(4) disclose previously undetected features of GPCR behavior. Significant impact of DMR is therefore anticipated in the emerging areas of systems biology and systems pharmacology but also for the discovery of mechanistically novel drugs.3

Selective modulation of cell function by G protein-coupled receptor (GPCR) activation is highly desirable for basic research and therapy but difficult to achieve. We present a novel strategy toward this goal using muscarinic acetylcholine receptors as a model. The five subtypes bind their physiological transmitter in the highly conserved orthosteric site within the transmembrane domains of the receptors. Orthosteric muscarinic activators have no binding selectivity and poor signaling specificity. There is a less well conserved allosteric site at the extracellular entrance of the binding pocket. To gain subtype-selective receptor activation, we synthesized two hybrids fusing a highly potent oxotremorine-like orthosteric activator with M(2)-selective bis(ammonio)alkane-type allosteric fragments. Radioligand binding in wild-type and mutant receptors supplemented by receptor docking simulations proved M(2) selective and true allosteric/orthosteric binding. G protein activation measurements using orthosteric and allosteric blockers identified the orthosteric part of the hybrid to engender receptor activation. Hybrid-induced dynamic mass redistribution in CHO-hM(2) cells disclosed pathway-specific signaling. Selective receptor activation (M(2)>M(1)>M(3)) was verified in living tissue preparations. As allosteric sites are increasingly recognized on GPCRs, the dualsteric concept of GPCR targeting represents a new avenue toward potent agonists for selective receptor and signaling pathway activation.

Seven transmembrane helical receptors (7TMRs) modulate cell function via different types of G proteins, often in a ligand-specific manner. Class A 7TMRs harbour allosteric vestibules in the entrance of their ligand-binding cavities, which are in the focus of current drug discovery. However, their biological function remains enigmatic. Here we present a new strategy for probing and manipulating conformational transitions in the allosteric vestibule of label-free 7TMRs using the M2 acetylcholine receptor as a paradigm. We designed dualsteric agonists as 'tailor-made' chemical probes to trigger graded receptor activation from the acetylcholine-binding site while simultaneously restricting spatial flexibility of the receptor's allosteric vestibule. Our findings reveal for the first time that a 7TMR's allosteric vestibule controls the extent of receptor movement to govern a hierarchical order of G-protein coupling. This is a new concept assigning a biological role to the allosteric vestibule for controlling fidelity of 7TMR signalling.

G protein-coupled receptors represent the largest superfamily of cell membrane-spanning receptors. We used allosteric small molecules as a novel approach to better understand conformational changes underlying the inactive-to-active switch in native receptors. Allosteric molecules bind outside the orthosteric area for the endogenous receptor activator. The human muscarinic M 2 acetylcholine receptor is prototypal for the study of allosteric interactions. We measured receptor-mediated G protein activation, applied a series of structurally diverse muscarinic allosteric agents, and analyzed their cooperative effects with orthosteric receptor agonists. A strong negative cooperativity of receptor binding was observed with acetylcholine and other full agonists, whereas a pronounced negative cooperativity of receptor activation was observed with the partial agonist pilocarpine. Applying a newly synthesized allosteric tool, point mutated receptors, radioligand binding, and a three-dimensional receptor model, we found that the deviating allosteric/orthosteric interactions are mediated through the core region of the allosteric site. A key epitope is M 2 Trp 422 in position 7.35 that is located at the extracellular top of transmembrane helix 7 and that contacts, in the inactive receptor, the extracellular loop E2. Trp 7.35 is critically involved in the divergent allosteric/orthosteric cooperativities with acetylcholine and pilocarpine, respectively. In the absence of allosteric agents, Trp 7.35 is essential for receptor binding of the full agonist and for receptor activation by the partial agonist. This study provides first evidence for a role of an allosteric E2/transmembrane helix 7 contact region for muscarinic receptor activation by orthosteric agonists. G protein-coupled receptors (GPCRs)4 have outstanding importance as targets for drug action (1, 2). Conformational changes underlying the inactive-to-active receptor switch in GPCRs are in the focus of current research. In general, the receptor transmembrane helices (TMs) rearrange, allowing the intracellular loop region to unfold and to stimulate neighboring G proteins (3, 4). Conformational changes include extracellular receptor regions, and a critical role of the second extracellular loop (E2) for GPCR activation and ligand binding has emerged (5-9). Rational development of agonistic drugs for GPCR activation requires deeper insight into such conformational changes. Because GPCRs are hardly accessible for crystallization, indirect approaches are applied that often involve modification of the receptor protein such as receptor mutagenesis, introduction of metal ion sites or disulfide bridges, or covalent linkage of moieties for fluorescence resonance energy transfer.Allosteric small molecules allow the study of native receptors. An increasing number of GPCRs is known to contain allosteric sites (10, 11); cinacalcet is the first allosteric GPCR modulator that has recently entered the market (12). Allosteric sites are located outside the orthosteric area that is occupied...

Increasing attention is paid in basic science and in drug discovery to pathway selective intracellular signaling as a novel approach to achieve precise control of cell function via G protein-coupled receptors (GPCRs). With respect to signaling, GPCRs are often promiscuous in that more than one intracellular biochemical pathway is activated upon receptor stimulation by the endogenous transmitter or by exogenous drugs. We studied signaling by a novel class of GPCR activators that were designed to bind simultaneously to the orthosteric transmitter-binding site and the allosteric site of muscarinic acetylcholine receptors. An optical biosensor technique was applied to measure activation-induced dynamic mass redistribution (DMR) in CHO cells stably expressing the muscarinic receptor subtype of interest. The use of tools to modulate signaling and measuring G protein activation directly proved that DMR is a valid and comfortable approach to gain real-time insight into intracellular signaling pathway activation and to identify signaling pathway-selective drugs.