BIOLOGICAL OVERVIEW

In the absence of mutations in any of the mammalian Homer genes, their in vivo roles remain unknown. A single gene has been identified in Drosophila encoding a protein homologous to the mammalian Homer proteins (Kato, 1998; Xiao, 1998 and Diagana, 2002). Drosophila Homer is enriched in the nervous system, where it is localized to the endoplasmic reticulum (ER) and targeted to dendritic processes. Genetic evidence is provided that homer is required for the function of the neural networks controlling locomotor activity and behavioral plasticity (Diagana, 2002).

Mammalian Homer proteins have been proposed to play a role in synaptogenesis, synapse function, receptor trafficking, and axon pathfinding. The Drosophila gene homer, the single Homer-related gene in fly, has been isolated and characterized. Using anti-Homer antibody it has been shown that Homer is expressed in a broad range of tissues but is highly enriched in the CNS. Similar to its mammalian counterpart, Drosophila Homer localizes to the dendrites and the endoplasmic reticulum (ER). This subcellular distribution is dependent on an intact Enabled/Vasp homology 1 domain, suggesting that Homer must bind to one or more of its partners for proper localization. Flies homozygous for a mutation in homer are viable and show coordinated locomotion, suggesting that Homer is not essential for basic neurotransmission. However, homer mutants display defects in behavioral plasticity and the control of locomotor activity. These results argue that in the CNS, Homer-related proteins operate in the ER and in dendrites to regulate the development and function of neural networks underlying locomotor control and behavioral plasticity (Diagana, 2002).

Proteins of the Homer family have been implicated in synaptogenesis, signal transduction, receptor trafficking, and axon pathfinding (Xiao, 2000; Foa, 2001; Thomas, 2002). In mammals, three independent genes (Homer-1, -2, and -3) encode at least six Homer proteins through differential splicing (Kato, 1998; Xiao, 1998). Members of the Homer family are expressed in various tissues but appear to be enriched in the CNS, where they have partially overlapping domains of expression (Brakeman, 1997; Xiao, 1998). Homer proteins are bipartite, consisting of an N-terminal Enabled/Vasp homology 1 (EVH1) domain and a C-terminal coiled-coil (CC) domain that mediates self-association (Brakeman, 1997; Kato, 1998; Tu, 1998; Xiao, 1998; Tadokoro, 1999). The EVH1 domain binds to group I metabotropic glutamate receptors (mGluRs) and their downstream effectors, the inositol-triphosphate receptor (InsP3R), by interacting with a proline-rich motif (PPxxF) found in these proteins (Brakeman, 1997; Tu, 1998, 1999; Diagana, 2002 and references therein).

One can envision a model in which Homer proteins, via their ability to self-associate, modulate group I mGluR function by mediating the formation of a multimolecular complex required for local and fast increase of Ca²⁺ concentration during mGluR activation (Xiao, 2000). Supporting this notion is the finding that Homer proteins regulate the intracellular trafficking of mGluRs (Roche, 1999; Ango, 2000). Further modulation is provided by Homer 1a, one of the proteins encoded by the Homer 1 gene, that consists of the EVH1 domain without the CC domain required for multimerization. Homer 1a is upregulated during synaptic activity (Brakeman, 1997; Xiao, 1998) and is capable of attenuating mGluR-evoked intracellular calcium release in vitro, presumably by disruption of a putative mGluR-Homer-InsP3R multimeric complex (Tu, 1998; Diagana, 2002 and references therein).

Homer proteins also bind to Shank/ProSAP, a postsynaptic protein that is part of a complex including the NMDA-type glutamate receptors. Recently, Shank has been implicated in the regulation of dendritic spine morphology and synaptic function, and this regulation is dependent on Shank binding to Homer (Sala, 2001). Therefore, Homer-related proteins may be part of a large multimolecular complex that modulates the structural and functional plasticity of glutamatergic synapses (Naisbitt, 1999; Sala, 2001; Diagana, 2002 and references therein).

It has been proposed that in vertebrates, Homer-related proteins might regulate synaptic plasticity possibly underlying learning and memory via the modulation of mGluR function. To test for a possible role of Homer in behavioral plasticity, the performance of homer^R102 mutant males was evaluated in a courtship conditioning assay, an associative learning paradigm in Drosophila. Courtship is a plastic behavior that can be conditioned by previous experience. Male flies display a complex and robust courtship behavior toward a female in response to olfactory, visual, and tactile cues. After exposure to a nonreceptive mated female, wild-type males will repress their level of courtship. This repression is sustained during subsequent exposure to a receptive, virgin female. Courtship repression is thus a conditioned behavior and is thought to depend on the association of positive stimuli with an aversive chemosensory signal from the mated female (Diagana, 2002).

In the courtship conditioning assay, individual males were placed with a nonreceptive mated female in a conditioning chamber for 1 hr. The mated female was then removed, and after 2 min, each male was individually tested for levels of courtship with an anesthetized virgin female. The amount of courtship displayed by these 'trained' males was monitored over a 10 min period and compared with courtship levels of 'naive' males that had been manipulated identically in the conditioning protocol, except without the nonreceptive mated female. In the assay, no defects were detected in the sequence or the length of the different steps of male courtship behavior of homer mutants, and thus homer is not essential for the execution of the courtship behavior (Diagana, 2002).

homer mutants display defects in behavioral plasticity. homer⁺ control males show a statistically significant reduction of courtship after training, as do homer^R102/homer⁺ heterozygotes. In contrast, homer^R102 trained males show no significant reduction of courtship. In addition, although there is no significant difference in the amount of courtship displayed by naive homer^R102 and naive homer⁺ flies, the trained homer^R102 mutant males show a significantly higher level of courtship when compared with the trained homer⁺ males. Collectively, these results demonstrate that homer^R102 mutants show behavioral plasticity deficits and fail to form and/or retain the conditioning by the nonreceptive mated female (Diagana, 2002).

To determine whether homer^R102 mutants show defects in the acquisition of conditioning during training, the amount of courtship displayed was monitored by tested males over the first and last 10 min of the conditioning period. Both homer^R102 mutant flies and homer⁺ controls show a statistically significant reduction of courtship level after conditioning by the mated female. homer^R102 mutant males show higher initial and final courtship levels when compared with homer⁺ controls. Median values for initial and final courtship levels are, respectively, 331 and 133 sec for homer^R102 mutants and 160 and 12 sec for the homer⁺ controls. These data demonstrate that homer^R102 mutants do suppress courtship behavior after conditioning by the mated female but show higher levels of initial and final courtship (Diagana, 2002).

homer mutant flies do not show olfactory defects but display deficits in the control of locomotor activity. The conditioned repression of male courtship is dependent on the perception of an aversive chemosensory cue secreted by the nonreceptive mated female. Thus, defects in olfaction could be an explanation for the poor conditioning of homer^R102 mutants in the courtship conditioning assay. To evaluate the olfactory competence of homer^R102 adult flies, the chemosensory jump assay was used. When suddenly exposed to chemical vapors, a wild-type fly exhibits an escape response consisting of a jump. The chemosensory jump response (CJR) of homer mutant flies to two different chemicals was tested. The CJR to propionic acid of homer^R102 mutant flies is similar to that of homer⁺ control flies, and the CJR of homer^R102 mutant flies to benzaldehyde is actually higher than that of homer⁺ control flies, demonstrating that homer^R102 mutant flies are not severely defective in olfaction. However, homer mutants do display deficits in their control of locomotor activity. homer^R102 mutant flies show a higher level of spontaneous locomotor activity when compared with homer⁺ homozygous flies, a feature that is consistent with homer^R102 flies showing higher courtship levels (Diagana, 2002).

The colocalization of Homer and Synaptotagmin (see Drosophila Syntaxin) in the dorsal-most region of the neuropil suggests that Homer is localized to regions containing a concentration of synapses. By expressing an epitope-tagged Homer, evidence has been provided that the Drosophila Homer is targeted to dendrites, similar to the targeting described for vertebrate Homer proteins. Within neuronal cell bodies, the Drosophila Homer colocalizes with a marker for the ER. In transfected cells, Homer 1b has similarly been shown to be localized in the ER compartment (Roche, 1999), a subcellular localization thought to be functionally relevant because Homer-related proteins are capable of binding the ER-resident receptor InsP3R (Tu, 1998). Thus, the evolutionary conservation of the subcellular localization of Homer-related proteins suggests that their functional roles might also have been conserved (Diagana, 2002 and references therein).

Mammalian Homer binds to several proteins, including Shank, Group I mGluRs, and the InsP3 receptor (Tu, 1998; Xiao, 1998; Tu, 1999). Homer specifically binds to a short proline-rich motif, PPxxF, present in each of these proteins (Tu, 1998). In support of a functional role for this binding is the finding that mutations in the PPxxF motif of mGluR5 that abolish the binding to Homer in vitro also abolish the ER retention of mGluR5 in cells cotransfected with mGluR5 and Homer (Roche, 1999). In addition, mutation of the Homer binding site of mammalian Shank disrupts the Shank-dependent targeting of Homer 1b to dendritic spines (Sala, 2001). Evidence is provided that Drosophila Homer likely requires binding to at least one of its putative partners to be properly localized. Mutation of amino acids within the EVH1 domain predicted to be required for PPxxF binding abolishes the localization of Homer to the ER and its targeting to dendrites. Similar mislocalization has been described in cell culture for deletion of the EVH1 domain of Homer 2a (Shiraishi, 1999). At present, it is not known which protein regulates Drosophila Homer subcellular localization. Given the finding that the EVH1 domain of Homer binds Drosophila Shank, it is possible that Shank might have a function similar to what has been implicated in vertebrates (Sala, 2001). Further genetic and biochemical studies will be required to address this question (Diagana, 2002).

In terms of candidate receptors in Drosophila to which Homer might bind, there are several putative mGluRs in the genome sequence database. Of these, only one, DmGluRA, has been characterized, and it has been pharmacologically classified as a group II mGluR (Parmentier, 1996). Consistent with this classification, the cytosolic domain of DmGluRA contains no Homer-binding motif and fails to bind Homer in yeast two-hybrid assay. In vertebrates, two ER-resident proteins, the ryanodine receptor (RyR) and the InsP3R, both control intracellular Ca²⁺ stores and contain Homer-binding motifs in their cytosolically disposed N termini (Tu, 1998). Moreover, the InsP3R coimmunoprecipitates with Homer proteins (Tu, 1998). There is a single InsP3R and a single RyR in Drosophila, both of which contain putative Homer-binding motifs. It will be of interest to determine whether Homer binds in vivo to either of these receptors (Diagana, 2002).

The finding that homer mutant flies are able to walk, fly, and exhibit an escape response to visual stimuli suggests that Homer has no essential role in vision or the performance of basic motor skills. However, homer mutants are hyperactive for both spontaneous locomotion and courtship behavior, implicating Homer in the control of locomotor activity. The homer mutants also exhibit deficits in behavioral plasticity, as assayed in a courtship conditioning paradigm. In this type of assay, the Drosophila memory mutant amnesiac shows specific defects in the retention of courtship conditioning. Similarly, homer mutants are capable of suppressing courtship behavior toward a nonreceptive mated female, but this suppression is not retained when subsequently tested with the virgin female. At present the possibility cannot be ruled out that the defective control of locomotor activity interferes somehow with the formation and retention of the conditioning. Addressing this will require the genetic separation of homer function in the control of locomotor activity and behavioral plasticity (Diagana, 2002).

In contrast to recent results suggesting a function for Homer-related proteins in axon pathfinding (Foa, 2001), no obvious pathfinding defects were detected in the CNS of homer mutant embryos. It is still possible, however, that Drosophila Homer could play a developmental role during synaptogenesis and that loss of Homer function results in structural defects undetectable at the light microscopy level used in this study. An alternative, but not mutually exclusive, possibility is that Drosophila Homer functions in the modulation of neuronal circuits by regulating synaptic plasticity, perhaps through the modulation of mGluR signaling. Loss-of-function of group I mGluRs in mice causes deficits in spatial learning and locomotor control, and although the basic synaptic physiology in these mutant animals is unaffected, aspects of synaptic plasticity are impaired. Vertebrate Shank and Homer have also been implicated in the regulation of the structure and function of the synaptic junction (Sala, 2001). Evidence for a physical interaction between Drosophila Homer and Shank raises the possibility of a similar synaptic function for these proteins in flies (Diagana, 2002).

GENE STRUCTURE

cDNA clone length - 1902

Bases in 5' UTR - 142

Exons - 7

Bases in 3' UTR - 575

PROTEIN STRUCTURE

Amino Acids - 394

Structural Domains

In the Berkeley Drosophila Genome Project sequence database, several expressed sequence tags (ESTs) have been identified encoding a gene product with high homology to the mouse protein Homer 1. These cDNAs encode a predicted protein of 394 amino acids. The same gene product has also been referred to as D-Homer or Dvh (Drosophila Ves-1 homolog). The N-terminal 120 amino acids of Drosophila Homer that contain the EVH1 domain show 73% amino acid identity to the rodent Homer 1 proteins. Although the Drosophila Homer C-terminal region overall shows only 25% identity with the Homer 1b protein, there are conserved amino acids within the CC domain and the two putative leucine zippers, which are thought to be involved in multimerization of Homer proteins (Tu, 1998; Xiao, 1998; Tadokoro, 1999). Alignment of homer cDNA and genomic sequences predicts a gene structure of seven exons (Diagana, 2002 and references therein).

date revised: 10 October 2002

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