Homer: a link between neural activity and glutamate receptor function

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Homer: a link between neural activity and glutamate receptor function Bo Xiao, Jian Cheng Tu and Paul F Worley* The proteins of the Homer family bind to proline-rich sequences in group I metabotropic glutamate receptors, inositol trisphosphate receptors, ryanodine receptors, and Shank family proteins. Homer proteins also self associate and function as adaptors to couple interacting proteins. Recent observations indicate a role for Homer complexes in signal transduction, synaptogenesis and receptor trafficking. Addresses Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA *e-mail: [email protected] Current Opinion in Neurobiology 2000, 10:370–374 0959-4388/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations CC coiled-coil EVH1 Ena/VASP homology 1 GKAP guanylate kinase associated protein IEG immediate early gene IP3 inositol trisphosphate IP3R IP3 receptor mGluR metabotropic glutamate receptor NMDA N-methyl-D-aspartate NMDAR NMDA receptor PPII type II polyproline PSD postsynaptic density PSD-95 PSD protein of 95 kDa RYR ryanodine receptor VASP vasodilator-stimulated phosphoprotein WASP Wiskott-Aldrich syndrome protein

Introduction Homer 1a is the product of an immediate early gene (IEG) and the original member of a family of proteins that function together as a regulated adaptor system. Current data support three broad hypotheses regarding the function(s) of Homer. The ‘signaling’ hypothesis envisions that Homer and Homer-related proteins regulate the coupling of membrane receptors with intracellular pools of releasable Ca2+ [1••–3••]. A related hypothesis is that Homer proteins play a role in synaptogenesis and spatial targeting of metabotropic glutamate receptors (mGluRs) [1••,2••]. Recent studies also indicate a role for Homer proteins in receptor trafficking [4,5•]. It currently appears that Homer proteins are involved in each of these functions, suggesting a shared cell biological mechanism that links signaling, synaptogenesis and trafficking. In this review, we will discuss the discovery of Homer and summarize current understanding of its function.

Cloning of Homer Homer 1a was first identified on the basis of the rapid upregulation of its mRNA in neurons from seizure-stimulated hippocampus [6,7]. In situ hybridization indicated

that Homer 1a is dynamically regulated in models of natural plasticity. As anticipated for an IEG product, Homer 1a protein is rapidly and transiently induced by neural activity [6]. The amino-terminal ~110 amino acids of Homer 1a protein show homology to the EVH1/WH1 (Ena/VASP homology 1/WASP homology 1) domains found in the Ena/vasodilator-stimulated phosphoprotein (VASP) protein family [7–9], as well as the Wiskott–Aldrich syndrome protein (WASP) and its neuronal homolog N-WASP [10–12]. A family of 12 Homer cDNAs has been cloned from rat, mouse, Drosophila, and human brain [13•,14•]. All of these cDNAs encode proteins with a similar structure and are the products of three independent mammalian genes (termed Homer 1, 2 and 3) and one Drosophila gene. Like Homer 1a, these new family members contain a conserved amino-terminal EVH1 domain [14•,15•]; however, in addition, they contain a carboxy-terminal domain with a predicted coiled-coil (CC) structure. These Homer CC domains mediate specific self-association [13•,14•,16•]. In contrast to Homer 1a, all CC-containing Homer family members (called CC-Homers) are constitutively expressed in brain [14•]. The domain structure and nomenclature of the Homer family have been reviewed recently [17] and are depicted in Figure 1. Homer cDNAs have been cloned from a variety of models of developmental and adult neural plasticity, and they have been variously named as Ania-3, Vesl, PSD-ZIP45, Cupidin [7,18,19•,20]. Vesl [7] and PSD-ZIP45 [20] are identical to Homer 1 proteins. Ania-3 is induced by dopamine-dependent signals in the rodent striatum and Figure 1

M-Homer 1a M-Homer 1b M-Homer 1c M-Homer 2a M-Homer 2b M-Homer 3 H-Homer 3 D-Homer

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Current Opinion in Neurobiology

Primary structure of mouse (M), human (H) and Drosophila (D) Homer proteins. For all Homer proteins, the conserved amino-terminal region is an EVH1 domain (light gray rectangles). For all CC-Homer proteins (i.e. all known Homer proteins aside from Homer 1a), the carboxyterminal region (dark gray rectangles) contains the CC domains mediating the multimerization of Homer proteins. The arrows indicate an insertion of a short amino acid stretch. aa, amino acid.

Homer: a link between neural activity and glutamate receptor function Xiao, Tu and Worley

represents a unique short form of Homer 1 that is identical to Homer 1a with the exception that it encodes a unique, short carboxyl terminus [18]. Cupidin [19•], which was cloned on the basis of its developmental increase in postnatal day 7 mouse cerebellum, is identical to Homer 2a. Additional Homer transcripts have been described, including EVH1-only forms of each of the Homer genes in brain and peripheral tissues; however, it remains to be determined whether these are translated in vivo [21•]. If so, EVH1-only forms of Homer may be broadly expressed. The mechanism by which neural activity leads to the expression of EVH1-only forms of Homer remains to be determined.

Homer selectively binds group 1 metabotropic receptors Using the yeast two-hybrid approach, we [6] found that Homer binds to the carboxyl terminus of mGluR5. Homer protein also binds to mGluR1α, but not to mGluR2, mGluR3, mGluR4, or mGluR7. This finding provided an interesting clue to the function of Homer because mGluR1 and mGluR5 (termed group 1 metabotropic receptors) couple to phospholipase C and activate hydrolysis of phosphoinositides to generate inositol trisphosphate (IP3) and diacylglycerol [22] (see Figure 2a). The mGluR1α and mGluR5 receptors also share sequence similarity in their long, cytosolic carboxyl terminus. Homer and mGluR5 naturally associate in brain, as confirmed by their co-immunoprecipitation from detergent extracts of rodent brain [6] and co-localization at excitatory synapses [6,14•]. Deletion analysis has identified a proline-rich sequence (PPSPF; single-letter amino-acid code used) approximately 50 amino acids from the carboxyl terminus of mGluR5 that is essential for Homer binding (see below). Binding of Homer to mGluRs has been confirmed by other reports on Homer family proteins [13•,19•,20].

Homer proteins function to link mGluR5 and the IP3 receptor in a signaling complex We [3••] recently identified a Homer-binding site in the IP3 receptor (IP3R) and ryanodine receptor (RYR). IP3R coimmunoprecipitates with each of the CC forms of Homer (i.e. Homer 1b/c, 2a/b and 3) in detergent extracts of rodent cerebellum [3••]. The crosslinking capacity of CC-Homers suggested that Homer proteins couple mGluRs and IP3Rs. This notion is appealing in that the IP3R is part of the signaling network that is activated upon glutamate stimulation of mGluR1/5. Data from ultrastructural analyses of synapses support the notion that the endoplasmic reticulum and plasma membranes can come in close apposition in neurons [23–25]. Remarkably, the smooth endoplasmic reticulum (SER) forms close appositions with the plasma membrane that are uniquely localized to the lateral margin of the postsynaptic density (PSD). These sites are precisely where the group 1 mGluRs are localized [26–28]. The IP3R is present in spines of cerebellar Purkinje neurons, where it is associated with the spine apparatus [29]. In hippocampal neurons, the RYR is present in the spine apparatus, whereas the IP3R

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appears to be restricted to the dendritic shaft (reviewed in [30]). Homer 1b/c and 3 are also enriched in the cytosol at the lateral margin of the PSD [14•]. Thus, synaptic mGluRs come in close apposition with SER-associated IP3Rs at sites that are enriched with CC-Homers. Biochemical data also support the hypothesis of a Homerdependent signaling complex. We [3••] found that IP3R antibody specifically co-immunoprecipitates Homer and mGluR1α from cerebellum. Because IP3Rs are not known to interact directly with mGluR1α, this result supports the hypothesis that Homer bridges these proteins to form a trimolecular signaling complex. A further prediction of the ‘signaling’ function is that Homer 1a uncouples the putative mGluR–CC-Homer–IP3R complex because it lacks the CC domain required for self-association and crosslinking of target proteins. We [3••] found that expression of a Homer 1a transgene in Purkinje neurons alters mGluRinduced Ca2+ release from intracellular stores, but that Homer 1b does not. This result is consistent with the notion that Homer 1a disrupts the physical linkage between mGluR1α and IP3R such that released IP3 must diffuse further (see Figure 2b), thereby resulting in a lower effective concentration of IP3 at the IP3R.

Homer interacts with Shank, suggesting a role in synaptogenesis and NMDA receptor function Studies to identify other natural partners of Homer in brain have identified a novel family of proteins termed Shank [1••] (also called ProSAP [31•] and SAPAP [32]). Shank proteins also interact with GKAP and PSD-95, and they function as part of the NMDA receptor (NMDAR)-associated PSD-95 complex [1••]. The Shank–Homer interaction is mediated by the EVH1 domain of Homer, which binds to a single Homer-ligand site present in the proline-rich domain of Shank proteins [2••]. A quaternary complex of Homer/Shank/GKAP/PSD-95 is assembled in heterologous cells, with Homer and PSD-95 co-localizing in large clusters [2••]. The Shank–Homer interaction also causes group 1 mGluRs to cluster [2••]. It is notable that the mechanism of clustering involves a linkage of mGluRs with the NMDAR scaffold. Thus, the Shank–Homer interaction could be relevant to synaptogenesis by docking mGluRs to a pre-established ‘core’ of NMDARs. By linking NMDAR and mGluR signaling pathways, the Shank–Homer interaction might also contribute to specific types of glutamate receptor crosstalk for which physical proximity of molecules may be important, such as activation of phospholipase C [33] or protein kinase C [34,35]. In addition, the Homer/Shank/GKAP/PSD-95 assembly may mediate physical association (and perhaps functional coupling) of the NMDAR with IP3R/RYR and intracellular Ca2+ stores. Consistent with such a functional interaction, recent studies indicate that NMDAR-dependent increases in Ca2+ in dendritic spines may derive from intracellular stores by a mechanism of Ca2+-induced Ca2+ release ([36]; for a review, see [37]). In this context, the monovalent Homer 1a product is anticipated to uncouple proteins that

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Current Opinion in Neurobiology

Homer regulates mGluR signaling. (a) Constitutively expressed Homer proteins (CC-Homers) are localized to the postsynaptic compartment, where they bind the carboxyl terminus of group I mGluRs and IP3Rs. These receptors are physically coupled by CC-Homers to form an efficient signaling complex that generates IP3 and releases Ca2+ from intracellular pools. (b) In response to specific forms of neural activity, Homer 1a is rapidly upregulated. It is hypothesized that Homer 1a

competes with CC-Homer and disassembles the signaling complex. This competition between Homer 1a and CC-Homer is anticipated to involve other targets of Homer, including Shank. Thus, neural activity evokes a feedback mechanism to reduce glutamate-induced release of Ca2+ from intracellular pools. C, carboxyl terminus; DAG, diacylglycerol; ER, endoplasmic reticulum; N, amino terminus; PIP2, phosphatidylinositol bisphosphate; PL-Cβ, phospholipase Cβ.

are linked via the constitutively expressed CC-Homer multimers, and thereby dynamically regulate the assembly of this postsynaptic network (see Figure 2b).

HeLa cells), the receptor reaches the plasma membrane where it is diffusely localized [5•]. This is also true when mGluR5 is co-expressed with Homer 1a. However, coexpression of mGluR5 with Homer 1b in HeLa cells results in retention of mGluR5 in the endoplasmic reticulum and a reduction of receptor at the plasma membrane [5•]. This effect is abolished by point mutations of mGluR5 that interrupt its interaction with Homer and is not seen with mGluR2. By contrast, co-expression of Homer 1c and mGluR1α is reported to result in increased cell surface expression of the receptor in HEK 293 cells [4], but has no effect on trafficking in COS-7 cells [16•]. Interestingly, Homer 1c increases transport of mGluR1α to the dendrites of transiently transfected CNS neurons [4].

Additional interactions of Homer with F-actin and small GTP-binding proteins has been reported [19•]. Such interactions could contribute to the structural plasticity of the synapse. However, the molecular basis and natural relevance of these interactions remain to be determined.

Homer alters trafficking of mGluR1α/5 Homer alters the cellular distribution of mGluRs expressed in heterologous cells. When wild-type mGluR5 is expressed in heterologous cells (e.g. HEK293, COS or

Homer: a link between neural activity and glutamate receptor function Xiao, Tu and Worley

Intracellular pools of both mGluR1α and mGluR5 have been reported in neurons [38], suggesting that Homer contributes to the targeting of metabotropic receptors to neuronal processes [5•].

(NIDA), National Institute of Mental Health (NIMH) and National Alliance for Research on Schizophrenia and Depression (NARSAD).

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest •• of outstanding interest

Homer EVH1 domain crystal structure A recent crystal structure of Homer elucidates its binding specificity [15•]. Homer EVH1 domains are highly conserved and bind a proline-rich motif present in mGluRs, IP3Rs, RYRs and Shank proteins [1••–3 ••]. EVH1 domains in other proteins also interact with polyprolinecontaining target sequences. The EVH1 domains of Ena and VASP recognize polyproline motifs in zyxin, vinculin, and the Listeria monocytogenes ActA protein [39,40]. The Homer ligand consensus sequence is PPXXF (single-letter amino-acid used, where ‘X’ is any amino acid) [3••] and contrasts with that of Ena and VASP, which bind the sequence FPPPP [39]. Crystal structures of Mena (a murine Ena homolog) and EVL (Ena/VASP-like) EVH1 domains complexed with proline-rich ligands have been determined [41,42]. These structures reveal the FPPPP sequence bound in virtually identical fashion with the ligand forming a type II polyproline (PPII) helix. The crystal structure of the Homer 1 EVH1 domain both alone and complexed with an mGluR-derived peptide reveals that the Homer EVH1 domain binds ligand in a unique manner that distinguishes it from other EVH1 domains, and indeed from other PPII-binding proteins [15•]. The orientation of the ligand and general sites of proline contact are similar between Homer and Ena/VASP; however, the phenylalanine in the Homer ligand contacts Gly89 directly in a manner that suggests any other residue at this site would interfere with this interaction. Indeed, mutation of this residue to either alanine or asparagine (as are present in all other EVH1 family members) eliminates detectable binding of Homer to its substrates. The distinctive mode of Homer ligand binding minimizes the potential for cross-reaction with the many available proline-rich target sequences.

Conclusions To date, all of the confirmed functions of Homer derive from the binding properties of its EVH1 and CC domains. CC-Homers are constitutively expressed and crosslink target proteins, whereas the IEG product (Homer 1a) is dynamically expressed and regulates the degree of coupling (see Figure 2). It is anticipated that additional interacting proteins will be identified that will expand the repertoire of Homer’s actions. As studies address increasingly systems-level functions, Homer should provide new insights into the relationship between neural activity and synaptogenesis and the trafficking and signaling of glutamatergic receptors.

Acknowledgements We thank our collaborators who have contributed much to the understanding of Homer. We also thank Jutta Benneken for preparation of Figure 2. This work was supported by grants from National Institute on Drug Abuse

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1. ••

Naisbitt S, Kim E, Tu J, Xiao B, Sala C, Valtschanoff J, Weinberg R, Worley P, Sheng M: Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin. Neuron 1999, 23:569-582. These two papers [1••,2••] define a novel family of proteins (the Shank family) that couples the mGluR and NMDAR receptor signaling pathways. Shank proteins are linked to Homer proteins, which are postulated to couple group 1 mGluRs with the release of Ca2+ from intracellular stores (see [3••]). Shank proteins define the first molecular link between NMDARs and mGluRs, and they may underlie aspects of crosstalk and synaptic structure. The paper by Naisbitt et al. [1••] defines the Shank family, whereas the one by Tu et al. [2••] defines the link between Shank and Homer. 2. ••

Tu JC, Xiao B, Naisbitt S, Yuan JP, Petralia RS, Brakeman PR, Aakalu VK, Lanahan AA, Sheng M, Worley P: mGluR/Homer and PSD-95 complexes are linked by the Shank family of postsynaptic density proteins. Neuron 1999, 23:583-592. See annotation [1••]. 3. ••

Tu JC, Xiao B, Yuan J, Lanahan A, Leoffert K, Li M, Linden D, Worley PF: Homer binds a novel proline rich motif and links group 1 metabotropic glutamate receptors with IP3 receptors. Neuron 1998, 21:717-726. Homer proteins are demonstrated to couple mGluRs with IP3 receptors in a signaling complex. The immediate early gene form of Homer protein (Homer 1a) is shown to regulate coupling efficiency, thereby defining a synaptic function for Homer 1a proteins. 4.

Ciruela F, Soloviev MM, Chan WY, McIlhinney RA: Homer-1c/Vesl-1L modulates the cell surface targeting of metabotropic glutamate receptor type 1α: evidence for an anchoring function. Mol Cell Neurosci 2000, 15:36-50.

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Roche KW, Tu JC, Petralia RS, Xiao B, Wenthold RJ, Worley PF: Homer 1b regulates the trafficking of group I metabotropic glutamate receptors. J Biol Chem 1999, 274:25953-25957. Presents an initial report of the effect of Homer on mGluR trafficking. CC-Homers cause the retention of group 1 mGluRs in the ER, whereas Homer 1a is permissive for insertion of the mature receptor into the plasma membrane. The action of CC-Homer requires direct protein–protein interaction with the receptor. Extensive pools of ER-associated mGluRs are demonstrated in CNS neurons, suggesting that Homer may play a similar ‘receptor trafficking’ role in in vivo systems. 6.

Brakeman PR, Lanahan AA, O’Brien R, Roche K, Barnes CA, Huganir RL, Worley PF: Homer: a protein that selectively binds metabotropic glutamate receptors. Nature 1997, 386:284-288.

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Kato A, Ozawa F, Saitoh Y, Hirai K, Inokuchi K: vesl, a gene encoding VASP/Ena family related protein, is upregulated during seizure, long-term potentiation and synaptogenesis. FEBS Lett 1997, 412:183-189.

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Ponting CP, Phillips C: Identification of homer as a homologue of the Wiskott-Aldrich syndrome protein suggests a receptor-binding function for WH1 domains. J Mol Med 1997, 75:769-771.

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13. Kato A, Ozawa F, Saitoh Y, Fukazawa Y, Sugiyama H, Inokuchi K: • Novel members of the Vesl/Homer family of PDZ proteins that bind metabotropic glutamate receptors. J Biol Chem 1998, 273:23969-23975. Describes the extended family of Homer/Vesl proteins. New members of the Homer/Vesl family are demonstrated to encode coiled-coil (CC) domains and self-associate. Because the IEG form (Homer 1a) lacks a CC domain, it was suggested that it functions as a natural dominant negative. 14. Xiao B, Tu JC, Petralia RS, Yuan J, Doan A, Breder C, Ruggiero A, • Lanahan AA, Wenthold RJ, Worley PF: Homer regulates the association of group 1 metabotropic receptors with multivalent complexes of Homer-related, synaptic proteins. Neuron 1998, 21:707-716. Describes the extended family of Homer/Vesl proteins and the current Homer nomenclature. CC-Homer proteins are visualized at the PSD and are demonstrated to self associate in vivo. Transgenic mice that overexpress Homer 1a demonstrate its ability to regulate the coupling of CC-Homer to group 1 mGluRs. 15. Beneken J, Tu JC, Xiao B, Nuriya M, Yuan JP, Worley PF, Leahy DJ: • Structure of the Homer EVH1 domain-peptide complex reveals a new twist in polyproline recognition. Neuron 2000, 26:143-154. Presents the structure of the Homer EVH1 domain, which elucidates the distinctive binding properties of the Homer subfamily. Mena/VASP EVH1 domains bind FPPPP, whereas Homer EVH1 binds PPXXF. The orientation of the peptides as they are bound to their respective EVH1 domains is identical, and binding specificity is conferred by distinct pockets for the phenylalanine. 16. Tadokoro S, Tachibana T, Imanaka T, Nishida W, Sobue K: • Involvement of unique leucine-zipper motif of PSD-Zip45 (Homer 1c/vesl-1L) in group 1 metabotropic glutamate receptor clustering. Proc Natl Acad Sci USA 1999, 96:13801-13806. The self-association properties of the Homer CC domain are examined and shown to be mediated by several different domains. This article anticipates the complexity of CC-Homer self-multimerization. 17.

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