Historically, the activation and inhibition of GPCR (G-protein-coupled receptor) function have been a very successful avenue for drug discovery and development. However, it is clear that receptors do not function in isolation but are impacted by other proteins. These proteins may alter either binding or functional responses. Identification and study of these interactions have grown rapidly in recent years and continue to do so, resulting in a plethora of potential receptor–protein connections. These associations can be regarded as alternative intervention points to modulate GPCR function and may not only provide alternative ways to modify receptor activity but also to exploit new chemical space for drug-like molecules. Such interactions may account for side-effects or undesirable properties associated with otherwise well-validated GPCR targets. Understanding and/or intervening in these interactions may allow scientists to progress those targets that may have been deemed unsuitable for therapeutic intervention. The present study reviews the opportunities for utilizing receptor interacting proteins as potential drug targets and the issues associated with them.

Historically, the activation and inhibition of GPCR (G-protein-coupled receptor) function have been a very successful avenue for drug discovery and development. Marketed drugs for both GPCR agonists (e.g. Sumatriptan; 5-HT1 receptor) and antagonists (e.g. Loratidine; H2 receptor) exist, and there are a number of compounds in clinical trials exploiting allosteric-binding sites present on GPCRs (e.g. Cinacalcet; Ca2+-sensing receptor, which has recently been launched in the U.S.A.).

However, it is clear that receptors do not function in isolation but are impacted by a myriad of other proteins. These proteins may alter not only ligand binding or functional responses but also such parameters as receptor localization and processing. The identification and study of these interactions and interacting proteins have grown rapidly in recent years and continue to do so, resulting in a plethora of potential receptor–protein connections. Whereas GPCRs constitute 15% of the druggable genome and 25% of molecular targets of experimental and marketed drugs, GPCR accessory proteins are an as yet unexplored area that may offer the opportunity to expand the druggable genome [1].

Proteins that interact with GPCRs can be segregated into groups depending on their role or function. These can be broadly categorized as proteins involved in GPCR homo- and heterodimerization, maturation and localization and alterations in pharmacology [2]. These associations may be regarded as alternative intervention points to modulate GPCRs and may not only provide alternative ways to modify receptor activity but also to exploit new chemical space for drug-like molecules. This review will outline, using key examples, the range of receptor interactions that are currently known and their potential for modulating GPCR indirectly.

As with any other class of proteins, a number of criteria must be satisfied before they can be considered as potential drug targets. These criteria include the physiological relevance of the interaction, evidence of involvement in a disease state or an animal model predictive for a disease, site(s) on the target that are amenable to small-molecule binding resulting in a change of activity and, related to this, the ability to develop an assay suitable for compound screening and testing.

Table 1
Summary of known interactions between D1-like dopamine receptors and accessory proteins

i3, third intracellular loop of the D1 receptor; COP, coatomer protein complex.

Interacting protein Interaction point on receptor Functional role Reference 
D1 receptor    
 NMDA NR1 C-terminus Decreased NMDA-mediated cell death; targets D1 receptor to membrane; prevents agonist-induced D1 receptor internalization [4,11
 NMDA NR2A C-terminus Inhibition of NMDA-mediated currents [5
 Calcyon C-terminus Allows D1 receptor to signal through Ca2+; regulates proportion of high-affinity state of D1 receptor [6,12,13
 NF-M i3 Modifies expression and regulation [7
 ADA  Implicated in heterodimerization of A1 and D1 receptors [10
 DRiP78 C-terminus ER export [14
 γCOP C-terminus ER export [17
 A1 adenosine receptor Not known Negative modulation by A1 receptor of D1 receptor cAMP signalling [9
 D3 dopamine receptor Not known Unclear, but cell-type-dependent [18
 AT1 angiotensin receptor  Unclear, but cell-type-dependent [19
D5 receptor    
 GABAA γ2 C-terminus Mutual inhibition of signalling function [5
Interacting protein Interaction point on receptor Functional role Reference 
D1 receptor    
 NMDA NR1 C-terminus Decreased NMDA-mediated cell death; targets D1 receptor to membrane; prevents agonist-induced D1 receptor internalization [4,11
 NMDA NR2A C-terminus Inhibition of NMDA-mediated currents [5
 Calcyon C-terminus Allows D1 receptor to signal through Ca2+; regulates proportion of high-affinity state of D1 receptor [6,12,13
 NF-M i3 Modifies expression and regulation [7
 ADA  Implicated in heterodimerization of A1 and D1 receptors [10
 DRiP78 C-terminus ER export [14
 γCOP C-terminus ER export [17
 A1 adenosine receptor Not known Negative modulation by A1 receptor of D1 receptor cAMP signalling [9
 D3 dopamine receptor Not known Unclear, but cell-type-dependent [18
 AT1 angiotensin receptor  Unclear, but cell-type-dependent [19
D5 receptor    
 GABAA γ2 C-terminus Mutual inhibition of signalling function [5

The question of physiological relevance is of utmost importance; the use of such techniques as the yeast two-hybrid system allows us to identify binding partners but in a highly artificial system. What is needed to confirm putative binding partners as true physiological interactors and, therefore, potential drug targets, is additional evidence such as expression in the same tissues or cell types and the demonstration of interactions in co-immunoprecipitation experiments using tissue preparations.

The identification of hit compounds requires firstly the identification of site(s) that are suitable for small molecule binding but also offers the opportunity for specificity/selectivity. Modulating receptor–interacting protein contacts by trying to design small molecules to interfere with the protein–protein interface(s) is considered by many as a less productive approach than inducing conformational change by small molecule intervention at a distal site. After identifying a target site, the development of a robust screening assay suitable for the testing of at least a focused set of compounds (∼50000 compounds) or a typically sized compound library (∼500000 compounds) is needed. The iterative chemical optimization of hits to lead compounds requires the development of not only primary assays to determine the affinity at, and/or efficacy of the accessory protein, but also requires selectivity assays against related proteins and possibly the associated GPCR. The optimization process may also offer significant challenges for the medicinal chemist. Whereas a great deal of knowledge has accumulated regarding preferred pharmacophores and privileged structures for the major target families, this would have to be developed for individual accessory proteins, e.g. calcyon or groups of proteins, such as RGS or homer proteins. Many accessory proteins, in particular cytosolic proteins, may be more amenable to crystal structure determination than GPCRs and so could benefit from this information for rational drug design. With the efforts currently being undertaken (see e.g. www.mepnet.org), it is clear that this information is seen as key to improving rational drug design approaches for GPCRs [3].

For proteins that are not sufficiently mature to have been directly implicated in a disease, hypothesis-based investigation using in vivo systems is the next milestone in progressing potential drug targets. For accessory proteins that interact with classical GPCRs, it may be possible to make use of established models, such as behavioural models, or biochemical techniques, such as microdialysis, to determine the effects of chemical modulators. On the other hand, where accessory proteins are being used as a means of progressing non-tractable or novel GPCRs, this may not be the case and may prove to be a major hurdle.

With evidence for GPCR-interacting proteins growing almost every day, one of the most ‘communicative’ receptors to date is the D1 dopamine receptor. D1-like dopamine receptors demonstrate a number of examples of physical interactions with a range of transmembrane and cytosolic proteins (Table 1). Both D1 and D5 receptors interact with ion channels [4,5], whereas the D1 receptor also interacts with other GPCRs, a single transmembrane protein, calcyon [6], as well as intracellular proteins such as NF-M (Neurofilament-M), a cytoskeletal protein [7].

The D1 dopamine receptor is expressed widely in the brain with high levels in the striatum, nucleus accumbens, olfactory tubercle and cortex. This receptor has a number of physiological functions (e.g. motor control). The D1 dopamine receptor agonism is a potential therapeutic approach for the treatment of Parkinson's disease and memory cognition. However, current D1 full agonists are subject to structural limitations, they have a catechol moiety that is pharmacokinetically unfavourable, and poor oral bioavailability [8]. Furthermore, long-term treatment of such conditions as Parkinson's disease and schizophrenia with D1 agonists has negative effects, such as memory impairment due to D1 receptor desensitization. Altogether, the D1 receptor is an important target for therapeutic intervention; alternative approaches to modulate receptor function may offer a means to progress further this target.

Ginés et al. [9] have shown that the D1 dopamine receptor can form heteromeric complexes with another GPCR, the A1 adenosine receptor. Furthermore, in transfected rat fibroblasts, acute exposure to D1 and A1 receptor agonists exhibited differential effects on these complexes. The A1 agonist r-PIA increased heteromerization, as seen by immunoprecipitation, and the D1 agonist SKF-38393 decreased this and promoted D1 receptor homomer formation. However, in rat primary cerebral cortical neurons, both compounds increased oligomerization. These differential effects may be due to receptor expression levels or different complements of membrane components. Furthermore, the D1-mediated functional activity through Gi could be uncoupled in the D1–A1 complex by pretreatment with both agonists. It is clear that the effects of agonist treatment are complex, and indicative of the problems that may be encountered if hetero-oligomerization is considered as an avenue for future drug discovery. Nonetheless, the authors suggest that this may be related to the functional antagonism in the brain and may offer an opportunity for novel intervention in such conditions as Parkinson's disease.

More recently the same collaborative group of researchers have demonstrated that the level of complexity of A1/D1 oligomerization is even greater, with the involvement of the cytosolic protein ADA (adenosine deaminase) [10]. They report that ADA, which is known to interact with the A1 receptor, is involved not only in A1 and D1 receptor aggregation in cultured neurons and transfected fibroblasts but also in the modulation of agonist binding to D1 receptors in these cells.

The D1 receptor has also been shown to form C-terminal contacts with both the ionotropic glutamate [NMDA (N-methyl-D-aspartate)] receptor subunits NR1 and NR2A [4,11]. Lee et al. [4] used a range of techniques, including immunoprecipitation and overexpression studies, to demonstrate that the D1 receptor has two sites on its C-terminus (t2 and t3) for interacting with NR1 and NR2A proteins respectively, although the question remains as to whether these interactions can both occur on the same receptor at the same time. These associations are affected by a D1 agonist, with agonist action resulting in decreased NMDA-mediated cell death through phosphoinositide 3-kinase-dependent survival mechanisms for the D1–NR1 pair. Furthermore, this effect was ablated by a t2 region blocking peptide. For the D1–NR2A interaction, an inhibition of NMDA-mediated currents was observed in hippocampal and striatal neurons as well as HEK-293 (human embryonic kidney 293) cells. This was blocked by a competitive peptide to the t3 sequence but was not affected by inhibitors of canonical G-protein signalling. Although the physiological importance of this interaction is being examined, the challenge for the pharmaceutical industry may be to integrate target family approaches rather than to maintain them is separate silos to progress them most optimally.

Another metabotropic–ionotropic receptor interaction is that between the D5 dopamine receptor and GABAA (where GABA stands for γ-aminobutyric acid) [5]. Liu et al. [5], using such techniques as filter overlay and co-immunoprecipitation, clearly demonstrate that the C-terminus of the D5 subtype, but not the D1 form, interacts with the second intracellular loop of the γ2 subunit of GABAA α1β2γ2. This association is dependent on the co-activation with both GABAA and D1 agonists. Treatment with either agonist had an inhibitory effect on the functions of the partner protein, e.g. pretreatment with GABA resulted in reduced maximal D1-mediated cAMP production; in contrast, GABAA-mediated whole-cell currents reduced dopamine acting through D5 but not D1. Furthermore, this group has been able to demonstrate that the D5 receptor was capable of modulating synaptic currents in cultured hippocampal neurons even in the presence of inhibitors of classical GPCR signalling. This provides an insight into the functional differences between D1 and D5 receptors, offering us the potential to discriminate between them in the hunt for treatments of psychomotor conditions.

Calcyon (named by its discoverers for ‘calcium on’) is a single transmembrane domain integral membrane protein that stimulates cross-talk between, specifically, the Gs-coupled D1 dopamine receptor and heterologous Gq/11-coupled GPCRs, thus allowing the D1 receptor to signal through both cAMP and Ca2+ [6]. Calcyon is expressed in the same prefrontal neurons as the D1 receptor, and results from studies on post-mortem samples of schizophrenic patients demonstrated an increase in dorsolateral prefrontal cortex calcyon levels, with no increase in other dopamine receptor interacting proteins filamin-A and spinophilin [12]. Samples from patients with major depression or bipolar disorder did not present any changes in the levels of any of the three proteins. In vitro, calcyon did not affect agonist or antagonist-binding affinities at the D1 receptor. It, however, appears to have an effect on the proportion of high- and low-affinity agonist-binding sites. In HEK-293 cells co-expressing both D1 and calcyon, the proportion of agonist high-affinity-binding sites was significantly reduced in a manner reversed by heterologous Gq/11-coupled receptor activation [13].

Altogether, calcyon represents a target with a specific and selective function, physiologically relevant distribution and disease association. It also has assayable effects in an in vitro format. By targeting calcyon and its effect on the prefrontal D1 receptor, it may be possible to reduce the peripheral side-effects mediated by the D1 receptor. However, it still remains to be determined whether the functions of calcyon can be modified by small-molecule ligands. Ultimately, if small-molecule modulators are identified, these will have to be demonstrated to be functional in a behavioural or biochemical model indicative of diseases such as Parkinson's disease or schizophrenia.

DRiP78 (dopamine receptor interacting protein 78) is an integral membrane protein located in the ER (endoplasmic reticulum) that is exquisitely involved in the export of the D1 receptor, through a C-terminal export signal, from the ER to the cell surface [14]. Both increase and decrease in the levels of DRiP78 in transfected HEK-293 cells affect receptor cell-surface expression, ligand binding and the kinetics of receptor glycosylation, suggesting its role in a number of functions. DRiP78 has also been shown to interact with other GPCRs, e.g. the M2 muscarinic receptor. It is known that ER-arrested receptor export can give rise to inherited diseases such as nephrogenic diabetes insipidus, which is due to a truncation of the V2 vasopressin receptor [15]. However, potential lack of selectivity of DRiP78 and lack of evidence for its involvement in D1-related disorders are indications of how much there is still to learn about this protein before it becomes a prospective drug target.

In addition to the interactions with integral membrane proteins there are also examples of D1-like receptor interactions with cytoskeletal proteins such as NF-M. Researchers identified this protein by yeast two-hybrid fishing using the D1 receptor third intracellular loop sequence as bait [7]. Co-expression of NF-M in D1-expressing HEK-293 cells resulted in decreased D1 cell-surface expression and cAMP production. Those receptors expressed at the cell surface were also not subject to agonist-mediated desensitization. Colocalization of NF-M and D1 in pyramidal cells and interneurons of rat frontal cortex was also established by immunohistochemistry. Although the evidence points to NF-M modifying D1 receptor expression and function in vivo, this has to be established before any role in disease or treatment can be elucidated.

A range of interactions between D1-like GPCRs and either other transmembrane proteins or intracellular proteins have been shown and discussed. Although these types of communication may account for some beneficial effects of current therapies, the converse that the side effects or undesirable properties associated with otherwise well-validated GPCR targets, may also apply. These interactions also highlight the gulf between the extremely complex physiological condition and the very simplistic recombinant systems employed particularly in the early stages of drug discovery. Understanding this connectivity may allow scientists to elucidate the physiological roles of these interactions. Furthermore, this may allow rational drug design to incorporate or remove such activities as appropriate, also potentially allowing us to progress those targets that may have been deemed unsuitable for direct therapeutic intervention.

In conclusion, GPCR accessory proteins represent potential future therapeutic targets for a range of conditions, however, their relative novelty and issues with the progressability of these proteins means that they may not enter the drug discovery pipeline in the short-term. Furthermore, with the exception of the RGS proteins, a number of which interact directly with GPCRs and constitute a family of related proteins, most accessory proteins may suffer the disadvantage of having to be dealt with as individual cases rather than as part of a family. The benefits of targeted family research, such as the concentration of knowledge and expertise in biology and chemistry, fits with the current trend in the pharmaceutical industry to target family-based research as a means to meet the ever-growing need to fill the drug discovery pipeline [16].

GPCR Allosterism and Accessory Proteins: New Insights into Drug Discovery: Focused Meeting and Satellite to BioScience2004, held at Organon Laboratories Ltd, Newhouse, Glasgow, U.K., 17 July 2004. Organized and Edited by A. Clark (Organon, U.K.), B. Henry (Organon, U.K.) and J. Presland (Organon, U.K.). Sponsored by Organon Laboratories and Invitrogen.

Abbreviations

     
  • ADA

    adenosine deaminase

  •  
  • DRiP

    dopamine receptor interacting protein

  •  
  • ER

    endoplasmic reticulum

  •  
  • GABA

    γ-aminobutyric acid

  •  
  • GPCR

    G-protein-coupled receptor

  •  
  • HEK-293

    human embryonic kidney 293 cells

  •  
  • NF-M

    Neurofilament-M

  •  
  • NMDA

    N-methyl-D-aspartate

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