**3. 7TMRs homo- and hetero-oligomerization**

82 Bioluminescence – Recent Advances in Oceanic Measurements and Laboratory Applications

seven hydrophobic stretches of 20-25 amino acids, predicted to form transmembrane -helices. Prediction of transmembrane folding was based largely on the method proposed by Kyte and Doolitle (Kyte & Doolittle, 1982). This method plots the hydrophobicity of the amino acids along the sequence, assigning each amino acids a hydrophobicity index. By summing this index over a window of nine residues, the transmembrane sequence is postulated when index reaches the value of 1.6 for a stretch of ~20 amino acids. This number is based on the assumption that the membrane spanning sequences of protein are -helical and that about six helical turns are required to span the lipid bilayer (Hucho & Tsetlin, 1996). The highly hydrophobic -helices that serve as transmembrane spanning domains (TMs) are connected by three extracellular (ECL) and three intracellular (ICL) hydrophilic loops. Amino (N) terminal fragment is extracellular and the carboxyl (C)-terminal tail is intracellular. In the recent years this common structural topology was also confirmed by three-dimensional crystal structure of some 7TMR members (reviewed by (Salon et al., 2011)). Additionally, 7TMRs may undergo a variety of posttranslational modifications such as N-linked glycosylation, formation of disulfide bonds, palmitoylation and phosphorylation. 7TMRs contain at least one consensus sequence for N-linked glycosylation (Asn-x-Ser/Thr), usually located near the N-terminus, although there are potential glycosylation sites in the intracellular loops. They also contain a number of conserved extracellular cysteine residues, some of which appear to play a role in stabilizing the receptor's tertiary structure. An additional highly conserved cysteine can be present within the C-terminal tail of many 7TMRs. When palmitoylated, it may anchor a part of cytoplasmic tail of the receptor to the plasma membrane, thus forming the fourth ICL and controlling the tertiary structure. Consensus sequences for potential phosphorylation sites (serine and threonine residues) are located in the second and third ICLs, and in particular, in the intracellular C-terminal tail. The most obvious structural differences between the receptors in subgroups are the length of their N-terminal fragment and the loops between TMs. Originally, 7TMRs were divided into six groups, A – F; families (also known as "groups" or "classes") A, B and C included all mammalian 7TMRs. Genome projects then generated numerous new 7TM sequences and more than 800 human 7TMRs were reclassified into five families, A – E (reviewed by

Family A (also known as the rhodopsin family) is by far the largest family of 7TMRs (containing ~700 members), and includes many of the receptors for biogenic amines and small peptides. It is characterized by very short N- and C-termini as well as several highly conserved amino acids. In most cases TMs serve as the ligand-binding site. This family contains some of the most extensively studied 7TMRs, the opsins and the β-adrenergic receptors. Recent structural information for a few family A 7TMR members (e.g. rhodopsin, opsin, human β2-adrenergic receptor, turkey β1-adrenergic receptor, human A2A-adenosine receptor, CXC chemokine receptor type 4 and D3-dopamine receptor) confirmed an obvious conservation of the topology and seven-transmembrane architecture (Salon et al., 2011). Family B (secretin-receptor family), which has considerably fewer members i.e. 15, is characterized by a long N-terminus (>400 amino acids) containing six conserved cysteine residues that contribute to three conserved disulfide bonds, which provide structural stability, and a conserved cleft for the docking of often helical C-terminal region of the peptide ligands. Natural ligands for family B 7TMRs are all moderately large peptides, such as calcitonin, parathyroid hormone and glucagon. Family C (metabotropic glutamate family) contains 15 members that are the metabotropic glutamate receptors (mGluRs), the

(Gurevich & Gurevich, 2006; Salon et al., 2011)).

In 1983, Fuxe et al. (Fuxe et al., 1983) formulated the hypothesis about the existence of homo-dimers for different types of 7TMRs and in the same year the first demonstration of 7TMRs homo-dimers and homo-tetramers of muscarinic receptors was published (Avissar et al., 1983). However, the evidence for dimerization existed even before that. Following classical radio-ligand studies on the insulin receptor (de Meyts et al., 1973), negative cooperativity, for which dimerization is a prerequisite, has also been demonstrated for β2 adrenergic receptor (β2-AR) (Limbird et al., 1975) and thyrotrophin-stimulating hormone (TSH) receptor (De Meyts, 1976) binding in the early 70's, before they were shown to be 7TMRs and this issue remained controversial for over two decades. 7TMRs can be either connected to identical partner(s), which results in formation of homo-dimers (or homooligomers), or to structurally different receptor(s), which results in formation of heterodimers (hetero-oligomers). 7TMR dimerization was proposed to play a potential role in i) receptor maturation and correct transport to the plasma membrane, ii) ligand-promoted regulation, iii) pharmacological diversity (e.g. positive and negative ligand binding cooperativity), iv) signal transduction (potentiating/attenuating signaling or changing Gprotein selectivity), and v) receptor internalization and desensitization (Terrillon & Bouvier, 2004). The first widely accepted demonstration of 7TMR hetero-dimerization came from the GABAB (GBBR) receptors that exclusively function in a heteromeric form (White et al., 1998).

There is now considerable evidence to indicate that 7TMRs can form and function as homodimers and hetero-dimers (reviewed by (Filizola, 2010; Gurevich & Gurevich, 2008a; Palczewski, 2010)) and that these dimers may have therapeutic relevance (Casado et al., 2009). Hetero-dimerization in the C family of receptors has been most extensively studied and for some experts in the field of 7TMRs the only one demonstrated to form real dimers (for recent review see (Kniazeff et al., 2011)). In this family of 7TMRs receptors hetero-dimerization is important for either receptor function, proper expression on the cell surface or enhancing receptor activity. In the most numerous family A 7TMRs dimerization was extensively studied, although with few exceptions functional role of receptor self-association is in most cases unclear. Compelling evidence for the dimerization in the family A 7TMR was only recently demonstrated *in vivo* by Huhtaniemi's group, who was able to rescue the LH receptor knockout phenotype by complementation i.e. co-expressing two nonfunctional receptor mutants in the knockout mice (Rivero-Muller et al., 2010). Members of the family B 7TMRs have also only recently been shown to associate as stable homo-dimers. The structural basis of this, at least for the prototypic secretin receptor, is the lipid-exposed face of TM4. This complex has been postulated as being important for the structural stabilization of the high affinity complex with G-protein (reviewed by (reviewed by (Kenakin & Miller, 2010)).

Quantitative Assessment of Seven Transmembrane Receptors

development process (reviewed by (Casado et al, 2009)).

**4. BRET principle and its application in the field of 7TMRs dimerization** 

BRET is a biophysical method that enables monitoring of physical interactions between two proteins fused to BRET donor and acceptor moieties, respectively, dependent on their intermolecular distance (10 – 100 Å) and on relative orientation due to the dipole-dipole nature of the resonance energy transfer mechanism (Zacharias et al., 2000). BRET is a nonradiative energy transfer, occurring between a bioluminescent donor that emits light in the presence of its corresponding substrate and a complementary fluorescent acceptor, which absorbs light at a given wavelength and re-emits light at longer wavelengths. To fulfill the condition for energy transfer, the emission spectrum of the donor must overlap with the excitation spectrum of the acceptor molecule (Zacharias et al., 2000). BRET occurs naturally in some marine species (e.g. in the sea pansy *Renilla reniformis*) and in 1999, Xu et al. (Xu et al., 1999) utilized this approach to study dimerization of the bacterial Kai B clock protein. Since then, several versions of BRET assays have been developed that use different substrates and/or energy donor/acceptor couples. The original BRET1 technology used the pairing of *Renilla luciferase* (Rluc) as the donor and yellow fluorescent protein (YFP) as the acceptor (Xu et al., 1999; Xu et al., 2003). The addition of coelenterazine h, the natural substrate of *Renilla luciferase* (Rluc), leads to a donor emission of blue light (peak at ~480 nm). When the YFP-tagged acceptor molecule, adapted to this emission wavelength, is in close proximity to the Rluc-tagged donor molecule, excitation of YFP occurs by resonance energy transfer resulting in an acceptor emission of green light (peak at ~530 nm). The substantial overlap in the emission spectra of Rluc and YFP acceptor emission (Stokes shift

(Schoneberg et al., 2004).

**4.1 BRET principle** 

(7TMRs) Oligomerization by Bioluminescence Resonance Energy Transfer (BRET) Technology 85

various subtypes of 5-HT receptors, are used to treat major depressive disorders

The increasing importance of dimerization for 7TMRs naturally suggests its possible relevance to drug discovery. It seems that the inclination to hetero-dimerize is common among the 7TM members and that the tissue-specific expression patterns probably underlay the creation of relevant receptor pairs. However, 7TMRs expression has been shown to be altered in some pathological situations. In support to the latter preeclampsia was the first disorder linked to alteration in the AT1−bradykinin B2 receptor hetero-dimerization (AbdAlla et al., 2001). Opioid and dopamine receptor hetero-dimerization has also been comprehensively studied, since their putative ligands are used in pathological conditions such as basal ganglia disorders, schizophrenia, drug addiction and pain. The increase in the dopamine D1-D3 hetero-dimer was shown to be involved in L-dopa-induced dyskinesia in patients with Parkinson's disease and the addition of an adenosine A2A receptor antagonist potentiates the anti-parkinsonian effect of L-dopa. Hetero-dimers of glutamate receptors mGluR2 and 5-HT2A have been specifically associated with hallucinogenic responses in schizophrenia. Furthermore, the opioid δ-μ receptor hetero-dimer is a better target than either μ or δ receptors alone, since blockade of the δ receptor decreases tolerance to the analgesic effects of the most used μ receptor agonist, morphine (reviewed by (Ferré & Franco, 2010; Kenakin & Miller, 2010)). These observations would probably led to broaden the therapeutic potential of drug targeting 7TMRs and it is also anticipated that the evolving concepts of 7TMR dimerization will be implemented in the BRET-based drug discovery and

In addition to widespread intra-family hetero-dimerization, inter-family hetero-dimerization has also been reported, at least between both of the family A members β2-AR and opsin and the family B member gastric inhibitory polypeptide receptor (GIP) (Vrecl et al., 2006), and between the family A serotonin 5-HT2A receptors and the family C mGluR2 (Gonzalez-Maeso et al., 2008). Both types of hetero-dimers were demonstrated to be functional, either by their ability to induce cAMP production upon agonist stimulation (family A/B hetero-dimer), or by their ability to modulate G-protein coupling (family A/C hetero-dimer).
