Metal Ion Site Engineering Indicates a Global Toggle Switch Model for Seven-transmembrane Receptor Activation

Abstract

Much evidence indicates that, during activation of seven-transmembrane (7TM) receptors, the intracellular segments of the transmembrane helices (TMs) move apart with large amplitude, rigid body movements of especially TM-VI and TM-VII. In this study, AspIII:08 (Asp 113 ), the anchor point for monoamine binding in TM-III, was used as the starting point to engineer activating metal ion sites between the extracellular segments of the ␤ 2 -adrenergic receptor. Cu(II) and Zn(II) alone and in complex with aromatic chelators acted as potent (EC 50 decreased to 0.5 M) and efficacious agonists in sites constructed between positions III:08 (Asp or His), VI:16 (preferentially Cys), and/or VII:06 (preferentially Cys). In molecular models built over the backbone conformation of the inactive rhodopsin structure, the heavy atoms that coordinate the metal ion were located too far away from each other to form high affinity metal ion sites in both the bidentate and potential tridentate settings. This indicates that the residues involved in the main ligand-binding pocket will have to move closer to each other during receptor activation. On the basis of the distance constraints from these activating metal ion sites, we propose a global toggle switch mechanism for 7TM receptor activation in which inward movement of the extracellular segments of especially TM-VI and, to some extent, TM-VII is coupled to the well established outward movement of the intracellular segments of these helices. We suggest that the pivots for these vertical seesaw movements are the highly conserved proline bends of the involved helices. Seven-transmembrane (7TM)2 receptors, which couple through G proteins but also through a number of G protein-independent signaling pathways such as arrestin-mediated kinase activation, constitute the largest family of proteins in the human genome (1-3). These receptors are activated by basically all kinds of chemical messengers in the body, and they act as chemosensors for a multitude of external chemical signals. 7TM receptors are also the target for the majority of drugs, which, however, address only a small fraction of the receptor repertoire (4). Because they are involved as regulators of key physiological functions throughout the body, 7TM receptors are one of the major focus areas of the pharmaceutical industry (4, 5).7TM receptors are activated by agonists that span a chemical diversity range from large glycoprotein hormones over neuropeptides, lipid messengers, and nucleotides to small monoamines, amino acids, and even metal ions such as calcium (1-3). Notably, although there apparently is no common "lock" for all these chemically highly diverse "keys" (6), it is nevertheless expected that there is a common molecular activation mechanism for 7TM receptors as such. The conformational changes that accompany receptor activation have been characterized in rather great detail for the intracellular segments of the seven-helix bundle (7)(8)(9)(10)(11)(12)(13)(14). This has been achieved through a number of different biochemi...

Ligand Modulation of the Epstein-Barr Virus-induced Seven-transmembrane Receptor EBI2

“…For example, the monoamine receptors contain an Asp at position III:08/3.32 that interacts with the quaternary amine of the monoamine ligand (30, 38 -40). Also, III:08/3.32 constitutes a crucial part of activating bi-and tridentate metal ion binding sites engineered into the ␤ 2 -adrenergic receptor (27) and also serves as an anchor point for C5a binding to the C5a receptor (41) as well as for most chemokines and small molecule agonists targeting chemokine receptors (42). We also found that Phe-substitution of Thr 115 at position III:12/3.36 and Met 297 at VII:09/ 7.42 completely destroyed the GSK682753A-mediated inhibition, whereas Ala substitution did not (Fig.…”

“…We initially focused on TM-III because it is known to be one of the key players in 7TM receptor activation, during which the top of this helix has been hypothesized to perform an inward see-saw movement, as described in the global toggle switch model (27)(28)(29). Specifically, position III:08/3.32 in TM-III has been shown to be important for ligand binding and helical movement as seen in, for example, the ␤-adrenergic subfamily (30).…”

Fluorescence/Bioluminescence Resonance Energy Transfer Techniques to Study G-Protein-Coupled Receptor Activation and Signaling

“…Multiple active states have been assumed on the basis of theoretical and modeling considerations (Kenakin, 1995;Leff et al, 1997; FRET TECHNIQUES FOR GPCR ACTIVATION AND SIGNALING Buranda et al, 2007;Onaran and Costa, 2009;Kenakin and Miller, 2010) and because of ligand-specific "biased" effects of various agonist/receptor systems (Violin and Lefkowitz, 2007;Drake et al, 2008;Shukla et al, 2008;Rajagopal et al, 2011). Metal chelating strategies, originally developed to prevent the activation of GPCRs (Sheikh et al, 1996;Elling et al, 1999Elling et al, , 2006, revealed for the PTH receptor that different conformations were involved in coupling to G-proteins versus ␤-arrestins (Vilardaga et al, 2001); furthermore, the determinants of this receptor for ␤-arrestin binding and internalization were shown to be different (Vilardaga et al, 2002). Similar distinctions between requirements for activation and endocytosis were seen for the complement 5a receptor (Whistler et al, 2002).…”

Crystal structure of the ligand-free G-protein-coupled receptor opsin

“…The change of the toggle switch, depending on cis/trans isomerisation, may produce a domino effect, i.e. a maximization of the proline-induced kink in TM6 to facilitate helices movements, a breakage of the "ionic lock", a rearrangement of cytoplasmic loops and helices, leading to an "open" cytoplasmic surface [71,72,73,169,170]. Moreover, the residue (Trp-265) may also act as a gatekeeper which is able to mediate the signal transfer from the extracellular region to the cytoplasmic surface for coupling to the G protein.…”