**2. G protein coupled receptors**

#### **2.1 A few examples**

The human genome contains at least 800 GPCRs, grouped in five main families (Fredriksson *et al.*, 2003). One of the best characterized GPCR, rhodopsin, is responsible for vision in the dark: it captures photons thanks to its prosthetic group (11-cis retinal), and leads to phosphodiesterase activation in retina rod cells. It is extremely abundant in the rod cell disks, comparatively easy to purify, and therefore has been very extensively studied for many years by biochemists. Other GPCRs allow us to taste and smell, control our appetite, fertility, stress, heart rate and breathing, etc. Adrenaline (the stress hormone), histamine (allergic reactions), glucagon (glycemia control), but also taste and odorant receptors, luteotropic and follicular stimulating hormone receptors (ovule and spermatozoid development), etc. recognize GPCRs and induce G protein activation.

#### **2.2 GPCR families**

All G protein coupled receptors possess a glycosylated extracellular amino-terminal (Nterm) and an intracellular carboxyl-terminal (C-term) domain, separated by 7 transmembrane helices (TM1 to TM7) joined by three intracellular (IC1 to IC3) and three extracellular (EC1 to EC3) loops (Figure 1).

Fig. 1. Ribbon representation of the X-ray structure of rhodopsin. Left: schematic representation of a GPCR, showing the TM helices, intracellular (IC) and extracellular (EC) loops. Right: ribbon representation of the crystal structure of rhodopsin (1GZM). The prosthetic group, retinal, is covalently bound to a lysine side chain in TM7 (sticks).

The first and second EC loops are joined by a conserved disulfide bridge. The C-term region begins by an intracellular α-helix, H8, which lies horizontally on the plasma membrane: it forms an aromatic cluster with a tyrosine side chains from TM7 and interacts with the phospholipid head groups through lysine and arginine side chains. The 7 helices are arranged in a bundle (Figure 1). GPCRs have been identified in animals, yeast, plants. They probably arise from a common ancestor (Fredriksson *et al.*, 2003). Several hundred putative GPCRs have been identified in the human genome where they represent 1-3% of the genes (Fredriksson *et al.*, 2003). The vast majority of GPCRs (including most odorant receptors) share "signature" amino acids with rhodopsin (see below). They have been grouped in "Family A" (Kolakowski, Jr., 1994) or "rhodopsin-like receptor family" (Fredriksson *et al.*, 2003). Other GPCR families do not possess these highly conserved amino acids, but share other signature amino acids. For instance, all "family B" (secretin-receptor like) receptors possess a typical N-terminal "sushi" domain with three conserved disulfide bridges and have very strong sequence homologies in the transmembrane domain. Fifteen of these receptors with a comparatively short N-term domain ("sushi" domain only) are specialized in recognition of peptide hormones and neurotransmitters (glucagon, Growth Hormone Releasing Hormone (GHRH or GRF), parathyroid hormone (PTH) and others).

All G protein coupled receptors possess a glycosylated extracellular amino-terminal (Nterm) and an intracellular carboxyl-terminal (C-term) domain, separated by 7 transmembrane helices (TM1 to TM7) joined by three intracellular (IC1 to IC3) and three

Fig. 1. Ribbon representation of the X-ray structure of rhodopsin. Left: schematic

representation of a GPCR, showing the TM helices, intracellular (IC) and extracellular (EC) loops. Right: ribbon representation of the crystal structure of rhodopsin (1GZM). The prosthetic group, retinal, is covalently bound to a lysine side chain in TM7 (sticks).

The first and second EC loops are joined by a conserved disulfide bridge. The C-term region begins by an intracellular α-helix, H8, which lies horizontally on the plasma membrane: it forms an aromatic cluster with a tyrosine side chains from TM7 and interacts with the phospholipid head groups through lysine and arginine side chains. The 7 helices are arranged in a bundle (Figure 1). GPCRs have been identified in animals, yeast, plants. They probably arise from a common ancestor (Fredriksson *et al.*, 2003). Several hundred putative GPCRs have been identified in the human genome where they represent 1-3% of the genes (Fredriksson *et al.*, 2003). The vast majority of GPCRs (including most odorant receptors) share "signature" amino acids with rhodopsin (see below). They have been grouped in "Family A" (Kolakowski, Jr., 1994) or "rhodopsin-like receptor family" (Fredriksson *et al.*, 2003). Other GPCR families do not possess these highly conserved amino acids, but share other signature amino acids. For instance, all "family B" (secretin-receptor like) receptors possess a typical N-terminal "sushi" domain with three conserved disulfide bridges and have very strong sequence homologies in the transmembrane domain. Fifteen of these receptors with a comparatively short N-term domain ("sushi" domain only) are specialized in recognition of peptide hormones and neurotransmitters (glucagon, Growth Hormone Releasing Hormone (GHRH or GRF),

**2.2 GPCR families**

extracellular (EC1 to EC3) loops (Figure 1).

parathyroid hormone (PTH) and others).

The majority of family B receptors possess several additional modules (EGF-like, Immunoglobulin-like, etc) before the sushi domain, suggesting that they might function as adhesion proteins (Mizuno and Itoh, 2011). Most of these are "orphan" receptors (that is: their ligand is unknown) and their ability to activate G proteins has not been proven yet. "Family C" receptors recognize amino acids (metabotropic receptors for glutamate and GABAB receptors for GABA) or calcium ions, through N-terminal "venus flytrap" domains (Jensen *et al.*, 2002; Wellendorph and Brauner-Osborne, 2009). Some of the taste receptors also are GPCRs: sweet and "umami" tasting molecules are recognized by "family C" GPCRs, and the "bitter" taste, by GPCRs that present very little homology with the other GPCRs, and form an additional receptor family (Fredriksson *et al.*, 2003).

#### **2.3 Conserved residues in "family A" receptors**

Rhodopsin and related receptors possess a few extremely conserved residues in each TM helix. In the Ballesteros and Weinstein nomenclature, the most conserved amino acid in each TM helix is numbered Xh.50 (where "h" is the helix number): for instance, R3.50 is the most conserved amino acid in TM3; D3.49 and Y3.51 are the two conserved amino acids immediately preceding and following this arginine.

Some of these very conserved side chains are involved in structural features, like the prolines in helices 5, 6 and 7 that induce kinks in the TM helices. In the different family A receptors crystal structures (rhodopsin but also β-adrenergic, adenosine, histamine H3 receptors), the conserved asparagine of the TM7 NPxxY(x)5-6F motif is part of a hydrogen bond network involving TM1, TM2 (D2.50) and TM7, while the tyrosine in this motif constrains TM7 in contact with aromatic side chains in the C term helix 8. Other conserved side chains play a role in the resting and/or active receptor conformation For instance, the arginine of the TM3 "DRY motif" (E/DR3.50Y) at the intracellular end of the third transmembrane helix forms in rhodopsin a H bond network with E3.49, E6.30 and T6.34. The "ionic lock" R3.50-E6.30 stabilizes the resting state: it is broken up in metarhodopsin II (the active rhodopsin conformation). In that structure, R3.50 folds back inside the G protein to interact with Y5.58, thereby creating an intracellular binding pocket, able to accommodate the G-protein. The ionic lock is less stable in the β-adrenergic receptors compared to rhodopsin, and this is perhaps responsible for their detectable constitutive activity (ability to activate G proteins in the absence of agonist) (Moukhametzianov *et al.*, 2011). W6.48 of the CWxP6.50 motif is in very close contact with the agonist ligands, and was thought to trip the switch of receptor activation by toggling between different rotamer conformations and thereby affecting the position of neighbouring aromatic side chains. Although this hypothesis is supported by computational mapping (Bhattacharya and Vaidehi, 2010), the toggle is not evident in the metarhodopsin II (Standfuss *et al.*, 2011; Choe *et al.*, 2011) or β2-adrenergic receptor crystal structures (Rasmussen *et al.*, 2011b; Rasmussen *et al.*, 2011a).
