The proteomes of neurotransmitter receptor complexes form modular networks with distributed functionality underlying plasticity and behaviour

Andrew J Pocklington¹, Mark Cumiskey^2,3, J Douglas Armstrong¹ & Seth G N Grant^2,3

Article highlights

Synopsis

We present an integrated analysis of molecular organisation, signal transduction, physiology and diseases of a neurotransmitter receptor signalling complex as a step toward synapse systems biology. We also present a new model for understanding the molecular complexity of the synapse proteome and its relationship to synapse physiology.

Within the brain, neurons encode information as patterns of electrical activity in the form of action potentials. Communication between neurons occurs at specialised junctions (synapses), where information is transferred via chemical messengers (neurotransmitters). Neurotransmitters released from the presynaptic cell cross the synaptic cleft and bind to receptors embedded in the postsynaptic membrane. Different receptors play different roles: while AMPA receptors are responsible for the onward transmission of electrical signals, NMDA and metabotropic glutamate (mGluR) receptors process the information contained in patterns of neurotransmitter release, activating intracellular biochemical pathways that lead to changes in the properties of the neuron. Changes in synaptic properties in response to patterns of stimulation (experience), collectively known as synaptic plasticity, are commonly thought to form the basis of memory and learning.

Receptors, signalling enzymes and scaffolding molecules are assembled into complexes embedded in the postsynaptic density (PSD), a dense layer of proteins associated with the intracellular surface of the postsynaptic membrane. Proteomic studies have identified over 1000 different proteins in the PSD, making it one of the most complex molecular structures known to cell biology (Husi et al, 2000; Husi and Grant, 2001; Farr et al, 2004; Collins et al, 2005). These studies have also shown that receptors associate with specific subclusters of the PSD. Of interest here, the NMDA and mGluR receptors are linked within large subcomplexes referred to as MASC (MAGUK-Associated Signalling Complex) that comprise some 186 proteins. Given the fundamental role of the synapse in information processing, behaviour and disease, it is important to understand the functional organization of the PSD and the macromolecular complexes within it.

As a first step towards unravelling the functional complexities of the synaptic proteome, we have made a detailed analysis of the MASC complex. Our approach consisted of three stages (Figure 1). The first stage, in which proteins present in the complex were isolated and identified, has been reported elsewhere (Husi et al, 2000; Collins et al, 2005). Briefly, the complexes were biochemically isolated from mouse brain and their protein composition identified using antibodies and mass spectrometry.

Figure 1

A three-step strategy for analysis of synapse proteome organisation. Step 1 (Proteomics) was the collection of proteomic data identifying specific proteins. Step 2 (Annotation) was the collection of specific structural and functional data on individual proteins from Step 1, which was followed by Step 3 (Analysis) using statistical and network approaches.

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Proteomic profiling was followed by systematic annotation, involving extensive literature searching and manual data curation. To investigate the involvement of proteins in biochemical pathways, they were annotated for structure and function. MASC proteins were highly enriched for domains associated with key elements of synaptic signalling such as calcium binding, scaffolding and phosphorylation. The functional roles of proteins reflected the presence of diverse signalling pathways, suggesting that these are co-ordinated within the complex.

To investigate the evolutionary conservation of MASC proteins, we searched for orthologues in yeast and fruit fly. While all protein families represented in the complex are evolutionarily conserved from yeast, most show significant expansion associated with the emergence of multicellularity. This clearly reflects specialisation of the complex for intercellular signalling.

To evaluate the role of the complex in synaptic function, behaviour and disease, an array of phenotypic data was collated from the scientific literature. This comprised of physiological data obtained from rodent studies, where mutations or drugs that specifically interfere with a given protein were tested for their effects on synapse electrophysiology or behaviour. Reports on the involvement of specific molecules in human diseases were also collated. A complete summary of the curated information and its provenance is provided in the supplementary material, allowing verification and reuse by other studies. In total, almost a quarter of MASC proteins were known to be essential for normal synaptic plasticity, with approximately the same number involved in rodent behaviour. Nearly a third of MASC proteins were implicated in human mental illness. Of these, more than twice as many were linked to cognitive (schizophrenia, mental retardation) than to affective disorders (bipolar disorder, depression). While the role of NMDA receptors in plasticity and learning was widely appreciated, it was not obvious that so many other proteins involved in synaptic plasticity, behaviour and disease would be brought into close association by the complex.

In the final stage, functional, phenotypic and phylogenetic annotations were first examined for significant molecular overlap. Statistical analysis of the number of proteins common to each pair of annotations uncovered an extremely high overlap between synaptic plasticity, behaviour and disease, pointing to a common molecular foundation. Of the psychiatric disorders, schizophrenia possessed a highly significant overlap with both synaptic plasticity and behaviour. Linking these higher-level processes to molecular function, NMDA receptor signalling was strongly associated with synaptic plasticity, behaviour and cognitive disorders (primarily schizophrenia), while metabotropic signalling (via mGluRs) showed a contrasting association with affective disorders. Although cognitive and affective disorders were associated with different input pathways, overall they possessed a high degree of molecular overlap.

We then investigated the organisation of MASC proteins, looking at their assembly into a functional complex via protein–protein interactions. Curating high-quality interaction data from the literature, we generated a network representation of the complex (in which proteins were represented as vertices and interactions as edges linking pairs of vertices) and examined its structure. The average number of interactions separating any two proteins was very low, allowing for rapid integration of information and coordination of responses. Protein connectivity followed an approximately power-law distribution—a property linked to structural and functional robustness (Albert et al, 2000; Jeong et al, 2001). To investigate higher-level structure, we sought to evaluate any clustering inherent in the network. The network possessed a clearly modular structure, with statistical analysis revealing significant overlap between individual modules and consistent sets of functional annotations.

Our analysis led us to propose that common principles underlie MASC organisation at all levels, with functions being distributed over proteins and modules. This modular organisation is summarised in Figure 6, with the complex as a whole coordinating the induction of synaptic plasticity. Several predictions of the model have been confirmed, most significantly the correlation between protein connectivity and quantitative perturbation of long-term potentiation of synaptic transmission. This study represents the first attempt at mapping synaptic organisation, and may now be extended to the synapse proteome as a whole.

Figure 6

Modular structure and functional organisation within MASC. MASC proteins are clustered into modules with well-defined functional roles. Primary signal reception modules (blue) are formed around ionotropic and metabotropic receptors. These inputs are integrated within a large signal-processing module (red) responsible for overall co-ordination of functional processes. Other sources of input ('other receptors') may feed into this module directly, or through smaller input/processing modules (such as cluster 10, Figure 3). Note that, within this general structure, individual modules may play multiple functional roles (e.g. regulation of effector mechanisms by input modules 1 and 2). In this way, information processing and regulation of effector pathways are distributed over multiple modules. The general principles underlying functional organisation within MASC are apparent in the co-ordinated regulation of common downstream effector pathways: a single, large module (red) is responsible for overall co-ordination; several intermediate modules (yellow) regulate overlapping sets of pathways, while numerous small modules (green) are specific to individual effector responses. Note that this is not a simple feed-forward mechanism, rather a dynamical balance between multiple functional processes. The resulting synchronisation of multiple cell-biological processes induces synaptic plasticity, manifest at a higher level of neurological function through behavioural learning. Numbering of the five largest clusters reflects that of Figure 3, as do the interactions between them (solid black lines). Internal/external modulation of MASC function and the regulation of effector mechanisms are denoted by dashed lines. The red line between clusters 4 and 5 denotes the fact that other interactions (e.g. phosphorylation) play an important role in MASC function.

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