**1. Introduction**

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Seven transmembrane receptors (7TMRs; also designated as G-protein coupled receptors (GPCRs)) form the largest and evolutionarily well conserved family of cell-surface receptors, with more than 800 members identified in the human genome. 7TMRs are the targets both for a plethora of endogenous ligands (e.g. peptides, glycoproteins, lipids, amino acids, nucleotides, neurotransmitters, odorants, ions, and photons) and therapeutic drugs and transduce extracellular stimuli into intracellular responses mainly via coupling to guanine nucleotide binding proteins (G-proteins) (McGraw & Liggett, 2006).

These receptors have traditionally been viewed as monomeric entities and only more recent biochemical and biophysical studies have changed this view. The idea that 7TMRs might form dimers or higher order oligomeric complexes has been formulated more than 20 years ago and since then intensively studied. In the last decade, bioluminescence resonance energy transfer (BRET) was one of the most commonly used biophysical methods to study 7TMRs oligomerization. This technique enables monitoring physical interactions between protein partners in living cells fused to donor and acceptor moieties. It relies on nonradiative transfer of energy between donor and acceptor, their intermolecular distance (10 – 100 Å) and relative orientation. Over this period the method has progressed and several versions of BRET have been developed that use different substrates and/or energy donor/acceptor couples to improve stability and specificity of the BRET signal. This chapter outlines BRET-based approaches to study 7TMRs oligomerization (e.g. BRET saturation and competition assays), control experiments needed in the interpretation i.e. establishing specificity of BRET results and mathematical models applied to quantitatively assess the oligomerization state of studied receptors.

## **2. Seven transmembrane receptors (7TMRs): Structure and characteristics**

Primary sequence comparisons reveal that 7TMRs share sequence and topology similarities allowing them to be classified as a super-gene family. These receptors are characterized by

Quantitative Assessment of Seven Transmembrane Receptors

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

family), with 24 members.

(7TMRs) Oligomerization by Bioluminescence Resonance Energy Transfer (BRET) Technology 83

Ca2+ sensing receptor, and the receptor for the major excitatory neurotransmitter in the central nervous system, the γ-aminobutyric acid (GABAB) receptor and orphan receptors. This family has a very large N-terminal domain (>600 amino acids), which bears the agonist binding site and also a long C-tail (Kenakin & Miller, 2010; McGraw & Liggett, 2006). Notably, family C members form obligatory dimers (Kniazeff et al., 2011). Two ancillary families consist of class D (adhesion family), containing 24 members, and class E (frizzled

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)).

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

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 Ca2+ sensing receptor, and the receptor for the major excitatory neurotransmitter in the central nervous system, the γ-aminobutyric acid (GABAB) receptor and orphan receptors. This family has a very large N-terminal domain (>600 amino acids), which bears the agonist binding site and also a long C-tail (Kenakin & Miller, 2010; McGraw & Liggett, 2006). Notably, family C members form obligatory dimers (Kniazeff et al., 2011). Two ancillary families consist of class D (adhesion family), containing 24 members, and class E (frizzled family), with 24 members.
