Current Drug Targets - Volume 13, Issue 1, 2012

G protein-coupled receptors (GPCRs) are heptaspanning membrane proteins that mediate physiological responses to a plethora of signals including hormones, neurotransmitters and exogenous sensory stimuli perceived by the senses (i.e. light, odor and taste) [1]. Classically, the mechanism by which GPCR transduce extracellular signals into cellular changes was envisaged as a plain linear model, thus the extracellular agonist ‘ligand’ binds to and switches the receptor from an inactive to an active state conformation, and the activated receptor catalyzes the activation of guanine nucleotide binding proteins (G proteins) [2]. The activation of the heterotrimeric G protein (Gαβγ) involved the exchange of a GDP for a GTP molecule on the α-subunit, thus leading to the G protein dissociation into its α and βγ subunits. Interestingly, both Gα and Gβγ subunits can activate or inhibit effector enzymes (i.e. adenylyl cyclases, phosphodiesterases, phospholipases) and ion channels which in turn trigger multiple intracellular signaling pathways that modulate cell functions in body systems as diverse as the skeletal, endocrine, cardiovascular and nervous systems, among others [3]. In cells, most of the functions are mediated by multiprotein complexes. Interestingly, apart from binding to and activating the G proteins, GPCRs associate with a large array of other GPCR-interacting proteins (GIPs). These, membrane associated or intracellular GIPs, contain structural interacting domains that allow, under certain circumstances, the formation of large functional multiprotein complexes that are essential for both G protein-dependent and -independent signalling. Eventually, these GIPs may simply act as scaffold proteins that anchor the GPCRs to specific plasma membrane domains, thus contributing to the subcellular GPCR compartmentalization, and also participating in the GPCR trafficking to and from the plasma membrane. Overall, GIPs play a key role in GPCR biology as they fine-tune the receptor's function by impinging in its trafficking, localization and/or pharmacological properties [4]. Recently, special attention has been paid to GIPs as they might potentially evolve as drug targets [4, 5]. Therefore, here we intended to focus on the emerging evidence of GPCR interactions with scaffolding, cytoskeletal and signalling proteins that will play a key role in the targeting, anchoring and functioning of these receptors in the plasma membrane, thus constituting a pharmacological target for drug intervention. Accordingly, the present issue of Current Drug Targets aims to bring together a number of leading experts in the field of GPCRs in order to summarize the main aspects of the state of the art GIPs of some representative GPCRs. Thus, ten excellent papers compose this special edition which revolves around the GIPs of the mammalian dim-light photoreceptor rhodopsin, the adrenergic receptors, the 5-hydroxytryptamine (5-HT) receptors, the muscarinic acetylcholine (mACh) receptors, the dopamine receptors, the parathyroid hormone (PTH) receptors, the opioid receptor, the γ-aminobutyric acid (GABA) type B receptor, the metabotropic glutamate (mGlu) receptors, and the small family B1 GPCR (secretin-like receptors) which includes within others the vasoactive intestinal peptide (VPAC) receptors, the secretin (SEC) receptors, and the corticotropin-releasing factor (CRF) receptors. Thus, we hope that this timely focused issue summarizing our current knowledge on GIPs will be of interest to a wide range of readers of the journal interested in the GPCR field. Finally, I would like to express my best thanks to all the contributing authors and co-authors of this issue for their commitment, time, experience and patience. Also, my special thanks to the anonymous reviewers for their excellent contributions to the peer-review process. In addition, I want to express my special gratitude to Professor Francis J. Castellino, editor-in-chief of Current Drug Targets, for giving me this opportunity, and to the staff at Bentham Science for their assistance and cooperation.

The mammalian dim-light photoreceptor rhodopsin is a prototypic G protein coupled receptor (GPCR), interacting with the G protein, transducin, rhodopsin kinase, and arrestin. All of these proteins interact with rhodopsin at its cytoplasmic surface. Structural and modeling studies have provided in-depth descriptions of the respective interfaces. Overlap and thus competition for binding surfaces is a major regulatory mechanism for signal processing. Recently, it was found that the same surface is also targeted by small molecules. These ligands can directly interfere with the binding and activation of the proteins of the signal transduction cascade, but they can also allosterically modulate the retinal ligand binding pocket. Because the pocket that is targeted contains residues that are highly conserved across Class A GPCRs, these findings imply that it may be possible to target multiple GPCRs with the same ligand(s). This is desirable for example in complex diseases such as cancer where multiple GPCRs participate in the disease networks.

The adrenergic receptors are among the best characterized G protein-coupled receptors (GPCRs) and knowledge on this receptor family has provided several important paradigms about GPCR function and regulation. One of the most recent paradigms initially supported by studies on adrenergic receptors is that both βarrestins and G proteincoupled receptors themselves can act as scaffolds binding a variety of proteins and this can result in growing complexity of the receptor-mediated cellular effects. In this review we will briefly summarize the main features of βarrestin binding to the adrenergic receptor subtypes and we will review more in detail the main proteins found to selectively interact with distinct AR subtype. At the end, we will review the main findings on oligomerization of the AR subtypes.

Serotonin (5-HT) is a phylogenetically ancient transmitter implicated in many vital functions in human such as sleep, food intake, reproduction, nociception, regulation of mood and emotions as well as cognitive functions. Correspondingly, dysfunction of serotonergic transmission has been implicated in numerous psychiatric disorders such as anxio-depressive states, psychoses and addiction, and serotonergic systems are targets for a large array of psychoactive compounds including antidepressants, antipsychotics and hallucinogens. 5-HT acts on numerous receptor subtypes (14). Except for 5-HT3 receptors, which are cationic channels, 5-HT receptors belong to the G protein-coupled receptor (GPCR) superfamily and allow an extraordinarily diverse and complex pattern of cellular signalling. Over the past ten years, the majority of metabotropic 5-HT receptors has been found to interact with specific protein partners in addition to the ubiquitous GPCR modulators, GPCR kinases and β-arrestins, mainly by mean of two-hybrid and proteomic screens. These proteins, called GPCR-interacting proteins (GIPs) were found to profoundly influence the targeting, trafficking and signal transduction properties of 5-HT receptors. This article first describes our current knowledge of the nature of GIPs that bind to the different metabotropic 5-HT receptor categories. It then focuses on their impact on receptor functional status at the cellular level and illustrates how GIPs permit G protein-independent signal transduction at G protein-coupled 5-HT receptors. Finally, it reports recent data dealing with the roles of GIPs in 5-HT-related behaviours and highlights the potential of manipulating 5-HT receptor-GIP interactions to design new treatments in psychiatric disorders related to perturbations of serotonergic systems.

Muscarinic acetylcholine receptors comprise a large family of G protein-coupled receptors that are involved in the regulation of many important functions of the central and peripheral nervous system. To achieve such a large range of physiological effects, these receptors interact with a large array of accessory proteins including scaffold molecules, ion channels and enzymes that operate as molecular transducers of muscarinic function in addition to the canonical heterotrimeric G proteins. Interestingly, as demonstrated for others G protein-coupled receptors, this type of receptor is also able to oligomerise, a fact that has been shown to play a critical role in their subcellular distribution, trafficking, and fine tuning of cholinergic signalling. On the other hand, the specificity of these receptor interactions may be largely determined by the occurrence of precise protein-interacting motifs, posttranslational modifications, and the differential tissue distribution and stoichiometry of the receptor-interacting proteins. Thus, the exhaustive cataloguing and documentation of muscarinic acetylcholine receptor-interacting proteins and the grasp of their specific function will explain key physiological differences in muscarinic-mediated cholinergic transmission. Overall, a better comprehension of the muscarinic receptor interactome will have a significant impact on the cholinergic pharmacology and thus provide previously unrealised opportunities to achieve greater specificity in muscarinic-related drug discovery and diagnostics.

D2 dopamine receptors (D2Rs) represent an important class of receptors in the pharmacological development of novel therapeutic drugs for the treatment of schizophrenia. Recent research into D2R signaling suggests that receptor properties are dependent on interaction with a cohort of dopamine receptor interacting proteins (DRIPs) within a macromolecular structure termed the signalplex. One component of this signalplex is neuronal calcium sensor 1 (NCS-1) a protein found to regulate the phosphorylation, trafficking, and signaling profile of the D2R in neurons. It has also been found that NCS-1 can contribute to the pathology of schizophrenia and may play a role in the efficacy of antipsychotic drug medication in the brain. In this review we discuss how the selective targeting of a DRIP, such as NCS-1, can be utilized as a novel strategy of drug design for the creation of new therapeutics for a disease such as schizophrenia. Using a fluorescence polarization assay we explore how the ability to detect changes in D2R/NCS-1 interaction can be exploited as an effective screening tool in the isolation and development of lead compounds for antipsychotic drug development. This line of work explores a novel direction in targeting D2Rs via their signalplex components and supports the notion that receptor interacting proteins represent an emerging new class of molecular targets for pharmacological drug development.

Opiate drugs mediate their analgesic, euphoriant, and rewarding effects by activating opioid receptors. Pharmacological and molecular studies have demonstrated the existence of three opioid receptor subtypes, μ, &delta, and κ- that couple predominantly to Gi/Go types of G proteins to regulate the activity of a diverse array of effector systems. Ample experimental evidence has demonstrated that these receptors can physically interact with a variety of accessory proteins, confirming that signal transduction of the opioid receptors is not restricted to heterotrimeric G protein activation. Such interactions can alter the effectiveness of agonist-driven cell signalling, determine the signals generated and alter the trafficking, targeting, fine tuning and cellular localization of these receptors by providing a scaffold that links the receptors to the cytoskeletal network. The current review will summarize opioid receptor interacting partners and their role as currently understood. Increasing knowledge of the mechanisms by which these interactions are regulated is expected to address problems related to phenomena such as pain perception, tolerance and dependence that occur upon chronic opiate administration and define whether disruption of such interactions may contribute to the development of novel therapeutic strategies.

G protein coupled receptors (GPCRs) play a crucial role in physiology and pathophysiology in humans. Beside the large family A (rhodopsin-like receptors) and family C GPCR (metabotropic glutamate receptors), the small family B1 GPCR (secretin-like receptors) includes important receptors such as vasoactive intestinal peptide receptors (VPAC), pituitary adenylyl cyclase activating peptide receptor (PAC1R), secretin receptor (SECR), growth hormone releasing factor receptor (GRFR), glucagon receptor (GCGR), glucagon like-peptide 1 and 2 receptors (GLPR), gastric inhibitory peptide receptor (GIPR), parathyroid hormone receptors (PTHR), calcitonin receptors (CTR) and corticotropin-releasing factor receptors (CRFR). They represent very promising targets for the development of drugs having therapeutical impact on many diseases such as chronic inflammation, neurodegeneration, diabetes, stress and osteoporosis. Over the past decade, structure-function relationship studies have demonstrated that the N-terminal ectodomain (N-ted) of family B1 receptors plays a pivotal role in natural ligand recognition. Structural analysis of some family B1 GPCR N-teds revealed the existence of a Sushi domain fold consisting of two antiparallel β sheets stabilized by three disulfide bonds and a salt bridge. The family B1 GPCRs promote cellular responses through a signaling pathway including predominantly the Gsadenylyl cyclase-cAMP pathway activation. Family B1 GPCRs also interact with a few accessory proteins which play a role in cell signaling, receptor expression and/or pharmacological profiles of receptors. These accessory proteins may represent new targets for the design of new drugs. Here, we review the current knowledge regarding: i) the structure of family B1 GPCR binding domain for natural ligands and ii) the interaction of family B1 GPCRs with accessory proteins.

The parathyroid hormone 1 receptor (PTH1R) is activated by parathyroid hormone (PTH) and parathyroid hormone related protein (PTHrP), hormones that mediate mineral ion homeostasis and tissue development, respectively. These diverse actions mediated by one receptor are likely due to the formation of cell-specific receptorsome complexes with cytosolic constituents. Through the second and third intracellular loops, the PTH1R couples to several G protein subclasses, including Gs, Gq/11, Gi/o and G12/13, resulting in the activation of many pathways. The PTH1R carboxy-terminal tail directs interactions with a plethora of binding partners. The WD1 and WD7 repeats of the G protein β subunit directly bind to a novel interaction domain located near the amino-terminal end of the PTH1R carboxy-terminal tail. This Gβγ binding site likely contributes to the promiscuous G protein coupling displayed by the PTH1R. Partially overlapping this site is an EF-hand binding domain that directs interactions with calpain, a calcium-activated protease, and calmodulin, a ubiquitous calcium sensor. A lysine-arginine-lysine motif located on the juxtamembrane region of the carboxy-terminal tail mediates interactions with ezrin, an actin-membrane cross-linking protein. The C-terminus of the PTH1R binds to the sodium-hydrogen regulatory factors (NHERFs) via a PDZ domain-mediated interaction, an association that influences signaling and membrane anchoring. Through direct interactions with ezrin and NHERF-1, a PTH1R receptorsome complex exists on apical membranes of the proximal tubule, an assembly that directs PTH-mediated regulation of phosphate transport. Targeting the PTH1R receptorsome will likely enhance therapies directed towards the treatment of osteoporosis and enhancing the hematopoietic stem cell niche.

γ-aminobutyric acid type B (GABAB) receptors play a critical role in neuronal excitability and modulation of synaptic neurotransmission in the central nervous system. They are G protein-coupled receptors that signal primarily through activation of G proteins (i.e. pertussis toxin sensitive Gαi/o family) to modulate the function of inwardly-rectifying K+ and voltage-gated Ca2+ channels, and to trigger cyclic adenosine monophosphate cascades. Functional GABAB receptors are obligated heterodimers formed by the co-assembly of two subunits, the GABAB1 and the GABAB2, which interact via coiled-coil domains in their C-terminal tails. It is now quite well established that GABAB receptors interact not only with heterotrimeric G proteins and effector ion channels but also with a plethora of accessory proteins that might impinge into the receptor's biology. Indeed, these proteins have been implicated in several key functional aspects of the receptor, namely to link functional GABAB receptors with components of the relevant signalling pathways, to target the receptor into specific subcellular compartments, to participate in their trafficking to and from the plasma membrane, and to regulate their signalling properties. Overall, in this review we focus on those proteins that interact with GABAB receptors. Thus, understanding how the interaction between GABAB receptors and its accessory proteins takes place will definitively open new opportunities for pharmacological tool assessment of novel therapeutic targets for the treatment of several neurological diseases involving these receptors.

The correct targeting, localization, regulation and signaling of metabotropic glutamate receptors (mGluRs) represent major mechanisms underlying the complex function of neuronal networks. These tasks are accomplished by the formation of synaptic signal complexes that integrate functionally related proteins such as neurotransmitter receptors, enzymes and scaffold proteins. By these means, proteins interacting with mGluRs are important regulators of glutamatergic neurotransmission. Most described mGluR interaction partners bind to the intracellular C-termini of the receptors. These domains are extensively spliced and phosphorylated, resulting in a high variability of binding surfaces offered to interacting proteins. Malfunction of mGluRs and associated proteins are linked to neurodegenerative and neuropsychiatric disorders including addiction, depression, epilepsy, schizophrenia, Alzheimer's, Huntington's and Parkinson's disease. MGluR associated signal complexes are dynamic structures that assemble and disassemble in response to the neuronal fate. This, in principle, allows therapeutic intervention, defining mGluRs and interacting proteins as promising drug targets. In the last years, several studies elucidated the geometry of mGluRs in contact with regulatory proteins, providing a solid fundament for the development of new therapeutic strategies. Here, I will give an overview of human disorders directly associated with mGluR malfunction, provide an up-to-date summary of mGluR interacting proteins and highlight recently described structures of mGluR domains in contact with binding partners.