Regulation of mglu7 receptors by proteins that interact with the intracellular C-terminus

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Regulation of mglu7 receptors by proteins that interact with the intracellular C-terminus Kumlesh K. Dev, Shigetada Nakanishi and Jeremy M. Henley The metabotropic glutamate type 7 (mglu7) receptor is a widely distributed, mainly presynaptic Group III mglu receptor that can regulate glutamate release. Recently, largely as a result of the identification of specific proteins that interact with the C-terminal domain of this receptor, considerable progress has been made towards understanding some of the mechanisms that underlie the regulation, signal transduction pathways and targeting of mglu7 receptors. This has led to the proposal that there are three distinct functionally relevant domains present in the intracellular C-terminus of this receptor: (1) a proximal intracellular signalling domain that interacts with G-protein βγ-subunits and the Ca2++ sensor Ca2++–calmodulin, and is phosphorylated by protein kinase; (2) a central domain thought to provide a signal for axonal targeting; and (3) an extreme PDZ-binding motif that interacts with the protein kinase C interacting protein, PICK1.

Kumlesh K. Dev Novartis Pharma AG, Nervous System Research, CH-4002 Basel, Switzerland. Shigetada Nakanishi Dept of Biological Sciences, Kyoto University, Faculty of Medicine, Kyoto, 606-8501, Japan. Jeremy M. Henley* Dept of Anatomy, MRC Centre of Synaptic Plasticity, Medical School, University of Bristol, Bristol, UK BS8 1TD. *e-mail: [email protected]

Metabotropic glutamate (mglu) receptors mediate their effects on ion channels and second messenger systems via G proteins1,2. Eight subtypes of mglu receptor (mglu1–mglu8) have been classified into three distinct groups3. Group I comprises the subtypes mglu1 and mglu5, which are coupled to phospholipase C (PLC), stimulate protein kinase C (PKC) and cause the release of Ca2+ from intracellular stores. By contrast, Group II (mglu2 and mglu3) and Group III (mglu4 and mglu6–mglu8) receptors are both negatively coupled to the cAMP cascade but are distinguished by their differing agonist–antagonist profiles and sequence homologies. However, recently, activation of mglu7 receptors has been shown to stimulate PLC in addition to reducing cAMP levels4. The different mglu receptor subtypes are differentially distributed in the mammalian brain and, depending on the subtype, can be localized extrasynaptically, presynaptically and postsynaptically, or show both pre- and postsynaptic localization. Functional roles of mglu7 receptors

Over recent years, an increasing number of proteins have been identified that interact with the C-termini of mglu receptors. There is now considerable evidence to suggest that the synaptic distributions and functional properties of mglu receptors can be regulated via these interactions. An overview of the C-terminal domains of mglu receptor splice variants, and the importance of both C-terminus splice variation and putative PDZ-binding motifs in specifying proteins that interact with mglu receptors are shown in Box 1. http://tips.trends.com

Group I mglu receptors (mglu1–mglu5) have been shown to interact with the Homer family of proteins. Homers contain an EVH domain that interacts with a proline-rich sequence (PPxxFR) located at the extreme C-termini of both Group I mglu receptors and the inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] receptor. Homer proteins can also dimerize via a coiled-coil motif, which allows Group I mglu receptors and Ins(1,4,5)P3 receptors to come into close proximity. Potentially, this provides a link between surface membrane Group I mglu receptors and the intracellular Ca2+-store Ins(1,4,5)P3 receptor5,6. The Ca2+ sensor Ca2+– calmodulin (CaM) has also been reported to interact with mglu5 receptors in a Ca2+dependent manner7. CaM binds at two distinct sites on the C-terminus of the mglu5 receptor (e.g. residues Val842–Arg869 and Lys922–Lys950 in mglu5b receptors). The binding of CaM is inhibited by PKC phosphorylation of the receptor and, in turn, phosphorylation is inhibited by the binding of CaM (Ref. 7). The C-terminal regions of Group I mglu receptors also interact with Siah-1A, a mammalian homologue of Drosophila seven in absentia8. This protein is involved in the differentiation of photoreceptor cells via a ubiquitin–proteasomedependent mechanism. The interaction occurs between the latter two-thirds of Siah-1A and the CaM-binding sites found in the C-termini of Group I mglu receptors (e.g. residues Lys905–Pro932 in the mglu1a receptor and Arg892–Pro918 in the mglu5a receptor)8. The binding of Siah-1A and CaM is competitive (Box 1). No proteins have yet been reported to interact directly with the C-termini of Group II mglu receptors (mglu2,3) (Box 1). Some proteins similar to those that interact with Group I mglu receptors have been shown to interact with mglu7 receptors, a representative of Group III mglu receptors. Although Homers do not interact with mglu7 receptors, other proteins have been identified that appear to be involved in the functional regulation and/or trafficking of mglu7 receptors. These include the G-protein βγ-subunits, the protein that interacts with PKC (PICK1), CaM and PKC (Refs 9–13) (Box 1 and Fig. 1). The mglu7 receptor has two alternative splice isoforms (mglu7a and mglu7b), of which the mglu7a receptor has been the most extensively studied. The

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Box 1. The importance of C-terminal regions of mglu receptors for binding proteins The C-terminal domains of Binding proteins Bindings sites on C-terminus Size of C-terminus metabotropic glutamate (mglu) Group I receptors and their a (α) K841–L1199 (359 residues) STL* splice variants are b (β) K841–L906 (66 residues) AQL* ct-mglu1 Homer shown in Fig. I. TVY* c K841–Y897 (57 residues) mglu1 receptors DGL* K841–L912 (72 residues) d CaM or are identical up to Siah-1A a K827–L1171 (345 residues) SSL* 46 residues into ct-mglu5 b K827–L1203 (377 residues) SSL* the C-terminus, and then differ in the following 313, Group II 20, 11 and 26 ct-mglu2 SSL* Q820–L872 (53 residues) residues in mglu1a, mglu1b, mglu1c and ct-mglu3 Q829–L879 (51 residues) SSL* mglu1d receptors, respectivelya–j. A Group III mglu1e receptor HAI* H848–I912 (65 residues) a splice form ct-mglu4 DGL* H848–L983 (136 residues) b encodes an PICK1 N-terminally ct-mglu6 H840–K871 (32 residues) DAK* truncated protein CaM or (not shown). LVI* H851–I915 (65 residues) a G protein ct-mglu7 Furthermore, a PTV* b H851–V922 (72 residues) mglu1f receptor H844–I908 (65 residues) HSI* splice isoform has ct-mglu8 a b H844–S908 (65 residues) STS* also been reported TRENDS in Pharmacological Sciences (not shown), which differs from the mglu1b receptor by Fig. I. The C-terminal domains of metabotropic glutamate (ct-mglu) receptor splice variants are shown, along with their interacting proteins. Asterisks denote stop codons, and the diagonal lines in ct-mglu1a, ct-mglu5a, ct-mglu5b receptors indicate sequence amino acids not shown. a deletion of Calmodulin (CaM) and Siah-1A bind competitively to ct-mglu1a, ct-mglu5a and ct-mglu5b, and CaM and G proteins bind competitively to ct-mglu7a 35 base pairs; and ct-mglu7b. however, this deletion occurs after the stop codon and (ct-mglu4a) receptor are replaced by 135 new seventh transmembrane domain, the thus the protein sequence of the mglu1f residues in the mglu4b receptor (Refs j,m–p). mglu5b receptor has a 32-amino-acid insertion in the mglu5a receptor proteink,l. Replacement of the last 16 amino acids of receptor remains the same as for the mglu1b The last 64 residues in the C-terminal mglu4a the mglu7a receptor by 23 different residues receptor. Forty-nine residues after the

splice variants of the mglu7 receptor differ by an out-offrame insertion of 92 nucleotides close to the C-terminus. The mglu7a receptor consists of 915 residues (the mglu7b receptor is seven amino acids longer) with a molecular weight of ~100 kDa but often migrates as a dimer on sodium dodecyl sulfate (SDS)–polyacrylamide gels11,14–16. The mglu7a receptor is widely distributed throughout the rat brain and shows a mainly presynaptic localization in asymmetric synapses14,15,17,18, being present at both glutamatergic and GABAergic synapses19,20. Consistent with its location predominantly at presynaptic active zones, the mglu7a receptor is thought to have an autoregulatory role in the inhibition of transmitter release at these synapses14,15,19,20. The lack of specific pharmacological tools has impeded the study of mglu7 receptor function but it is clear that this receptor represents an important http://tips.trends.com

potential therapeutic target because it plays a role in the regulation of normal synaptic activity. Consequently, impairment of mglu7 receptor responses could play a role in increased neuronal excitability and neurodegeneration. Consistent with this hypothesis, mglu7 receptor knockout mice exhibit increased cortical excitability, and lateonset epilepsy has been observed in mglu7 receptor knockout mice at 10–12 weeks of age (T.J. Bushell and H. van der Putten, pers. commun.). However, in the CA1 region of the hippocampus of mglu7 receptor knockout mice, there were no reductions in normal synaptic transmission, paired-pulse facilitation or long-term potentiation. Nevertheless, short-term potentiation was decreased in the knockout mice, which suggests that there is a reduction in high-frequency synaptic transmission. Studies using knockout mice also suggest that the

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generates the C-terminus of the mglu7b receptor p–t. An exchange of the extreme 16 residues of the mglu8a receptor with 16 different residues forms the mglu8b receptor (Refs t,u). Inter-species variation also exists in the C-termini of mglu receptors. For example, in humans, the mglu1a receptor has a sevenresidue deletion (QPQQPPPP) in the C-terminus compared with the rat protein. The human sequence of the mglu1b receptor ends with VQL whereas the rat sequence of this receptor splice variant ends with AQL. Furthermore, the rat mglu1d receptor is 912 residues long and ends with DGL, whereas the human mglu1d receptor is 908 amino acids in length and ends with residues EDQ. By contrast, for mglu2 (Refs j,v), mglu4, mglu7 and mglu8 receptors there is either none or only 1–3 residue differences between the C-terminal domains in rats and humans. Whether these differences or conservations in amino acids are important for mglu-receptor–protein interactions is, as yet, unclear. C-terminal splice variance is important in the determination of mglureceptor–protein interactions and where PDZ-domain-containing proteins can putatively bind (Fig. I). Homer, calmodulin (CaM) and Siah-1A have been identified as proteins that interact with Group I mglu receptors; no proteins have yet been reported to bind to the C-terminal domains of Group II mglu receptors. For Group III mglu receptors, ct-mglu7 has been reported to interact with PICK1, CaM and the G-protein βγ-subunits.

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References a Houamed, K.M. et al. (1991) Cloning, expression, and gene structure of a G proteincoupled glutamate receptor from rat brain. Science 252, 1318–1321 b Masu, M. et al. (1991) Sequence and expression of a metabotropic glutamate receptor. Nature 349, 760–765 c Pin, J-P. et al. (1992) Alternative splicing generates metabotropic glutamate receptors inducing different patterns of calcium release in Xenopus oocytes. Proc. Natl. Acad. Sci. U. S. A. 89, 10331–10335 d Mary, S. et al. (1998) A cluster of basic residues in the carboxyl-terminal tail of the short metabotropic glutamate receptor 1 variants impairs their coupling to phospholipase C. J. Biol. Chem. 273, 425–432 e Stephan, D. et al. (1996) Human metabotropic glutamate receptor 1: mRNA distribution, chromosome localization and functional expression of two splice variants. Neuropharmacology 35, 1649–1660 f Laurie, D.J. et al. (1996) HmGlu1d, a novel splice variant of the human type I metabotropic glutamate receptor. Eur. J. Pharmacol. 296, R1–R3 g Berthele, A. et al. (1998) Differential expression of rat and human type I metabotropic glutamate receptor splice variant messenger RNAs. Neuroscience 85, 733–749 h Pin, J-P. and Duvoisin, R. (1995) The metabotropic glutamate receptors: structure and functions. Neuropharmacology 34, 1–26 i Soloviev, M.M. et al. (1999) Identification, cloning and analysis of expression of a new alternatively spliced form of the metabotropic glutamate receptor mGluR1 mRNA1. Biochim. Biophys. Acta 1446, 161–166 j Tanabe, Y. et al. (1992) A family of metabotropic glutamate receptors. Neuron 8, 169–179 k Abe, T. et al. (1992) Molecular characterization of a novel metabotropic glutamate receptor mGluR5 coupled to inositol phosphate/Ca2+ signal transduction. J. Biol. Chem. 267, 13361–13368 l Minakami, R. et al. (1993) A variant of metabotropic glutamate receptor subtype 5: an

mglu7 receptor is involved in two separate types of amygdala-dependent learning and memory behaviours: fear response and conditioned taste aversion21. In these experiments, the knockout mice showed a reduction in fear-related freezing responses induced by electric shock and a failure to associate taste (saccharin) with a negative stimulus (an injection of LiCl, which induces toxicity). Pain sensitivity, locomotor activity and taste preference were not altered21. Interaction domains on the C-terminus of the mglu7 receptor

Several recent studies have shown that several proteins can interact with the C-terminus of mglu7 receptors. On the basis of the nature of these interacting proteins, the C-terminus can be divided into three domains (Fig. 1). http://tips.trends.com

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evolutionally conserved insertion with no termination codon. Biochem. Biophys. Res. Commun. 194, 622–627 Flor, P.J. et al. (1995) Molecular cloning, functional expression and pharmacological characterization of the human metabotropic glutamate receptor type 4. Neuropharmacology 34, 149–155 Makoff, A. et al. (1996) Molecular characterization and localization of human metabotropic glutamate receptor type 4. Mol. Brain Res. 40, 55–63 Thomsen, C. et al. (1997) Cloning and characterization of a metabotropic glutamate receptor, mGluR4b. Neuropharmacology 36, 21–30 Wu, S. et al. (1998) Group III human metabotropic glutamate receptors 4, 7 and 8: molecular cloning, functional expression, and comparison of pharmacological properties in RGT cells. Mol. Brain Res. 53, 88–97 Okamoto, N. et al. (1994) Molecular characterization of a new metabotropic glutamate receptor mGluR7 coupled to inhibitory cyclic AMP signal transduction. J. Biol. Chem. 269, 1231–1236 Saugstad, J.A. et al. (1994) Cloning and expression of a new member of the L-2-amino-4phosphonobutyric acid-sensitive class of metabotropic glutamate receptors. Mol. Pharmacol. 45, 367–372 Flor, P.J. et al. (1997) A novel splice variant of a metabotropic glutamate receptor, human mGluR7b. Neuropharmacology 36, 153–159 Corti, C. et al. (1998) Cloning and characterization of alternative mRNA forms for the rat metabotropic glutamate receptors mGluR7 and mGluR8. Eur. J. Neurosci. 10, 3629–3641 Duvoisin, R.M. et al. (1995) A novel metabotropic glutamate receptor expressed in the retina and olfactory bulb. J. Neurosci. 15, 3075–3083 Flor, P.J. et al. (1995) Molecular cloning, functional expression and pharmacological characterization of the human metabotropic glutamate receptor type 2. Eur. J. Neurosci. 7, 622–629

The proximal region of the C-terminus: an intracellular signalling domain

The coupling of adenylyl cyclase to Group III mglu receptors via pertussis-toxin-sensitive inhibitory G proteins has been described in heterologous expression systems14,15. Activation of mglu7 receptors causes inhibition of cAMP formation2, opening of KIR K+ channels9 and inhibition of voltage-gated Ca2+ channels5. More recently, the activation of mglu7 receptors has been suggested to selectively inhibit P/Q-type Ca2+ channels in cultured cerebellar granule cells4. Surprisingly for a Group III mglu receptor, mglu7 has been suggested to couple to PLC via G-protein αo- and βγ-subunits. Through this interaction, mglu7 receptors can increase intracellular Ca2+ levels and activate PKC (Ref. 4). Two groups of researchers have fused the entire C-terminal domain of the mglu7a receptor to

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γ 890

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Fig. 1. Proteins that interact and/or modulate metabotropic glutamate type 7 (mglu7) receptors. The 65 residues of the intracellular C-terminus of the mglu7 receptor are shown. The mglu7 receptor is reported to couple to adenylyl cyclase (AC) and phospholipase C (PLC) via G proteins. The binding domains on the proximal region of the C-terminus for calmodulin (CaM) and the phosphorylation site for protein kinase C (PKC) overlap. Gγβ shows some, but not complete, binding overlap with the CaM-binding site on the mglu7 receptor. Binding between the PDZ domain of PICK1 and the extreme PDZ-binding motif of the mglu7 receptor is indicated. The fact that other proteins are likely to interact with the C-terminus of the mglu7 receptor is represented by ‘protein X’. The model suggests mutual inhibitory effects between PKC phosphorylation and both CaM and Gγβ binding, and the inhibitory effects of PICK1 binding on PKC phosphorylation. Green proteins are those that are thought to interact directly with the C-terminus of mglu7 (ct-mglu7), whereas red proteins are those that might phosphorylate the mglu7 receptor. Abbreviations: GRK, G-protein-coupled receptor kinases; PKA, protein kinase A.

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glutathione-S-transferase (GST– ct-mglu7a) and used this construct for affinity chromatography9,10. These GST–ct-mglu7a pull-down experiments demonstrate that the G-protein βγ-subunits bind to the proximal region of the C-terminus of the mglu7a receptor9. Gα i alone does not bind directly to mglu7a receptors but is retained in the presence of βγ-subunits. CaM has also been shown to bind to mglu7 receptors9,10. CaM binding was abolished by deletion of a short region (residues KAVVTAATMSSRL) in the proximal section of ct-mglu7a (mglu7–∆CaM)9,10. CaM binding was Ca2+ dependent9,10 and independent of the binding of Gβγ. The CaM-binding domain of mglu7a receptors can be phosphorylated by PKC, consistent with the overlapping CaM and PKC consensus sequences9,10. CaM itself was not phosphorylated by PKC but similar to reports for Group I mglu receptors7,8, PKC elicited phosphorylation that inhibited the binding of CaM, whereas CaM binding prevented mglu7a receptor phosphorylation9,10. The site of interaction for Gβγ appears distinct from the CaM–PKC-binding domain because mglu7–∆CaM still bound Gβγ, whereas CaM binding was completely abolished9,10. Electrophysiological experiments have shown that CaM antagonists attenuate the presynaptic inhibition of glutamate release elicited by L-2-amino4-phosphonobutyrate (L-AP4)-induced activation of Group III mglu receptors in hippocampal autapses (synapses)9. Consistent with these data, in HEK293 cells co-transfected with mglu7a receptors and KIR K+ channels, L-AP4 enhanced the inward currents caused by hyperpolarizing voltage steps; this effect was lost on application of the CaM antagonist ophiobolin A. L-AP4 had no effect on mglu7–∆CaM but it enhanced the binding of [35S]GTPγS to HEK cell membranes that expressed either wild-type mglu7 receptors or mglu7–∆CaM (Ref. 9). These results demonstrate that CaM is not necessary for G-protein activation; rather CaM appears to promote dissociation of Gβγ from mglu7a receptors, thereby releasing Gβγ, which can then inhibit voltage-gated Ca2+ channels. Collectively, these findings indicate a http://tips.trends.com

link between PKC, CaM and Gβγ, and suggest that PKC phosphorylation can disrupt the coupling of mglu7 receptors to G proteins. Presynaptic mglu2-receptor-mediated responses are inhibited by protein kinase A (PKA), owing to phosphorylation of Ser843 in the C-terminal domain of the receptor22. However, it has been reported that the C-terminal domain of mglu7 receptors is not a target for PKA phosphorylation, although it remains possible that the intracellular loop regions of mglu7 receptors could be phosphorylated by PKA (Ref. 10). G-protein-coupled receptor kinases (GRKs) generally phosphorylate G-protein-coupled receptors after agonist activation, which leads to receptor desensitization and internalization of receptors via arrestin and clathrin-coated pits23. Recently, Group I mglu receptors have been shown to be substrates for GRKs (Ref. 24). Although it seems likely that other mglu receptors, including mglu7, can be phosphorylated by GRKs, confirmation of this requires further investigation. Moreover, it remains unclear whether GRKs, PKA or PKC mediate phosphorylation-dependent receptor desensitization and/or internalization, and whether, in turn, CaM [or other ct-mglu7-interacting proteins (see below)] is involved in these events. The central region of the C-terminus: an axonal targeting signal

The correct trafficking of receptors to the relevant cellular compartments is of fundamental importance for neuronal function. To investigate trafficking of these receptors further, Stowell and Craig used immunostaining procedures and virus-mediated expression of MYC-tagged mglu2 and mglu7 receptors in cultured rat hippocampal neurones25. The MYCtagged mglu2 receptor was targeted to dendrites but was excluded from axons, whereas the MYC-tagged mglu7 receptor was trafficked to both dendrites and axons. Given that the mglu7 receptor is believed to be predominantly presynaptic, its presence in dendrites was unexpected. To account for these observations it was suggested that axonal proteins could be transported to both axons and dendrites, but then degraded in dendrites and/or stabilized in axons. The exclusion of mglu2 receptors from axons in hippocampal pyramidal cells differs from previous work showing that mglu2 receptors localize to axons and dendrites in dentate granule cells26. The different distribution observed in hippocampal pyramidal cells, where mglu2 receptors are not endogenously expressed, could suggest that the targeting of receptors depends not only on a signal present in the receptor protein itself but also on the type of neurone in which it is expressed. In the same study, receptor chimeras were constructed that contained the C-termini of both mglu2 and mglu7 receptors, and in which the C-termini of mglu2 and mglu7 receptors were exchanged. Although no interacting proteins

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responsible for the specific targeting of mglu2 or mglu7 receptors have yet been identified, these experiments suggested that their C-termini carried information for axonal exclusion and targeting, respectively25. The axonal targeting sequence of mglu7a receptors was confined to residues 883–912, which lie between the proximal CaM–G-protein–PKC recognition domain and the extreme PDZ-binding motif 25. Furthermore, the C-terminus of the mglu7 receptor was dominant because its presence caused the axonal targeting of the mglu2 receptor. Interestingly, a C-terminal truncation of the mglu7 (∆-mglu7) receptor was excluded from axons, whereas ∆-mglu2 receptors retained the capacity to migrate to both axons and dendrites, which suggests that additional recessive targeting signals, perhaps in the intracellular loops of these receptors, might also be present. However, based on the results obtained with the mglu7–mglu2 chimeras and double-tail constructs, it seems unlikely that these recessive targeting signals play a dominant role in native receptors. A caveat of the MYC-tagged mglu7 receptor study is that the virally expressed receptors, although targeted to synaptic surface membranes, showed no evidence of synaptic clustering. The failure to cluster was not due to a lack of specific aggregation molecules in dissociated cultures because untagged receptors expressed in these cells using nonviral techniques clustered correctly11. It was therefore probably due to the high level, short-term expression of receptors from the viral vector and/or the epitope tagging of the receptor. The distal region of the C-terminus: presynaptic clustering

PDZ domains comprise ~90 amino acids that function as independent modules for protein interaction and bind proteins that contain appropriate consensus sequences. PDZ domains are present, either singly or as repeats, in over 100 otherwise unrelated proteins27. PDZ-domaincontaining proteins play a role in ion channel and receptor synaptic clustering and in targeting kinases and phosphatases towards their substrates. Some members of the PDZ-domain-containing family of proteins are present at high concentrations in neurones and have been implicated in neuronal receptor targeting to, and anchoring at, the postsynaptic membrane28. A current view is that the PDZ proteins can act as adapters that mediate the formation of protein complexes. In this way it is hypothesized that the PDZ-domain-containing proteins might be intimately involved in the spatial organization of synaptic proteins. PICK1 is a 46.5-kDa single PDZ-domaincontaining protein that was originally isolated as a binding partner for the catalytic region of PKC-α (Refs 29,30) and is also an efficient substrate for PKC phosphorylation. PICK1 has been shown to interact with several proteins, including AMPA http://tips.trends.com

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receptor subunits31,32, ephrin-B ligands, Eph receptor tyrosine kinases33 and class I ADPribosylation factors34. PICK1 can dimerize through a site distinct from its PDZ motif, which gives it the option of linking its interacting proteins to one another30. Three research groups have reported that PICK1 interacts with a PDZ-binding motif located at the extreme C-terminus of mglu7 receptors11–13. PICK1 and mglu7 receptors were co-immunoprecipitated from solubilized brain preparations and showed considerable overlap in their immunocytochemical distributions in cultured hippocampal neurones13. It has been shown that in in vitro phosphorylation assays, PICK1 attenuates PKC-elicited phosphorylation of mglu7 receptors. The attenuation did not occur in a mglu7 receptor construct that lacked the PDZ-binding motif but retained phosphorylation sites, which suggests that, rather than inhibiting the PKC-elicited phosphorylation by substrate competition, PICK1 might mediate this effect through steric hindrance of the PKC phosphorylation sites on mglu7 receptors. Another group, using quantitative β-galactosidase assays and the yeast two-hybrid system, identified mglu7a receptors as a major PICK1 interaction partner12. PICK1 (unlike CaM) was reported to have no effect on mglu7 receptor G-protein-mediated regulation of KIR K+ channels12. PDZ domains have been divided into type I and type II classes according to their binding specificities and sequence homologies. Class I PDZ domains interact with proteins that contain the consensus motif E-S/T-x-V/I (where x is any amino acid) at their extreme C-termini. Type II PDZ domains bind sequences that contain residues with hydrophobic or aromatic side-chains at the extreme C-terminal (consensus motif F/Y-x-F/V/A). Such binding occurs because the type II PDZ domains contain a secondary binding pocket and can tolerate a hydrophobic residue at position −2 (Ref. 35). Betz and co-workers have reported that the C-terminal domains of all Group III mglu receptor family members, except mglu4b (i.e. mglu4a, mglu7b, mglu8a and mglu8b), bind PICK1 (Ref. 12). On the basis of this observation, these authors suggest that PICK1 might possess a novel type III PDZ domain that can tolerate hydrophobic and basic, but not acidic, residues at the −2 position. It should be noted, however, that the affinities of the interactions with mglu4a, mglu7b, mglu8a and mglu8b receptors were much lower, were not confirmed outside the yeast two-hybrid system and have not been reported by other groups. Therefore, the case for a type III PDZ domain remains to be fully established. Craig and co-workers have demonstrated that under conditions where mglu7 receptors are clustered at presynaptic terminals in hippocampal neurones, a mutant receptor, mglu7a∆3, which lacks the last three amino acid residues and does not bind to PICK1, was

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Fig. 2. The PDZ-binding motif of metabotropic glutamate type 7a (mglu7a) receptors has a role in receptor clustering. Cultured hippocampal neurones were co-transfected with a membrane-bound form of green fluorescent protein (mGFP; green) and either mglu7a (a) or mglu7a∆3, a mutant that lacks the last three amino acids and does not interact with PICK1 (b). Neurones were fixed and immunostained for mglu7a receptors (red) and the synaptic marker SV2 (blue). (a) Co-transfection with mGFP demonstrates preserved morphology. The mglu7a receptor clusters are in the GFPexpressing axons and are largely colocalized with SV2 (white and arrows). (b) By contrast, mglu7a∆3 is mostly diffusely distributed along the axons with no colocalization with SV2. Scale bar = 8 µm. Reproduced, with permission, from Ref. 11.

Acknowledgements Our research was supported in part by research grants from the MRC (UK), the Wellcome Trust (UK) and the Ministry of Education, Science and Culture of Japan. We are grateful to Steve Fitzjohn, Herman van der Putten, Guido Meyer, Trevor J. Bushell, Anne Marie Craig, Helene Boudin and Yoshiaki Nakajima for their useful comments.

correctly targeted to axons but did not cluster at synapses11. These data suggest that the PDZ-binding motif is required for synaptic aggregation of the mglu7 receptor, presumably via an interaction with PICK1. Significantly, however, mglu7∆3 can be trafficked to axons in the absence of PICK1, although whether PICK1 might affect the route of trafficking remains unclear (Fig. 2). In hippocampal pyramidal cells, mglu7 receptors are differentially expressed at the presynaptic grid depending on the target cell36. Pyramidal cells that synapse onto mglu1α-receptor-expressing interneurones have a significantly higher level of presynaptic mglu7 receptors than those making synapses with pyramidal cells or interneurones. Clearly, the control of mglu7 receptor expression levels at different synapses provides a mechanism for

References 1 Nakanishi, S. (1994) Metabotropic glutamate receptors: synaptic transmission, modulation, and plasticity. Neuron 13, 1031–1037 2 Pin, J-P. and Duvoisin, R. (1995) Review: neurotransmitter receptors I. The metabotropic glutamate receptors; structure and functions. Neuropharmacology 34, 1–26 3 Conn, P.J. and Pin, J-P. (1997) Pharmacology and functions of metabotropic glutamate receptors. Annu. Rev. Pharmacol. Toxicol. 37, 205–237 4 Perroy, J. et al. (2000) Selective blockade of P/Q-type calcium channels by the metabotropic glutamate receptor type 7 involves a phospholipase C pathway in neurons. J. Neurosci. 20, 7896–7904 5 Fagni, L. et al. (2000) Complex interactions between mGluRs, intracellular Ca2+ stores and ion channels in neurons. Trends Neurosci. 23, 80–88 http://tips.trends.com

differentially regulating transmitter release, depending on individual synaptic connections. However, the molecular mechanisms that underlie the aggregation of presynaptic mglu7 receptors are unclear. One possible mechanism is an activitydependent expression of presynaptic proteins that interact with the intracellular domains of mglu7 receptors and regulate its surface expression. In this respect it would be interesting to determine the relative concentrations of PICK1 at these different synapses. Another possibility is that postsynaptic neurones expressing mglu1α receptors could specifically release a protein that clusters mglu7 receptors by interacting with its extracellular regions. An example of a protein that can act in this way is NARP, which clusters AMPA receptors by interacting with their extracellular regions37. A third possibility is that pyramidal cells and interneurones without clustered mglu7 receptors at the presynaptic terminals that synapse on them release a retrograde signal or protein that inhibits the aggregation of presynaptic mglu7 receptors at these synapses. Concluding remarks

The regulation of glutamate receptors at synaptic membranes is a dynamic process that is dependent on a complex series of protein–protein interactions. Three regions of the C-terminus of mglu7 receptors have been designated as interacting with different sets of proteins (Fig. 1): (1) the proximal region that is potentially involved in intracellular signalling mechanisms; (2) the central region that is potentially involved in axonal–dendritic targeting; and (3) the distal amino acid residues that are involved in presynaptic clustering and intracellular signalling through interactions with at least one PDZ-domaincontaining protein. Future studies aimed at revealing the biological roles of identified, and as of yet unidentified, receptorinteracting proteins and the signalling–scaffolding cascades to which they belong will provide insight into the mechanisms that underlie receptor localization and function.

6 Xiao, B. et al. (2000) Homer: a link between neural activity and glutamate receptor function. Curr. Opin. Neurobiol. 10, 370–374 7 Minakami, R. et al. (1997) Phosphorylation and calmodulin binding of the metabotropic glutamate receptor subtype 5 (mGluR5) are antagonistic in vitro. J. Biol. Chem. 272, 20291–20298 8 Ishikawa, K. et al. (1999) Competitive interaction of seven in absentia homolog-1A and Ca2+/calmodulin with the cytoplasmic tail of group 1 metabotropic glutamate receptors. Genes Cells 4, 381–390 9 O’Connor, V. et al. (1999) Calmodulin dependence of presynaptic metabotropic glutamate receptor signaling. Science 286, 1180–1184 10 Nakajima, Y. et al. (1999) A relationship between protein kinase C phosphorylation and calmodulin binding to the metabotropic glutamate receptor subtype 7. J. Biol. Chem. 274, 27573–27577

11 Boudin, H. et al. (2000) Presynaptic clustering of mGluR7a requires the PICK1 PDZ domain binding site. Neuron 28, 485–497 12 El Far, O. et al. (2000) Interaction of the C-terminal tail region of the metabotropic glutamate receptor 7 with the protein kinase C substrate PICK1. Eur. J. Neurosci. 12, 1–9 13 Dev, K.K. et al. (2000) PICK1 interacts with and regulates PKC phosphorylation of mGLUR7. J. Neurosci. 20, 7252–7257 14 Okamoto, N. et al. (1994) Molecular characterization of a new metabotropic glutamate receptor mGluR7 coupled to inhibitory cyclic AMP signal transduction. J. Biol. Chem. 269, 1231–1236 15 Saugstad, J.A. et al. (1994) Cloning and expression of a new member of the L-2-amino-4phosphonobutyric acid-sensitive class of metabotropic glutamate receptors. Mol. Pharmacol. 45, 367–372

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16 Flor, P.J. et al. (1997) A novel splice variant of a metabotropic glutamate receptor, human mGluR7b. Neuropharmacology 36, 153–159 17 Ohishi, H. et al. (1995) Presynaptic localization of a metabotropic glutamate receptor, mGluR7, in the primary afferent neurons: an immunohistochemical study in the rat. Neurosci. Lett. 202, 85–88 18 Kinoshita, A. et al. (1998) Immunohistochemical localization of metabotropic glutamate receptors, mGluR7a and mGluR7b, in the central nervous system of the adult rat and mouse: a light and electron microscopic study. J. Comp. Neurol. 393, 332–352 19 Lafon-Cazal, M. et al. (1999) mGluR7-like metabotropic glutamate receptors inhibit NMDA-mediated excitotoxicity in cultured mouse cerebellar granule neurons. Eur. J. Neurosci. 11, 663–672 20 Lafon-Cazal, M. et al. (1999) mGluR7-like receptor and GABA(B) receptor activation enhance neurotoxic effects of N-methyl-Daspartate in cultured mouse striatal GABAergic neurons. Neuropharmacology 38, 1631–1640 21 Masugi, M. et al. (1999) Metabotropic glutamate receptor subtype 7 ablation causes deficit in fear response and conditioned taste aversion. J. Neurosci. 19, 955–963 22 Schaffhauser, H. et al. (2000) cAMP-dependent protein kinase inhibits mGluR2 coupling to

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Therapeutic implications of human endothelial nitric oxide synthase gene polymorphism Suvara Kimnite Wattanapitayakul, Michael J. Mihm, Anthony P. Young and John Anthony Bauer Vascular endothelial dysfunction is now recognized as a common phenomenon in an array of cardiovascular disorders. Production of nitric oxide via the endothelial isoform of nitric oxide synthase [eNOS (previously termed NOS3 or ecNOS)] is vital for a healthy endothelium; several polymorphic variations of the gene encoding eNOS (NOS3 ) are now known and have been investigated with respect to disease risk. Surprisingly, only approximately half of these studies have demonstrated significant associations between NOS3 polymorphisms and cardiovascular disease, and many reports are contradictory. Central issues include adequate statistical power, appropriateness of control cohorts, multigene interactions and plausible biological consequences. So far, the inconsistencies are not unique to the NOS3 polymorphisms, but probably represent the broad challenges in defining genetic aspects of complex disease processes.

The vascular endothelium is now recognized as an important participant in a healthy cardiovascular system, and dysfunction of this cellular monolayer might be an initiating event in many or most http://tips.trends.com

cardiovascular disease states1. A hallmark feature of endothelial function is the synthesis and release of nitric oxide (NO), which provides local regulation of vasomotor tone and anti-thrombotic actions1. This readily diffusible gas has limited stability in biological settings (t1/2 of several seconds) and is known to participate in a variety of chemical reactions with metals, thiols and other reactive oxygen species2. Given the numerous physiological roles for NO and its rapid reaction and inactivation in cellular systems, strict control of NO production is crucial for its selective actions. Mammalian NO production is governed by the expression and activities of the nitric oxide synthase (NOS) enzyme family. The three primary NOS isoforms have a high degree of sequence homology and their biochemical catalysis is

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