BIOLOGICAL OVERVIEW

Four ionotropic glutamate receptors had been identified in Drosophila: DGluRI, a kainate-type receptor expressed in the CNS (Ultsch, 1992); DNMDAR, an NMDA-like receptor expressed in brain (Ultsch, 1993), and DGluRIIA and DGluRIIB, the subjects of this overview. DGluRIIA and DGluRIIB are closely linked genes encoding muscle-specific glutamate receptors (Schuster, 1991 and Petersen, 1997). Receptors are classified by whether or not they serve as ion channels, and by the type of chemical that serves to stimulate receptor activity. The two DGluRII receptors are ionotropic receptors, referring to the fact that they serve as ion channels. Although they are classified as non-NMDA types, they cannot be classified as either an AMPA or kainate type. These two genes are adjacent in the genome, have similar genomic organization, and are more closely related to each other than to any other glutamate receptors. Nonetheless, DGluRIIA and DGluRIIB share only 44% amino acid identity. The sequence of DGluRIIA is identical to vertebrate channels in the putative pore region, an area that is critical for Ca2+ permeability, while the sequence of DGluRIIB is divergent. These genes are expressed in all somatic muscles and are excluded from the nervous system (Currie, 1995 and Petersen, 1997). Such a configuration is known as postsynaptic, referring to the location of the glutamate channel on muscular synaptic boutons (Saitoe, 1997 and Petersen, 1997) targeted by neurons. By analogy with other channel proteins, the ligand-gated glutamate channels are likely to be multimeric assemblies of individual subunits.

Of major importance in synapse biology is the role of retrograde signaling: postsynaptic activity in the regulation of presynaptic structure. Activity-dependent mechanisms play a central role in shaping the pattern and strength of synaptic connections as they form during development and are modified during learning and memory throughout life. Evidence has begun to accumulate that suggests a role for postsynaptic activity in the regulation of presynaptic structure and function during development. The neuromuscular synapse can be used as model to dissect the molecular mechanisms of retrograde signaling that control synaptic strength during development. Such a mechanism might be used more generally in the regulation of synaptic plasticity. The Drosophila neuromuscular junction (NMJ) is used for these studies because it is possible to independently manipulate the genotype of the presynaptic or postsynaptic cell, and to study the consequences on both the structure and the function of the synapse in vivo (Peterson, 1997 and references).

The Drosophila NMJ shares several important features with central excitatory synapses in the vertebrate brain: it is glutamatergic, with homologous ionotropic glutamate receptors, and it is organized into a series of boutons that can be added or eliminated during development and plasticity. In addition, both the Drosophila NMJ and vertebrate central synapses exhibit dynamic functional plasticity. In Drosophila, this plasticity is revealed by genetic manipulations that alter (1) neuronal excitability (ether a go-go and Shaker: Budnik, 1990), (2) second messengers (dunce: Zhong, 1991), (3) protein kinases (Calcium/calmodulin dependent protein kinase II: Wang, 1994), (4) linker proteins (Discs large: Budnik, 1996), (5) cell adhesion molecules (Fasciclin II: Schuster, 1996a; Schuster, 1996b; Stewart, 1996 and Fasciclin I: Zhong and Shanley, 1995), and (6) transcription factors (CREB: Davis, 1996). All of these previous genetic manipulations have altered both the presynaptic and postsynaptic cells, so it has not been possible to assess the role of the target cell in synaptic plasticity. Alteration of the glutamate receptor allows for the exclusive targeting of postsynaptic cells, allowing an assessment of the role of the postsynaptic cell in determination of synaptic function (Peterson, 1997 and references).

The developmental history of the Drosophila NMJ makes it a good candidate synapse for retrograde regulation. As the Drosophila larvae develops from the first to third instar over a period of several days, there is at least a 100-fold increase in the surface area of the postsynaptic muscle. This increase in size leads to a dramatic decrease in input resistance, so that a larger synaptic current is required to depolarize the muscle. During this developmental period, there is a concomitant growth of the presynaptic nerve terminal, resulting in an increased number of both boutons and active zones per bouton. In fact, there is a tight correlation between muscle size and the number of synaptic boutons (Schuster, 1996a).

Before discussing the necessity for postulating a retrograde signal to compensate for postsynaptic receptor deficiencies (remember these glutamate receptors are postsynaptic), this discussion will focus on the effects of mutation of the glutamate receptor on postsynaptic function. Loss-of-function DGluRIIA mutants have been generated that lead to a decreased postsynaptic sensitivity to transmitter. Gain-of-function mutants, in which DGluRIIA is overexpressed, have been engineered that have an increased postsynaptic sensitivity to transmitter. What is the mechanism by which changes in the amount of DGluRIIA lead to differences in postsynaptic sensitivity to transmitter? The relationship between gene dosage of DGluRIIA and the miniature extrajunctional potential (mEJP) amplitude suggests that the density of channels may be an important determinant of quantal size (the amplitude of elementary synaptic currents). This implies that receptors are the limiting factor determining mEJP amplitude and is consistent with the idea that glutamate receptors are saturated by a single quantum (Wang, 1994). Alternatively, DGluRIIA and DGluRIIB may form channels with different properties. Relative levels of DGluRIIA could regulate the conductance of the channel, with higher proportions of DGluRIIA favoring higher conductance channels. In addition to changing quantal size, deletion of DGluRIIA leads to a reduction in the measured frequency of spontaneous mEJPs. Because there is a change in quantal size, conclusions cannot be drawn about the actual presynaptic rate of spontaneous vesicle fusions. However, it is likely that the postsynaptic response in DGluRIIA mutants has become so small that some events are lost in the noise of the recording. These events have become functionally silent (Peterson, 1997).

Analysis of DGluRIIA mutants reveals that a decreased postsynaptic sensitivity is compensated for by an increase in transmitter release from the neuron. The presynaptic neuron is thus regulated in response to a physiological change in the postsynaptic cell, indicating the existence of a retrograde signaling mechanism. This mechanism may be used during normal development to ensure that the muscle receives adequate amounts of transmitter during its rapid growth from embryonic to larval stages. As the muscle grows and its input resistance drops, a much larger synaptic current is required to depolarize the muscle and allow for efficient contraction. A retrograde signal would ensure a match between postsynaptic requirements for transmitter and presynaptic release characteristics. During normal development, the muscle requires increasing amounts of transmitter; thus, there may be no need for a mechanism to down-regulate the average number of released transmitter packets (quantal content) in the face of increased postsynaptic activity. This is consistent with the finding that increased amplitude of elementary synaptic currents (quantal size) does not lead to a down-regulation of quantal content (Peterson, 1997).

The identification of the existence of an unknown retrograde signal at the Drosophila NMJ leaves a number of open questions. (1) What is being sensed by the muscle that initiates the generation of this signal? The muscle could respond to synaptic depolarization, or it could be sensitive to a second messenger that is regulated by glutamate receptor function, such as calcium influx. (2) What is the presynaptic target of the postsynaptic signal? No sprouting of synaptic boutons or change in the calcium dependence of transmitter release is observed, suggesting that the increase in transmitter release is not secondary to gross structural plasticity or to a change in the function of the calcium sensor. The increase in presynaptic release may be due to an increase in calcium influx into the presynaptic terminal or to changes in the function of the release machinery. (3) What is the nature of the retrograde signal initiated by activity in the muscle? In Drosophila, unlike in vertebrates, each muscle is not regulated by a sensory neuron-to-motor neuron circuit, so the mechanism is unlikely to be cellular. Precedent exists for diffusible signals such as nitric oxide and arachidonic acid to function as retrograde signals for synaptic plasticity. Since the presynaptic and postsynaptic cell are in tight apposition throughout development, the signal could also involve membrane-bound molecules. These questions will be the subject of future genetic and physiological analyses (Peterson, 1997).

A retrograde signal from muscle to motoneuron could influence presynaptic release by increasing presynaptic structure. Bouton number was quantified for the junction at muscles 6 and 7 in abdominal segment A3. There was no change in bouton number when comparing wild type with larvae expressing increased PKA in muscle. Similarly, there is no change in presynaptic bouton number in larvae in which PKA is inhibited in muscle. Thus, a retrograde signal most likely regulates either the number of presynaptic active zones present in each presynaptic bouton or regulates some aspect of the presynaptic release mechanism (Davis, 1998).

PROTEIN STRUCTURE

Amino Acids - 883

Structural Domains

Insects and other invertebrates use glutamate as a neurotransmitter in the central nervous system and at the neuromuscular junction. A complementary DNA from Drosophila melanogaster, designated DGluR-II, has been isolated that encodes a distant homolog of the cloned mammalian ionotropic glutamate receptor family and is expressed in somatic muscle tissue of Drosophila embryos. Electrophysiological recordings made in Xenopus oocytes that express DGluR-II revealed depolarizing responses to L-glutamate and L-aspartate but low sensitivity to quisqualate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate. The DGluR-II protein may represent a distinct glutamate receptor subtype, which shares its structural design with other members of the ionotropic glutamate receptor family (Schuster, 1991).

Comparison of DGluR-IIA amino acid sequence with the rat glutamate receptor subtypes reveals overall amino acid identities between 26% and 28%. In all of the glutamate receptors analyzed, the highest conservation is found in the C-terminal half of the polypeptides, the putative ionotropic glutamate receptor 'core' domain (residues 408 to 822 of DGluR-IIA). Core sequence identities of 37% to 38% suggest that the DGluR-IIA protein is a distantly related member of the glutamate receptor subfamily. Hydropathy plot analysis results in a profile similar to that of rat GluR5. Although up to seven potential membrane spanning regions may be assigned to the mature polypeptide, more precise topology predictions remain highly speculative. However, some sequence features (including the distribution of charged residues around four putative core transmembrane regions; the distribution of conserved potential NH2-linked glycosylation sites in the hypothetical extracellular non-core sequence, and the distribution of conserved cysteine residues) are consistent with a four-transmembrane model proposed for the superfamily of ligand-gated ion channel proteins (Schuster, 1991)

DGluRIIA and DGluRIIB share 44% amino acid identity overall, with 51% identity in the highly conserved transmembrane region. Sequence analysis indicates that they are members of the AMPA/kainate supergroup but does not clearly classify them as either AMPA or kainate subtypes. The two receptors are more closely related to each other than to any other known glutamate receptor. In vertebrates, the calcium permeability of AMPA/kainate receptors is determined by the presence of a glutamine or arginine within the putative pore region. The sequence around this region (MQQ) is highly conserved and is present in DGluRIIA. However, in DGluRIIB, this sequence is LNQ, suggesting that the two receptors may differ in their physiological properties. Both receptors have numerous potential phosphorylation sites in their intracellular cytoplasmic tails; however, only DGluRIIA contains the ideal consensus site (RRXS) for protein kinase A. The clustering of some synaptic proteins is mediated by interactions between their C-terminal tails and a class of proteins containing protein-protein interaction modules known as PDZ domains. Neither DGluRIIA nor DGluRIIB contains a C-terminal sequence indicative of such an interaction (Peterson, 1997).

A retrograde signal from muscle to motoneuron could influence presynaptic release by increasing presynaptic structure. Bouton number was quantified for the junction at muscles 6 and 7 in abdominal segment A3. There is no change in bouton number when comparing wild type with larvae expressing increased PKA in muscle. Similarly, there is no change in presynaptic bouton number in larvae in which PKA is inhibited in muscle. Thus, a retrograde signal most likely regulates either the number of presynaptic active zones present in each presynaptic bouton or regulates some aspect of the presynaptic release mechanism (Davis, 1998).

date revised: 12 May 99

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