Posts tagged receptors

I am going to reproduce some of the term papers that I wrote for the spring semester in school. Hopefully I can pull myself back to writing more posts this year especially over the summer now that I have some breathing space from school.

(PS. Unfortunately, I could not post any of the images/figures I used in the actual paper here for obvious reasons. If you see any mention of them in the text just ignore them and I am hoping that the content still makes sense to you guys.)

Abstract:

Gamma amino butyric acid (GABA) is one of the major inhibitory neurotransmitters found in the mammalian central nervous system. Until recently, GABA was known to function by binding to a ligand-gated chloride ion channel known as GABA A receptors, thus producing its inhibitory effects. GABA B receptors are newly discovered metabotropic subtype of GABA receptors and belong to the Class C family of G-protein coupled receptors (GPCRs). There are the first and only GPCRs that have been identified to show obligate oligomerization. They have been identified to modulate the release of a number of key neurotransmitters in the CNS by indirectly activating ion-channels and therefore are considered to be a key member in a diverse set of physiological pathways. Therefore, understanding these proteins could help in the on-going drug discovery efforts to treat conditions such as pathological pain, muscle spasms, drug craving, anxiety, schizophrenia, depression and autism.

Furthermore, investigating the structural and functional aspects of these proteins can reveal answers to some of the fundamental questions of GPCR oligomerization. They can provide an insight into the integral molecular mechanisms involved in neurotransmitter recognition and signal transduction in receptor oligomers and can unravel the unexpected level of pharmacological diversity. In this review, I will address the key structural features and molecular mechanisms that make these receptors unique and how the knowledge gained from studying these receptors can be generalized to all other G-protein coupled receptors.

Introduction:

G-protein coupled receptor (GPCR) superfamily comprises of more than 800 protein and represent the largest family of integral membrane proteins in the human genome¹. They are characterized by 7 transmembrane-spanning helices (H1-H7), which are interconnected by 3 extracellular loops (ECL1-3) and 3 intracellular loops (ICL1-3). Based on a comprehensive phylogenetic analysis, GRAFS classification categorizes the mammalian GPCR repertoire into five main families; Glutamate (Class C), Rhodopsin (Class A), Adhesion (Class B), Frizzled (Class F), and Secretin (Class B).

GPCRs mediate a large number of vital cellular responses and therefore are of extremely importance as a drug target and for translational research. Currently about 50% of all small molecule drugs approved by the FDA target these receptors and are one of the most targeted families of proteins in the human body³. They are therefore of enormous pharmacotherapeutic importanceand it is vital that we better understand these complex proteins and how they function in order to develop better pharmacological agents.

Histroy of GPCR oligomerization:

Traditionally, the function of ligand binding and signal transduction was assumed to be through monomeric receptor units and through interactions with a single guanine binding nucleotide protein once activated by the ligand. But in 1978-82, Dr. Nigel Birdsall from the National Institute of Medical research, London raised the earliest questions about receptor-receptor interactions. His hypothesis that receptors interact with each other was based on his observations in muscarinic receptor membrane preparations when treated with beta-adrenergic receptor ligands⁴. Seeman and collaborators further confirmed the existence of dopamine receptor dimers by positron emission tomography (PET) in human brain⁵. Other biochemical studies, such as photo-affinity labeling assays of the muscarinic receptor, and radiation quenching studies of beta-2 adrenergic, gonadotropin and adenosine A1 receptors further provided evidence of the existence of GPCR oligomers⁶. The crosslinking of the glucagon receptors²², and hydrodynamic properties of cardiac muscarinic receptors,further supported this idea. But unfortunately, these early studies did not gain much interest from the scientific community.

In 1993, Dr. Wess et al. confirmed the intermolecular interactions between muscarinic receptors using chimeric muscarinic/adrenergic receptors overexpressed in CHO cells⁷. They observed that the chimeric receptors were either not expressed alone or showed no ligand binding and functional activity. But when the two chimeras were co-expressed, they exhibited ligand binding and functional signaling to both muscarinic and adrenergic ligands. They also proposed a hypothetical model for receptor dimerization, which resulted in much excitement in the field of receptor pharmacology. For the first time, the importance of oligomerization in ligand affinity and functional activity was being discussed. Until then, only the hetero-dimerization of two nonfunctional (Chimeric) receptors had been confirmed. But in 1999, Dr. Lakshmi Devi and collaborators identified the presence of hetero dimers of two fully functional opioid receptors, κ and δ. They also characterized the changes in their ligand affinities and observed the presence of receptor synergism⁸.

Over the past two decades, tremendous research has been done on GPCR dimerization and one of the most well studied receptors is the GABA receptor. These studies have been able to paint a vivid picture of the interactions between receptors and their implications in their activity and function. In this critical review, I plan to address the progress made in the field of GPCR oligomerization using GABA receptor as the template.

GABA receptor:

            Gamma amino butyric acid (GABA), an inhibitory neurotransmitter found mostly in the central nervous system (CNS), binds to the membrane proteins known as GABA receptors. Over the years, a number of different classes of GABA receptors have been identified and are currently broadly classified into three groups – GABA A, GABA B and GABA C receptors. GABA A and C receptors are ionotropic receptors, whereas the GABA B receptors are of the metabotropic nature.

GABA A and C receptors are chloride-ion channels and each receptor is composed of 5 hetero-oligomeric subunits that are arranged into a cylindrical channel with the central pore of chloride ion entry¹⁰. Each subunit is made up of 4 transmembrane domains and is folded such that the 5 2nd transmembrane domains form the wall of the ion pore. The GABA binding domain has been identified at the outer surface of the receptor. The allosteric binding sites for drugs such as Benzodiazepines and Barbiturates that mediate through the GABA receptors have also been identified at the interface between subunits. To date, there are 16 human GABA A receptor subunits (α1-6, β1-4, γ1-4, δ, ε) and two human GABA C receptor subunits (ρ1 and ρ2) have been identified⁹. 

            GABA B receptors are seven transmembrane metabotropic G-protein coupled receptors and belong to the family C GPCR. Family C GPCRs are characterized by the unusually long N-terminal loop of transmembrane domain connected with with a large Venus Flytrap domain (VFT) through a Cysteine rich domain (CRD)¹⁰. GABA B receptors mediate the second messenger systems - phospholipase C and adenylate cyclase, through the guanine-nucleotide binding proteins (G-proteins) and thus activate K⁺ and Ca²⁺ ion channels. Unlike the GABA A receptors that produce rapid inhibitory signals through chloride influx through the ion channel, the GABA B receptors produce slow, prolonged inhibitory signals. They are localized at both the pre-synaptic and post-synaptic neurons and function to modulate the release of neurotransmitters and key post-synaptic cell signals respectively⁹.

Dimerization of GABA B receptor:

To date, three subtypes of GABA B receptors have been cloned and are termed as GABA B1a, GABA B1b and GABA B2. The GABA B receptors resemble the metabotropic glutamate receptors (mGluR) and were found to be functionally active only as heterodimers of either GABA B1a or GABA B1b subunit when combined with the GABA B2 subunit. It has ben identified that GABA_B1 contains the GABA binding site¹¹, while GABA_B2 is responsible for G_i/o protein activation^12.

Due to the close sequence homology among the Class C GPCR family of receptors, a number of homology models have been constructed for the GABA B receptor based on the published crystal structure of the mGluR1. The structure as shown below consists of two key domains – Venous Flytrap domain and Heptahelical transmembrane domain similar to the other Class C GPCRs. Interestingly, unlike the other Class C GPCRs, the Cysteine-rich domain connecting these two domains is absent in GABA B receptors.

The Venous Flytrap domain (VFT) is large hydrophobic bi-lobed structure found extending from the surface of the cell membrane. The ligand has been identified to be binding at the cleft between the two lobes of the domain and when bound it stabilizes its closed structure. It has also been observed that the lobe 1 of each monomer interacts with each other and form constitutive dimers¹³. It has also been shown that the absence of this interaction results in the loss of activity of the GABA B receptor¹⁴. Mutational studies have shown that the key residues in lobe 1 involved in Agonist binding are Ser246 and Asp 471. The key residues for antagonist binding are Ser246, Ser265, Tyr266, Phe463 and Tyr470. Although most of the key residues for ligand binding are mostly present in the lobe 1 some residues such as the Tyr366 in lobe 2 are also considered important. Most of these residues are absent in the VFT domain of the GABA B2 and therefore this monomer is not involved in ligand binding¹⁵.

The Heptahelical transmembrane domain (HTD), similar to other GPCRs, consists of 7 alpha helices that are linked by 3 intracellular loops (ICL 1-3) and 3 extracellular loops (ECL 1-3), which are less than 30 residues in length¹⁶. The two C-termini are considered to be in the form of a coiled-coil and are not involved in signaling but involved in receptor trafficking and also in holding the two monomers together¹⁷. The Class 3 GPCRs have a low sequence homology to the Class A GPCRs whose 3D structural features have been well studied. Of the 19 conserved residues in Class C receptors, only 7 are found in the rhodopsin-family of GPCRs. But there still a few key residues and interactions similar to the ones seen in rhodopsin-like receptors. GABA B1 receptor has a PKxR and GABA B2 has a PKxI motif similar to the NPxxP motif in the TM7 of Family A receptors¹⁸. The disulfide bond that connects the top of TM3 with the ECL2 is also conserved in Family C receptors¹⁶. The highly conserved DRY region in Family A receptors is present only in GABA B2 receptor which may explain why only the B2 monomer is involved in G-protein signilaing¹⁸. Also the C-terminal end forms an 8th helix that is related to G-protein coupling similar to the Class A family of GPCRs. But one of the key differences between the GABA B receptor and Rhodopsin-like GPCRs is the absence of ligand binding at the extracellular domains of the seven transmembrane helices. However, a conserved binding pocket has still been identified and could be of potential use to develop allosteric drugs targeting this site to bind and modulate receptor activity¹⁹.

Mechanism of activation of GABA B dimers:

            The ligand binding at the VFT cleft is followed by a series of conformational changes that are translated to the intracellular surface from the VFT through the heptahelical tansmenbrane domain resulting in the activation of the G-protein. The exact mechanism of the transduction of these signals from the extracellular domains to the G-protein is still unclear. But the mGluR1 crystal structure provides us some insight into what must be the mechanism.

            It has identified that the receptor dimer in the resting state exists such that both the lobe 2 VFT domains are far away from each other. Once bound by the ligand at the cleft, the lobes are stabilized into a closed conformation and move to the active state. In this state, the lobe 2 of the GABA B1 VFT domain move closer and make contact with the lobe 2 of the GABA B2 VFT domain¹⁹. Rondard et al. confirmed this that by observing a decrease in receptor activity in response to orthosteric ligands, when a N-glycan wedge was introduced to the lobe 2¹⁴. It can be hypothesized that signal is translated from the VFT domains to the transmembrane domains in a direct interaction as the GABA B receptors lack the cysteine-rich domain. Owing to the lack of a crystal structure of the membrane-bound domain, the exact mechanism of signal transduction across the membrane to the G-protein is still a mystery. But based on the phylogenetic studies, it can be predicted that similar to the Class A GPCRs, the GABA B transmembrane domains show conformational changes in the helices 3, 5 and 6 and thus causing changes in the interactions with the G-protein at the ICL 2 and 3.

            Apart from the orthosteric binding pocket in the cleft of the VFT domain, a number of allosteric sites have also been identified and are considered to be of interest for drug discovery and development¹⁹. Currently only one drug – Baclofen, an anti-spasmodic agent is known to interact at the orthosteric site at GABA B1. It is a GABA derivative and currently in clinical trials for the indication of alcoholism treatment. Recently, a positive allosteric modulator - CGP7930 has been discovered to increase both potency and efficacy of GABA. It has been shown to bind to the 7-transmembrane domain and enhance the binding to the Go protein. A model has been proposed that the binding of the agonist at the VFT site causes a increase in the size of the cleft between the two transmembrane domain and CGP7930 is expected to further widen the cleft by binding between the 2 heptahelical domain²⁰. Other positive allosteric inhibitors that have been discovered and synthesized are GS39783 and analogs of rac-BHFF²¹. Unfortunately, none of these ligands have been clinical viable due to their low potency and unfavorable pharmacokinetics.

Role of dimerization in GABA-B function:

The GABA B receptor is the first GPCR identified to be an obligatory heterodimer composed of two subunits, GABA B1 and GABA B2. The role of GABA-B receptor in the central nervous system synaptic transmission has been well studied over the past two decades. The activated GABA-B receptors have been identified to reduce transmission at excitatory and inhibitory synapses, as a result of an increase in K+ conductance through adenylyl cyclase or through inwardly rectifying potassium channels (GIRKs), or decrease the voltage-dependent Ca2+ currents. The GB1 monomer binds to the ligand and GB2 monomer has been identified to interact with the G-protein and does not bind to a ligand. In general, this system can therefore be considered as an excellent model to study the functional significance of GPCR dimerization.

Dr. Jean-Philippe Pin’s group has demonstrated that GABA B receptors can also exist as stable tetramers²³. They also established that the interaction between heterodimers to form tetramers does not result just from overexpression in in-vivo cell cultures and that they exist as tetramers even in the human brain. They further showed that the tetramers exhibited a lower G-protein coupling efficacy than the heterodimers produced by mutations at the contact area. GABA B receptor heterodimer GB1–GB2 could be compared with a monomeric class A receptors in that higher oligomers show reduced G-protein efficacy. But unlike the Class A receptors where this phenomenon was due the steric forces not allowing the interaction of two G-proteins at once, in case of GABA-B receptor it is hypothesized that the it is due to the fact that only one of the dimer in each tetramer can reach the active conformation at one instance.

The exact physiological significance for this inclination to form tetramers despite being less efficient than dimers is unclear. But we could speculate that the higher oligomer formation may be a receptor storage mechanism, as in the case of Rhodopsin and that they form dimers when needed for activation. Also tetramers provide better stability during receptor translocation, desensitization/internalization and turnover. They could also provide a method to alter the strength of signaling by segregating the pre-synaptic and post-synaptic receptors accordingly²³.

Clinical implications of GABA-B dimierization:

            GABA B receptors have been observed in high density in the thalamic nuclei, the molecular layer of the cerebellum, the cerebral cortex, the interpeduncular nucleus, and the dorsal horn of the spinal cord²⁴. Other studies have shown that GABA B1a receptors are also present in peripheral tissues such the adrenals, pituitary, spleen, and prostate, whereas GABA B1b receptors have been found in the rat kidney and liver. Both the pre- and post-synaptic receptors influence the release and effects of a number of key neurotransmitters such as dopamine, nor-adrenaline, adenosine, glycine, glutamate and serotonin. Thus, the GABA_B receptor agonists display a number of pharmacological effects, including anti-spasticity, antitussive action, bronchiolar relaxation, alteration of micturition, gastrointestinal motility, epileptogenesis, cognition, gastric acid secretion, and hormone release, drug craving suppression, anti-nociception, induction of hypotension and brown fat thermogenesis²⁵. Therefore, there is tremendous potential for developing drugs that could help treat a number of pathological conditions.          

            Furthermore, obligatory oligomerization of receptors opens new avenues to drug discovery. A number of new techniques such as dimer ligands, or ligands disrupting or enhancing oligomerization have become popular among pharmaceutical and biotech companies²⁶. STX209, is an oral selective GABA-B receptor agonist developed by Seaside Therapeutics, Inc. is currently being studied in a Phase 2b study in autism spectrum disorders and two Phase 3 studies in fragile X syndrome²⁷.

Conclusion:

GABA B receptor is unique in that it is the only Family C GPCR known to show obligatory dimerization and signaling through transactivation. This makes GABA B receptor an ideal model for studying GPCR oligomerization. In recent years, allosteric modulation has become an attractive approach and theoretically can overcome some undesirable side effects, tolerance and provide subtype selectivity easily unlike orthosteric ligands. Apart from the orthosteric site at the VFT domain, a large number of binding pockets have been identified on these receptors, which makes it an attractive target for allosteric drugs. A number of drug discovery projects focusing on the development of allosteric modulators, targeting Class C receptors, are currently underway both in the pharmaceutical industry and academia. Until now, very few drugs have been successful in clinical studies and it calls for further investigation of the precise structural dynamics and allosteric modulation mechanisms. Further studies using advanced functional assays, such as BRET (bioluminescence resonance energy transfer) or FRET (fluorescence resonance energy transfer) could reveal details about conformational changes during dimer or oligomer formation. Understanding the mechanisms of dimerization could be important in the development and screening of drugs that act through not only Class C receptors but also give vital information about all GPCR dimers. The changes in ligand-binding and signaling properties due to hetero-dimerization could also open doors to novel pharmacological interventions.

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