Showing papers on "Protein–protein interaction published in 2009"

Identification of direct residue contacts in protein-protein interaction by message passing.

Abstract: Understanding the molecular determinants of specificity in protein–protein interaction is an outstanding challenge of postgenome biology. The availability of large protein databases generated from sequences of hundreds of bacterial genomes enables various statistical approaches to this problem. In this context covariance-based methods have been used to identify correlation between amino acid positions in interacting proteins. However, these methods have an important shortcoming, in that they cannot distinguish between directly and indirectly correlated residues. We developed a method that combines covariance analysis with global inference analysis, adopted from use in statistical physics. Applied to a set of >2,500 representatives of the bacterial two-component signal transduction system, the combination of covariance with global inference successfully and robustly identified residue pairs that are proximal in space without resorting to ad hoc tuning parameters, both for heterointeractions between sensor kinase (SK) and response regulator (RR) proteins and for homointeractions between RR proteins. The spectacular success of this approach illustrates the effectiveness of the global inference approach in identifying direct interaction based on sequence information alone. We expect this method to be applicable soon to interaction surfaces between proteins present in only 1 copy per genome as the number of sequenced genomes continues to expand. Use of this method could significantly increase the potential targets for therapeutic intervention, shed light on the mechanism of protein–protein interaction, and establish the foundation for the accurate prediction of interacting protein partners.

Yeast Two-Hybrid, a Powerful Tool for Systems Biology

Abstract: A key property of complex biological systems is the presence of interaction networks formed by its different components, primarily proteins. These are crucial for all levels of cellular function, including architecture, metabolism and signalling, as well as the availability of cellular energy. Very stable, but also rather transient and dynamic protein-protein interactions generate new system properties at the level of multiprotein complexes, cellular compartments or the entire cell. Thus, interactomics is expected to largely contribute to emerging fields like systems biology or systems bioenergetics. The more recent technological development of high-throughput methods for interactomics research will dramatically increase our knowledge of protein interaction networks. The two most frequently used methods are yeast two-hybrid (Y2H) screening, a well established genetic in vivo approach, and affinity purification of complexes followed by mass spectrometry analysis, an emerging biochemical in vitro technique. So far, a majority of published interactions have been detected using an Y2H screen. However, with the massive application of this method, also some limitations have become apparent. This review provides an overview on available yeast two-hybrid methods, in particular focusing on more recent approaches. These allow detection of protein interactions in their native environment, as e.g. in the cytosol or bound to a membrane, by using cytosolic signalling cascades or split protein constructs. Strengths and weaknesses of these genetic methods are discussed and some guidelines for verification of detected protein-protein interactions are provided.

Induction of protein-protein interactions in live cells using light

Abstract: Protein-protein interactions are essential for many cellular processes. We have developed a technology called light-activated dimerization (LAD) to artificially induce protein hetero- and homodimerization in live cells using light. Using the FKF1 and GIGANTEA (GI) proteins of Arabidopsis thaliana, we have generated protein tags whose interaction is controlled by blue light. We demonstrated the utility of this system with LAD constructs that can recruit the small G-protein Rac1 to the plasma membrane and induce the local formation of lamellipodia in response to focal illumination. We also generated a light-activated transcription factor by fusing domains of GI and FKF1 to the DNA binding domain of Gal4 and the transactivation domain of VP16, respectively, showing that this technology is easily adapted to other systems. These studies set the stage for the development of light-regulated signaling molecules for controlling receptor activation, synapse formation and other signaling events in organisms.

Design of protein-interaction specificity gives selective bZIP-binding peptides.

Abstract: Interaction specificity is a required feature of biological networks and a necessary characteristic of protein or small-molecule reagents and therapeutics. The ability to alter or inhibit protein interactions selectively would advance basic and applied molecular science. Assessing or modelling interaction specificity requires treating multiple competing complexes, which presents computational and experimental challenges. Here we present a computational framework for designing protein-interaction specificity and use it to identify specific peptide partners for human basic-region leucine zipper (bZIP) transcription factors. Protein microarrays were used to characterize designed, synthetic ligands for all but one of 20 bZIP families. The bZIP proteins share strong sequence and structural similarities and thus are challenging targets to bind specifically. Nevertheless, many of the designs, including examples that bind the oncoproteins c-Jun, c-Fos and c-Maf (also called JUN, FOS and MAF, respectively), were selective for their targets over all 19 other families. Collectively, the designs exhibit a wide range of interaction profiles and demonstrate that human bZIPs have only sparsely sampled the possible interaction space accessible to them. Our computational method provides a way to systematically analyse trade-offs between stability and specificity and is suitable for use with many types of structure-scoring functions; thus, it may prove broadly useful as a tool for protein design.

Tissue specificity and the human protein interaction network.

Abstract: A protein interaction network describes a set of physical associations that can occur between proteins. However, within any particular cell or tissue only a subset of proteins is expressed and so only a subset of interactions can occur. Integrating interaction and expression data, we analyze here this interplay between protein expression and physical interactions in humans. Proteins only expressed in restricted cell types, like recently evolved proteins, make few physical interactions. Most tissue‐specific proteins do, however, bind to universally expressed proteins, and so can function by recruiting or modifying core cellular processes. Conversely, most ‘housekeeping’ proteins that are expressed in all cells also make highly tissue‐specific protein interactions. These results suggest a model for the evolution of tissue‐specific biology, and show that most, and possibly all, ‘housekeeping’ proteins actually have important tissue‐specific molecular interactions. Mol Syst Biol. 5: 260

An integrated workflow for charting the human interaction proteome: insights into the PP2A system

Abstract: Protein complexes represent major functional units for the execution of biological processes. Systematic affinity purification coupled with mass spectrometry (AP-MS) yielded a wealth of information on the compendium of protein complexes expressed in Saccharomyces cerevisiae. However, global AP-MS analysis of human protein complexes is hampered by the low throughput, sensitivity and data robustness of existing procedures, which limit its application for systems biology research. Here, we address these limitations by a novel integrated method, which we applied and benchmarked for the human protein phosphatase 2A system. We identified a total of 197 protein interactions with high reproducibility, showing the coexistence of distinct classes of phosphatase complexes that are linked to proteins implicated in mitosis, cell signalling, DNA damage control and more. These results show that the presented analytical process will substantially advance throughput and reproducibility in future systematic AP-MS studies on human protein complexes.

Targeted tandem affinity purification of PSD-95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins

Abstract: The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD-95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD-95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including glutamate receptors, K+ channels, scaffolding and signaling proteins, were recovered. Network clustering of protein interactions generated five connected clusters, with two clusters containing all the major ionotropic glutamate receptors and one cluster with voltage-dependent K+ channels. Annotation of clusters with human disease associations revealed that multiple disorders map to the network, with a significant correlation of schizophrenia within the glutamate receptor clusters. This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease.

Visualization of molecular interactions using bimolecular fluorescence complementation analysis: Characteristics of protein fragment complementation

Abstract: Investigations of the molecular processes that sustain life must include studies of these processes in their normal cellular environment. The bimolecular fluorescence complementation (BiFC) assay provides an approach for the visualization of protein interactions and modifications in living cells. This assay is based on the facilitated association of complementary fragments of a fluorescent protein that are fused to interaction partners. Complex formation by the interaction partners tethers the fluorescent protein fragments in proximity to each other, which can facilitate their association. The BiFC assay enables sensitive visualization of protein complexes with high spatial resolution. The temporal resolution of BiFC analysis is limited by the time required for fluorophore formation, as well as the stabilization of complexes by association of the fluorescent protein fragments. Many modifications and enhancements to the BiFC assay have been developed. The multicolor BiFC assay enables simultaneous visualization of multiple protein complexes in the same cell, and can be used to investigate competition among mutually exclusive interaction partners for complex formation in cells. The ubiquitin-mediated fluorescence complementation (UbFC) assay enables visualization of covalent ubiquitin family peptide conjugation to substrate proteins in cells. The BiFC assay can also be used to visualize protein binding to specific chromatin domains, as well as other molecular scaffolds in cells. BiFC analysis therefore provides a powerful approach for the visualization of a variety of processes that affect molecular proximity in living cells. The visualization of macromolecular interactions and modifications is of great importance owing to the central roles of proteins, nucleic acids and other macromolecular complexes in the regulation of cellular functions. This tutorial review describes the BiFC assay, and discusses the advantages and disadvantages of this experimental approach. The review will be of interest to scientists interested in the investigation of macromolecular interactions or modifications who need exquisite sensitivity for the detection of their complexes or conjugates of interest.

Human Cancer Protein-Protein Interaction Network: A Structural Perspective

Abstract: Protein-protein interaction networks provide a global picture of cellular function and biological processes. Some proteins act as hub proteins, highly connected to others, whereas some others have few interactions. The dysfunction of some interactions causes many diseases, including cancer. Proteins interact through their interfaces. Therefore, studying the interface properties of cancer-related proteins will help explain their role in the interaction networks. Similar or overlapping binding sites should be used repeatedly in single interface hub proteins, making them promiscuous. Alternatively, multi-interface hub proteins make use of several distinct binding sites to bind to different partners. We propose a methodology to integrate protein interfaces into cancer interaction networks (ciSPIN, cancer structural protein interface network). The interactions in the human protein interaction network are replaced by interfaces, coming from either known or predicted complexes. We provide a detailed analysis of cancer related human protein-protein interfaces and the topological properties of the cancer network. The results reveal that cancer-related proteins have smaller, more planar, more charged and less hydrophobic binding sites than non-cancer proteins, which may indicate low affinity and high specificity of the cancer-related interactions. We also classified the genes in ciSPIN according to phenotypes. Within phenotypes, for breast cancer, colorectal cancer and leukemia, interface properties were found to be discriminating from non-cancer interfaces with an accuracy of 71%, 67%, 61%, respectively. In addition, cancer-related proteins tend to interact with their partners through distinct interfaces, corresponding mostly to multi-interface hubs, which comprise 56% of cancer-related proteins, and constituting the nodes with higher essentiality in the network (76%). We illustrate the interface related affinity properties of two cancer-related hub proteins: Erbb3, a multi interface, and Raf1, a single interface hub. The results reveal that affinity of interactions of the multi-interface hub tends to be higher than that of the single-interface hub. These findings might be important in obtaining new targets in cancer as well as finding the details of specific binding regions of putative cancer drug candidates.

Bayesian Modeling of the Yeast SH3 Domain Interactome Predicts Spatiotemporal Dynamics of Endocytosis Proteins

Abstract: SH3 domains are peptide recognition modules that mediate the assembly of diverse biological complexes. We scanned billions of phage-displayed peptides to map the binding specificities of the SH3 domain family in the budding yeast, Saccharomyces cerevisiae. Although most of the SH3 domains fall into the canonical classes I and II, each domain utilizes distinct features of its cognate ligands to achieve binding selectivity. Furthermore, we uncovered several SH3 domains with specificity profiles that clearly deviate from the two canonical classes. In conjunction with phage display, we used yeast twohybrid and peptide array screening to independently identify SH3 domain binding partners. The results from the three complementary techniques were integrated using a Bayesian algorithm to generate a high-confidence yeast SH3 domain interaction map. The interaction map was enriched for proteins involved in endocytosis, revealing a set of SH3-mediated interactions that underlie formation of protein complexes essential to this biological pathway. We used the SH3 domain interaction network to predict the dynamic localization of several previously uncharacterized endocytic proteins, and our analysis suggests a novel role for the SH3 domains of Lsb3p and Lsb4p as hubs that recruit and assemble several endocytic complexes.

Evolutionarily Conserved Herpesviral Protein Interaction Networks

Abstract: Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1), murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H), and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species.

EIN2, the central regulator of ethylene signalling, is localized at the ER membrane where it interacts with the ethylene receptor ETR1.

Abstract: Genetic studies have identified the membrane protein EIN2 (ethylene insensitive 2) as a central component of ethylene signalling in Arabidopsis. In addition, EIN2 might take part in multiple hormone signalling pathways and in response to pathogens as demonstrated by recent genetic and biochemical studies. Here we show, by an integrated approach using in vivo and in vitro fluorescence techniques, that EIN2 is localized at the ER (endoplasmic reticulum) membrane where it shows specific interaction with the ethylene receptor protein ETR1.

14-3-3 and FHA Domains Mediate Phosphoprotein Interactions

Abstract: Many aspects of plant growth and development require specific protein interactions to carry out biochemical and cellular functions. Several proteins mediate these interactions, two of which specifically recognize phosphoproteins: 14-3-3 proteins and proteins with FHA domains. These are the only phosphobinding domains identified in plants. Both domains are present in animals and plants, and are used by plant proteins to regulate metabolic, developmental, and signaling pathways. 14-3-3s regulate sugar metabolism, proton gradients, and control transcription factor localization. FHA domains are modular domains often found in multidomain proteins that are involved in signal transduction and plant development.

Structure of protein interaction networks and their implications on drug design.

Abstract: Protein-protein interaction networks (PINs) are rich sources of information that enable the network properties of biological systems to be understood. A study of the topological and statistical properties of budding yeast and human PINs revealed that they are scale-rich and configured as highly optimized tolerance (HOT) networks that are similar to the router-level topology of the Internet. This is different from claims that such networks are scale-free and configured through simple preferential-attachment processes. Further analysis revealed that there are extensive interconnections among middledegree nodes that form the backbone of the networks. Degree distributions of essential genes, synthetic lethal genes, synthetic sick genes, and human drug-target genes indicate that there are advantageous drug targets among nodes with middle- to low-degree nodes. Such network properties provide the rationale for combinatorial drugs that target less prominent nodes to increase synergetic efficacy and create fewer side effects.

Toward a quantitative theory of intrinsically disordered proteins and their function

Abstract: A large number of proteins are sufficiently unstable that their full 3D structure cannot be resolved. The origins of this intrinsic disorder are not well understood, but its ubiquitous presence undercuts the principle that a protein's structure determines its function. Here we present a quantitative theory that makes predictions regarding the role of intrinsic disorder in protein structure and function. In particular, we discuss the implications of analytical solutions of a series of fundamental thermodynamic models of protein interactions in which disordered proteins are characterized by positive folding free energies. We validate our predictions by assigning protein function by using the gene ontology classification--in which "protein binding", "catalytic activity", and "transcription regulator activity" are the three largest functional categories--and by performing genome-wide surveys of both the amount of disorder in these functional classes and binding affinities for both prokaryotic and eukaryotic genomes. Specifically, without assuming any a priori structure-function relationship, the theory predicts that both catalytic and low-affinity binding (K(d) greater, >or= 0(-7) M) proteins prefer ordered structures, whereas only high-affinity binding proteins (found mostly in eukaryotes) can tolerate disorder. Relevant to both transcription and signal transduction, the theory also explains how increasing disorder can tune the binding affinity to maximize the specificity of promiscuous interactions. Collectively, these studies provide insight into how natural selection acts on folding stability to optimize protein function.

The determination of protein-protein interactions by the mating-based split-ubiquitin system (mbSUS).

Abstract: Dynamic and reversible protein-protein interactions have a pivotal function in all living cells. For instance, protein-protein interactions are involved in the assembly and regulation of multimeric enzymes and transcription factors, various signal response pathways, intracellular sorting and movement of proteins and membrane vesicles, cell-to-cell protein transport, and many others. Here we provide a detailed protocol for the mating-based split-ubiquitin system (mbSUS), which is a sensitive and user-friendly alternative to the classical yeast two-hybrid system in particular. mbSUS relies on the ubiquitin-degradation pathway as a sensor for protein-protein interactions. Thus, mbSUS is predominantly suitable for the determination of full-length proteins localized in the cytoplasm and in or at membrane compartments, without the need for their truncation and nuclear mislocation. In addition, we present a set of Gateway compatible mbSUS vectors that allow the rapid generation of constructs for fast and efficient interaction studies. An additional vector is introduced that allows the extension of mbSUS for the analysis of oligomeric protein complex formation and competition assays in vivo. In summary, mbSUS provides an additional versatile tool for protein-protein interaction studies, which is complementary to in planta assays such as BiFC and FRET.

Dynamic map of protein interactions in the Escherichia coli chemotaxis pathway

Abstract: Protein–protein interactions play key roles in virtually all cellular processes, often forming complex regulatory networks. A powerful tool to study interactions in vivo is fluorescence resonance energy transfer (FRET), which is based on the distance-dependent energy transfer from an excited donor to an acceptor fluorophore. Here, we used FRET to systematically map all protein interactions in the chemotaxis signaling pathway in Escherichia coli, one of the most studied models of signal transduction, and to determine stimulation-induced changes in the pathway. Our FRET analysis identified 19 positive FRET pairs out of the 28 possible protein combinations, with 9 pairs being responsive to chemotactic stimulation. Six stimulation-dependent and five stimulation-independent interactions were direct, whereas other interactions were apparently mediated by scaffolding proteins. Characterization of stimulation-induced responses revealed an additional regulation through activity dependence of interactions involving the adaptation enzyme CheB, and showed complex rearrangement of chemosensory receptors. Our study illustrates how FRET can be efficiently employed to study dynamic protein networks in vivo.

Proteomic analysis of an alpha7 nicotinic acetylcholine receptor interactome.

Abstract: The alpha7 nicotinic acetylcholine receptor (nAChR) is well established as the principal high-affinity alpha-bungarotoxin-binding protein in the mammalian brain. We isolated carbachol-sensitive alpha-bungarotoxin-binding complexes from total mouse brain tissue by affinity immobilization followed by selective elution, and these proteins were fractionated by SDS-PAGE. The proteins in subdivided gel lane segments were tryptically digested, and the resulting peptides were analyzed by standard mass spectrometry. We identified 55 proteins in wild-type samples that were not present in comparable brain samples from alpha7 nAChR knockout mice that had been processed in a parallel fashion. Many of these 55 proteins are novel proteomic candidates for interaction partners of the alpha7 nAChR, and many are associated with multiple signaling pathways that may be implicated in alpha7 function in the central nervous system. The newly identified potential protein interactions, together with the general methodology that we introduce for alpha-bungarotoxin-binding protein complexes, form a new platform for many interesting follow-up studies aimed at elucidating the physiological role of neuronal alpha7 nAChRs.

Copper(I)-mediated protein-protein interactions result from suboptimal interaction surfaces.

Abstract: The homoeostasis of metal ions in cells is the result of the contribution of several cellular pathways that involve transient, often weak, protein-protein interactions. Metal transfer typically implies the formation of adducts where the metal itself acts as a bridge between proteins, by co-ordinating residues of both interacting partners. In the present study we address the interaction between the human copper(I)-chaperone HAH1 (human ATX1 homologue) and a metal-binding domain in one of its partners, namely the P-type copper-transporting ATPase, ATP7A (ATPase, Cu+ transporting, alpha polypeptide). The adduct was structurally characterized in solution, in the presence of copper(I), and through X-ray crystallography, upon replacing copper(I) with cadmium(II). Further insight was obtained through molecular modelling techniques and site-directed mutagenesis. It was found that the interaction involves a relatively small interface (less than 1000 A(2), 1 A=0.1 nm) with a low fraction of non-polar atoms. These observations provide a possible explanation for the low affinity of the two apoproteins. It appears that electrostatics is important in selecting which domain of the ATPase is able to form detectable amounts of the metal-mediated adduct with HAH1.

The Modular Organization of Protein Interactions in Escherichia coli

Abstract: Escherichia coli serves as an excellent model for the study of fundamental cellular processes such as metabolism, signalling and gene expression. Understanding the function and organization of proteins within these processes is an important step towards a ‘systems’ view of E. coli. Integrating experimental and computational interaction data, we present a reliable network of 3,989 functional interactions between 1,941 E. coli proteins (∼45% of its proteome). These were combined with a recently generated set of 3,888 high-quality physical interactions between 918 proteins and clustered to reveal 316 discrete modules. In addition to known protein complexes (e.g., RNA and DNA polymerases), we identified modules that represent biochemical pathways (e.g., nitrate regulation and cell wall biosynthesis) as well as batteries of functionally and evolutionarily related processes. To aid the interpretation of modular relationships, several case examples are presented, including both well characterized and novel biochemical systems. Together these data provide a global view of the modular organization of the E. coli proteome and yield unique insights into structural and evolutionary relationships in bacterial networks.

Native electrophoretic techniques to identify protein–protein interactions

Abstract: Permanent protein-protein interactions are commonly identified by co-purification of two or more protein components using techniques like co-immunoprecipitation, tandem affinity purification and native electrophoresis. Here we focus on blue-native electrophoresis, clear-native electrophoresis, high-resolution clear-native electrophoresis and associated techniques to identify stable membrane protein complexes and detergent-labile physiological supercomplexes. Hints for dynamic protein-protein interactions can be obtained using two-hybrid techniques but not from native electrophoresis and other protein isolation techniques except after covalent cross-linking of interacting proteins in vivo prior to protein separation.

Residues in a Highly Conserved Claudin-1 Motif Are Required for Hepatitis C Virus Entry and Mediate the Formation of Cell-Cell Contacts

Abstract: Claudin-1, a component of tight junctions between liver hepatocytes, is a hepatitis C virus (HCV) late-stage entry cofactor. To investigate the structural and functional roles of various claudin-1 domains in HCV entry, we applied a mutagenesis strategy. Putative functional intracellular claudin-1 domains were not important. However, we identified seven novel residues in the first extracellular loop that are critical for entry of HCV isolates drawn from six different subtypes. Most of the critical residues belong to the highly conserved claudin motif W 30 -GLW 51 -C 54 -C 64 . Alanine substitutions of these residues did not impair claudin-1 cell surface expression or lateral protein interactions within the plasma membrane, including claudin-1-claudin-1 and claudin-1-CD81 interactions. However, these mutants no longer localized to cell-cell contacts. Based on our observations, we propose that cell-cell contacts formed by claudin-1 may generate specialized membrane domains that are amenable to HCV entry.

Amyloidogenic regions and interaction surfaces overlap in globular proteins related to conformational diseases.

Abstract: Protein aggregation underlies a wide range of human disorders. The polypeptides involved in these pathologies might be intrinsically unstructured or display a defined 3D-structure. Little is known about how globular proteins aggregate into toxic assemblies under physiological conditions, where they display an initially folded conformation. Protein aggregation is, however, always initiated by the establishment of anomalous protein-protein interactions. Therefore, in the present work, we have explored the extent to which protein interaction surfaces and aggregation-prone regions overlap in globular proteins associated with conformational diseases. Computational analysis of the native complexes formed by these proteins shows that aggregation-prone regions do frequently overlap with protein interfaces. The spatial coincidence of interaction sites and aggregating regions suggests that the formation of functional complexes and the aggregation of their individual subunits might compete in the cell. Accordingly, single mutations affecting complex interface or stability usually result in the formation of toxic aggregates. It is suggested that the stabilization of existing interfaces in multimeric proteins or the formation of new complexes in monomeric polypeptides might become effective strategies to prevent disease-linked aggregation of globular proteins.

Identifying Novel Protein-Protein Interactions Using Co-Immunoprecipitation and Mass Spectroscopy

Abstract: Proteomics has evolved from genomic science due to the convergence of advances in protein chemistry, separations, mass spectroscopy, and peptide and protein databases. Where identifying protein-protein interactions was once limited to yeast two-hybrid analyses or empirical data, protein-protein interactions can now be examined in both cells and native tissues by precipitation of the protein complex of interest. Coupling this field to receptor pharmacology has recently allowed for the identification of proteins that differentially and selectively interact with receptors and are integral to their biological effects. It is becoming increasingly apparent that receptors in neurons do not exist as singular independent units, but rather are part of large macromolecular complexes of interacting proteins. It is a primary quest of neuroscience to piece together these interactions and to characterize the regulatory signalplexes of all proteins. This unit presents co-immunoprecipitation-coupled mass spectroscopy as one way of identifying signalplex partners.