**6. "Resting" and "active" receptor structures**

When rhodopsin is illuminated, its conformation passes through a number of intermediates (bathorhodopsin (t1/2 50ns), lumirhodopsin (t1/2 50µs), followed by metarhodopsin I and II) before releasing the all-trans retinal. Metarhodopsin II is biologically active: it catalyses G protein (transducin) activation.

The three dimensional structure of rhodopsin and several "family A" GPCRs has been elucidated by X-ray crystallography in the absence and presence of antagonists, agonists, G protein surrogates, or of the trimeric-Gs protein (Choe *et al.*, 2011; Lebon *et al.*, 2011; Rasmussen *et al.*, 2011a; Rasmussen *et al.*, 2011b; Rosenbaum *et al.*, 2011; Standfuss *et al.*, 2011; Warne *et al.*, 2011; Xu *et al.*, 2011). A conserved ionic bond between rhodopsin arginine 135 (R3.50) (in the conserved E/DRY motif at the intracellular end of TM3) and glutamate 247 (E6.30) , at the end of the third intracellular loop-TM6 junction tethers rhodopsin TM3 to TM6. In antagonist-bound β2-adrenergic receptors this hydrogen bond between the conserved arginine and glutamate is not visible in the crystal and β2-adrenergic receptors are known to activate slightly their cognate GS G protein even in the absence of agonist; the ionic bridge is less stable than in rhodopsin (Moukhametzianov *et al.*, 2011).

Fig. 8. Resting and activated GPCR conformations. Ribbon representation of the IC and TM regions of rhodopsin (1GZM), metarhodopsin II (3PXO), metarhodopsin II – Gt Cterm complex (3PQR), antagonist-bound β2-adrenergic receptor (2RH1), agonist-bound β2 adrenergic receptor (3PDS) and agonist-GS (Cterm) bound β2-adrenergic receptor (3SN6), seen from the cytosol. The conserved TM3 arginine (R3.50), TM5 tyrosine (Y5.58) and TM6 glutamate (E6.30) side chains are shown in red, yellow and blue respectively. The Cterminal transducin peptide (top right) and the C-terminal region of Gα<sup>S</sup> (bottom right) are shown as green ribbons.

In contrast with (dark adapted) rhodopsin, a large intracellular binding pocket is present between the TM helices of metarhodopsin II (Choe *et al.*, 2011): the distance between the conserved arginine (R3.50) at the intracellular end of TM3 (E/DRY motif) and the conserved glutamate at the junction between the third intracellular loop and TM6 (E6.30) increases from less than 3.3Å in rhodopsin (PDB 1GZM), bathorhodopsin (PDB 2G87) and lumirhodopsin (PDB 2HPY) to >15 Å in opsin (PDB 3CAP, 3DBQ) and metarhodopsin II (PDB 3PQR, 3PXO) (Figure 8). Arginine R3.50 forms in opsin and metarhodopsin II a strong ion-dipole interaction with the conserved tyrosine, Y5.58 in TM5. The G-protein binding pocket is created by the rotation of TM 5 and 6. It is large enough to accommodate the C-terminal helix of the transducin Gα subunit or of GαS, at almost 40° from the membrane surface (Park *et al.*, 2008; Scheerer *et al.*, 2008; Choe *et al.*, 2011) (Figure 8): this movement forces the opening of the GDP binding pocket and release GDP from the G protein (Rasmussen *et al.*, 2011b).

The sixth transmembrane helix (TM6) of the crystallized β1-, β2-adrenergic and adenosine A2A receptors remains very close to TM3 even in the presence of agonists (Rasmussen *et al.*, 2011a; Rosenbaum *et al.*, 2011; Warne *et al.*, 2011); and the conserved E/DRY motif arginine folds towards the cytoplasm, in the direction of the conserved TM6 glutamate: the G protein binding pocket is unavailable (Figure 8). An open, "metarhodopsin II-like" structure is achieved by β2-adrenergic receptors only in the presence of a G protein surrogate or of GS (Rasmussen *et al.*, 2011a; Rosenbaum *et al.*, 2011; Rasmussen *et al.*, 2011b): TM5 and TM6 rotate away from TM3, and the arginine side chain R3.50 toggles away from the IC loop E6.30 towards the conserved tyrosine, Y5.38.
