Univeristy no.

: 0527680

Dimerization of G Protein Coupled Receptors

Abstract
G-protein coupled receptors have been known to exist for over 100 years, but it was always thought that they could only exist as monomers. Advancements in biophysical and biochemical techniques over the past 10 years have led to the discovery that GPCRs can exhibit either dimeric or oligomeric structures as well. The physiological consequences of dimerization still remain elusive although some dimers have been well characterized. This essay will focus on homo and hetero dimerization of different GPCRs. I will first discuss the different methods of detecting dimers in cells and then go on to discussing the mechanisms and functional consequences of dimerization. ------------------------------------------------------------------------

Introduction
A cell is highly responsive to its constantly changing environment. Signal transduction cascades are responsible for the sensing and processing of the stimuli generated from the extracellular environment. These transduction cascades detect external signals which generate an internal response such as a change in enzyme activity, gene expression or ion concentration. Most signalling molecules are too large and too polar to pass the plasma membrane so a transport system and signalling system must be available. The information that these signalling molecules contain must be transmitted into the cell without the molecule entering the cell and does so through a receptor protein. The receptors are integral membrane proteins with extracellular binding sites that recognise specific ligands or agonists. The ligands are known as the primary messengers and when bound alter the structure of the receptor. Secondary messengers carry the information forward. They diffuse freely in the cell and influence gene expression, protein kinases etc... The seven transmembrane helix receptors are one of the largest protein families in the body [1]. Also known as G- protein coupled receptors, they regulate virtually all known physiological processes in mammals. Although GPCRs were discovered over 100 years ago, most of the information on the receptors has been discovered during the past 30 years and we now have a greater understanding about the properties of the receptors, how they signal and how their functions are regulated. All GPCRs have a core domain made of seven transmembrane helices. They are connected by three intracellular loops and three extracellular loops. The N terminus is 1

University no.: 0527680 extracellular and the C terminus is intracellular [2]. When ligands bind to GPCRs, guanyl nucleotide binding proteins (G proteins) are activated. G proteins are intermediary in signal transduction and stimulate other targets such as adenylate cyclase. Dimerization of GPCRs is quite a recent discovery and the presence of a dimeric GPCR was first implicated by experiments carried out 20 years ago. This experiment involved the use of bivalent antibodies and was used against an antagonist of the gonadotropin releasing hormone receptor. It was not until the 1990s however that the phenomenon of dimerization was more widely considered due to technological advancements in biochemical and biophysical techniques [3]. Studies have shown that GPCRs may exhibit either dimeric or oligomeric structures. This evidence has led scientists to question the classical view of hormone-receptor interaction. Although a lot of work has been done to demonstrate this phenomenon, the physiological effects of hetero and homo dimerization still remains somewhat of a mystery.

Detecting dimers in cells

The methods for detecting dimers in cells have advanced a great deal in the last 10 years. The most widely used biochemical technique in recent years has been Coimmuniprecipitation. This technique was first used to detect intermolecular interactions between 2-adrenergic receptors (2A) [4]. Since then other homo-dimers have been found using the same method, e.g. the calcium receptor, the mGlu5 receptor, the opioid receptors, and the M3 muscarinic receptors. Hetero dimers such as the GABAB receptors and the SST3 and the SST2a receptors have also been found using Coimmuniprecipitation. Although this is has been a very successful method for studying dimerization is GPCRs there are some downsides to the technique; the membrane receptors need to be solubilised in detergents and this could cause problems when dealing with hydrophobic proteins. A way to deal with this is to treat the cells with hydrophilic cross-linking agents before being treated with detergents. This has shown to stabilize dimers and suggesting that these complexes do exist on the surface of cells [5]. Biophysical methods based on light resonance energy transfer are used to detect dimers in living cells. This technique is based on the nonradiative transfer of energy between the electro magnetic dipoles of an energy donor and acceptor. The first method is called Bioluminescence Resonance Energy Transfer (BRET) and is a natural phenomenon which occurs in many marine organisms. The 2A receptor has been studied using BRET [6]. Green fluorescent protein (GFP) and Renilla luciferase (Rluc) are genetically linked to the C-terminus of the 2A receptor. Energy transfer between the 2ARRluc and the 2AR-GFP is observed when co-expressed which indicates that these receptors can form constitutive homo-dimers. When the 2A receptors are stimulated with a selective agonist, an increased level of energy transfer was detected. This could be an indication that receptor activation increases dimer formation or simply that it leads to a conformational

University no.: 0527680 change in the receptor, bringing the GFP and the Rluc closer together which increases the efficiency of energy transfer. Therefore the results obtained from BRET experiments can be quite inconclusive because it is hard to distinguish whether the increase in energy transfer is due to a conformational change in a pre-existing dimer or and increase in their numbers [5]. Another biophysical technique that is widely used is Fluorescence Resonance Energy Transfer (FRET). There are many types of FRET, for example FRET with GFP. GFP from jelly fish has been isolated and have spectral properties that make them good for measuring protein-protein interactions. Studies using this kind of FRET have shown that dimerization is not a uniform process but that it actually occurs in specific regions on the surface of the cell. It has also shown that some dimerization is dependant upon agonist stimulation [5]. Other FRET techniques include photobleaching FRET, homogenous time resolved FRET, and the use of fluorescent ligands. Using resonance energy transfer methods is a unique approach to studying GPCR dimerization and has a clear advantage over Coimmuniprecipitation in that they do not disrupt the natural environment in which the cells occur.

Mechanisms of GPCR dimerization

GPCRs can be divided into five main classes based on their sequence similarity. Rhodopsin like receptors form class A, secretin like and metabotropic glutamate like receptors form class B and C. Finally, frizzled and a subgroup of pheromone receptors from the remaining classes. These different classes of GPCRs have different mechanisms by which they dimerize. A well known mechanism of GPCR dimerization of the interactions between the coiled coil domains within the C-terminal tails of the GABAB receptors (Figure 1). This type of interaction is present between many types of proteins but is not a general mechanism of GPCR dimerization [7].

Figure 1: Coiled coil

interaction between GABAB receptor C-terminal tails, vital for membrane delivery (Graeme Milligan, 2001)

University no.: 0527680 Class C GPCRs have a large extracellular tail that contains several cysteine residues. Disulphide cross links has been shown to form between GPCRs which shows they contribute to dimerization (Figure 2). An experiment carried out with the mGlu1 rector showed that dimerization cannot occur if the extracellular domain is removed. A mutation in the Cys140 residue showed that it is involved in dimerization but also that it is not the only point of the domain that forms disulphide bonds [8].

Figure 2: Disulphide
bonds between extracellular tails. Dimer stabilisation is also provided by the intramolecular bonds. (Graeme Milligan, 2001)

Class A GPCRs also form dimers but with different mechanisms compared to class C GPCRs. One proposed mechanism is the idea of domain swapping between GPCRs. The receptors can split between helices V and VI and functional receptors can the re-formed (Figure 3). Since the two segments are derived from different GPCRs the receptors will have distinct ligand binding properties. This proposed domain swapping mechanism is supported by an experiment with the muscarine M3 acetylcholine receptor carried out by Maggio et al [9].

Figure 3: Domain swapping;

Functional GPCRs can be formed by expressing separated fragments of a GPCR including helices I-V and VI-VII. (Graeme Milligan, 2001)

University no.: 0527680 A second proposed mechanism for class I GPCRs involves lateral packing of individual polypeptides (Figure 4). Studies have shown that in this type of dimer, contact occurs between helices V and VI. This type of dimer is likely to be formed as a result of ligands being too large and lateral packing is required to initiate signal transduction [7].

Figure 4: Lateral packing.

Required for receptor activation if ligand is too large to bind to only one receptor. (Graeme Milligan, 2001)

Dimerization of class A GPCRs

The Rhodopsin-like receptors, class A GPCRS, comprises roughly 90% of all GPCRs making it the largest family of membrane proteins in the human genome [10]. Although they are not as structurally complex as class C GPCRs, they induce some of the most important signalling pathways in cells.

1 - adrenoceptors
The adrenoceptors are a type of GPCR that respond to adrenaline and noradrenalin. They can be classified as 1, 2 or based on their affinity to bind agonists as well as synthetic ligands. The 1-adrenoceptors can be divided into three subtypes (1a, 1b, 1d) but all subtypes seem to have similar affinity for adrenaline and noradrenalin [11]. The 1-adrenoceptors can exist as both homo-dimers and hetero-dimers. Studies using FRET have shown that the 1a and the 1b receptors both form homo-dimers. Time resolved FRET has been used to demonstrate that a large portion of the homo-dimers exist intracellularly, suggesting that they form constitutive dimers [11]. It is not fully known what effect homo-dimerization has on the 1-adrenoceptors, it might enhance ligand binding or could be a necessity to transport to the surface of the cell. Univeristy no.: 0527680 5

Since the 1-adrenoceptors are highly homologous within the transmembrane, it can be predicted that the three subtypes do from hetero-dimers. This is true to some extent. The 1d adrenoceptors is normally only expressed on the cell surface if dimerized to the 2A receptor showing that this dimer plays an important role in trafficking receptors from the ER to the cell surface [13]. Interactions between the 1-adrenoceptors and the H1 Histamine and DOP opioid receptors have also been seen but the affinity for these dimers to form is very low [13].

2 adrenergic receptors
BRET studies have shown that 80% of 2A receptors exist as homo-dimers [14]. The dimers seem to be constitutive as the dimerization occurs during receptor synthesis in the ER. This process is necessary for the transport of the receptors to the cell surface. The receptors interact between the transmembrane helices and interference with these interactions has shown to reduce ligand binding. This is supported by an experiment carried out by Ali Salahpour et al [15]. The 2A receptors can also form hetero-dimers and this has an effect on receptor trafficking and internalization. The 2A receptor can interact with the -opioid and the opioid receptors. If the 2A receptor is co-expressed with the -opioid receptor then agonist activation causes internalization of the -opioid receptor. In contrast, if the 2A receptor is bound the -opioid receptor then internalization does not occur when activated by agonists [14]. The 2A receptor also plays an important role in the airway smooth muscle; it induces bronchodilatation. The prostanoid EP1 receptor has the opposite effect; it causes bronchoconstriction. Under normal conditions, when an agonist activates the 2A receptor the G-protein (Gs) is activated, stimulating adenylyl cyclase thus increasing the concentration of cAMP and bronchodilatation occurs. When the EP1 receptor is activated by its agonist PGE2 the G-protein (Gq) is activated leading to an increase in IP3 causing bronchoconstriction. However when the 2A and the EP1 receptor dimerize a different result is obtained. McGraw et al carried out an experiment supporting this [16]. In the experiment, Coimmuniprecipitation and BRET were used to detect EP1- 2A heterodimeric receptors. The heterodimer was then stimulated with PGE2, and instead of causing bronchoconstriction as expected, the 2A receptor was uncoupled from its G-protein. This diminished the bronchodilator response of the 2A agonist (Figure 5). This reduced response to the 2A agonist is maybe was occurs in people with asthma and demonstrates that hetero-dimers have important functional consequences.

Figure 5: A) Activation of the EP1 receptor causes bronchoconstriction. Activation of the

2A receptor causes bronchodilatation. B) Dimerization is promoted by PGE2 and causes to the G-protein to uncouple from the 2A receptor. When the 2A receptor is stimulated by its agonist bronchodilatation is reduced as a result of the dimerization. (Peter J. Barnes, 2006 [14])

Dimerization of class C GPCRs

Class C receptors are made of three main structural domains. These domains include the cysteine rich domains (CRP), the heptahelical domain (HD) which is involved in Gprotein activation, and the Venus Flytrap Domain (VFT) which bind the ligands [17]. The VFT is a bilobule domain and studies have shown that both agonists and antagonists bind to the cleft that separates both lobes. Studies have also shown the VFTs of class C GPCRs can adopt either an open or closed conformation. When an antagonist is bound, only the open conformation has been observed. The closed conformation has been observed when a ligand is bound and in the absence of one. The VFT is therefore stabilized by agonists in the closed state and antagonists prevent this closure [17]. Dimerization is an important factor in the activation of class C GPCRs. They have shown to form constitutive dimers, the receptors being linked mostly by disulphide bridges. The mGlu5 receptor has shown to form homo-dimers using western blot and Coimmuniprecipitation. The homo-dimers can exist in both transfected cells as well as in native tissue. The CaS receptor has also shown to exist as homo-dimers and the dimer is stabilized by the Cys129 residue which is found in the VFT. This residue has also been identified in the mGlu1 and mGlu5 receptors and since this residue is conserved in all mGlu receptors, they are all expected to be disulphide linked dimers [18]. The exact role of the disulphide bridge is not yet known although it does prevent dissociation of the constitutive dimers under normal conditions. One of the receptors of the class C GPCRs that is not a disulphide-linked dimer is the gamma-amonobutynic acid B receptor (GABAB) involved in the activation of GIRK

Univeristy no.: 0527680 potassium channels. The GABAB receptor is an obligatory heterodimer and is only fully functional when composed of its two distinct subunits GABAB1 and GABAB2. The GABAB1 receptor cannot reach the cell surface on its own because it is prevented by an endoplasmic retention signal on its intracellular tail. Heterodimerization occurs in the endoplasmic reticulum and when GABAB1 is bound to GABAB2 it can reach the surface. Within the dimeric complex the two receptors have different functions. GABAB2 activates the G protein and GABAB1 is responsible for binding the ligand [2, 17]. Homo-dimers of class C GPCRs require two agonists to be fully activated. In contrast, only a single agonist is sufficient to activate a hetero-dimeric receptor complex. For example, in the GABAB1-GABAB2 complex the agonist GABA only binds to the GABAB1 VFT. The GABAB2 does posses a binding site but no ligands bind to this domain. However, the GABAB2 VFT is important in receptor activation and agonist affinity. When the heterodimer is formed the affinity for agonists increases while the affinity for antagonists decreases. This occurs as a result from a stabilization of the closed state of the agonist-bound GABA B1 VFT by the GABAB2 VFT [19]. Similar to the GABAB receptor, the sweet and umami receptors only require one agonist to activate its heterodimeric complex [17]. The sweeteners aspartame and neotame bind to the VFT of the T1R2 receptor of the T1R2-T1R3 dimer. In the T1R2-T1R3 dimer, glutamate binds to the T1R2 VFT. As described earlier, the GABAB2 VFT has no known binding activity. In contrast, the T1R3 VFT has been very well conserved throughout evolution which suggests that it is capable of ligand binding. Studies have shown that if T1R3 is co-expressed with T1R2 other sweet tasting molecules such as sucrose and saccharin are recognised [2].

Hetero-dimerization affects -arrestin interactions

Hetero-dimerization regulates the endocytotic processing of GPCRs. Receptor activation promotes the involvement of -arrestin and is responsible for signal termination by blocking G-protein interactions and initiating receptor internalization. It does this with the help of clathrin and the adaptor protein AP-2 [20]. GPCRs can the divided into 2 classes based on their interactions with -arrestin. Class I GPCRs have a higher affinity for -arrestin 2 than -arrestin 1. Class II GPCRs have the same affinity for both -arrestins. Soon after -arrestin has been recruited to a Class I GPCR it rapidly dissociates during internalization. The receptor is also rapidly dephosphorylated and recycled back to the plasma membrane. -arrestin forms a more stable complex with a class II GPCR which leads to co localization in the endosomes. A class II GPCR is recycled very slowly back to the plasma membrane [2]. An experiment carried out by Terrillon et al [20] shows that a class I V1a vasopressin receptor can dimerize to a class II V2 vasopressin receptor. This heterodimer has effects on arrestin binding, endocytosis and recycling of the receptors.

University no.: 0527680 When the two receptors are expressed separately with agonists, internalization occurs with both the V1a and V2 receptor. When the receptors are co-expressed, internalization of V2 remains the same whereas the internalization of V1a is significantly reduced. Since the V2 receptor is brought into the endosomes with -arrestin, the availability of -arrestin in the cytosol is decreased significantly therefore inhibiting V1a internalization. When the receptors are co-expressed with non selective agonists, the V2 receptor promotes a change in the behaviour of the V1a receptor so that it follows an endocytotic pathway similar to a class II GPCR. This is a result of dimerization. A V1a receptor that is not dimerized to a V2 receptor will internalize like a class I GPCR whereas with it forms a heterodimeric complex with V2 the complex in internalized like a class II GPCR. Internalization of V1a and V2 is largely dependant upon the stability of their interactions between -arrestin. Therefore, if the stability of the complex can be predicted then it could be determined whether a class I or II endocytotic pathway will occur. When a V1a receptor is selectively activated it leads a weak -arrestin interaction. This is because of the lack of cross activation of the V2 receptor and class I endocytosis will occur. In the case of non-selective activation of the receptors causes a more stable interaction between -arrestin and the V2 receptor and class II endocytosis would occur.

Conclusion
Protein-protein interactions are an important aspect of biology and GPCR dimerization will definitely play a major role in studies to come. Key aspects that will need to be addressed will be the regulation of dimerization and the functional consequences in signal transduction mechanisms. Since it is now known that GPCRs form dimers, this phenomenon can be exploited to improve drug specificity. It is already known that the hetero-dimerization in the CCR2-CCR5 receptors prevents HIV from interacting with CCR5 [2]. This delays the progression to AIDS by 2-4 years. In order to understand how ligands bind to dimers the structure and organization GPCR dimers in the membrane need to be better understood and the possibility of developing selective ligands for hetero-dimeric GPCRs is going to play a major role in future drug development.

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References
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