. Although optineurin H486R retains the capacity to bind to mGluR1a, optineurin H486R-dependent attenuation of mGluR1a signaling is not enhanced by the expression of mutant Htt. Because G protein-coupled receptor kinase 2 (GRK2) protein expression is relatively low in striatal tissue, we propose that optineurin may substitute for GRK2 in the striatum to mediate mGluR desensitization. Taken together, these studies identify a novel mechanism for mGluR desensitization and an additional biochemical link between altered glutamate receptor signaling and Huntington disease.     

The C terminus of the metabotropic glutamate receptor subtypes 2 and 7 specifies the receptor signaling pathways

There is accumulating evidence that the specificity of the transduction cascades activated by G protein-coupled receptors cannot solely depend on the nature of the coupled G protein. To identify additional structural determinants, we studied two metabotropic glutamate (mGlu) receptors, the mGlu2 and mGlu7 receptors, that are both coupled to G(o) proteins but are known to affect different effectors in neurons. Thus, the mGlu2 receptor selectively blocks N- and L-type Ca(2+) channels via a protein kinase C-independent pathway, whereas the mGlu7 receptor selectively blocks P/Q-type Ca(2+) channels via a protein kinase C-dependent pathway, and both effects are pertussis toxin-sensitive. We examined the role of the C-terminal domain of these receptors in this coupling. Chimeras were constructed by exchanging the C terminus of these receptors and transfected into neurons. Different chimeric receptors bearing the C terminus of mGlu7 receptor blocked selectively P/Q-type Ca(2+) channels, whereas chimeras bearing the C terminus of mGlu2 receptor selectively blocked N- and L-type Ca(2+) channels. These results show that the C terminus of mGlu2 and mGlu7 receptors is a key structural determinant that allows these receptors to select a specific signaling pathway in neurons.