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.

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...

In general, the M 2 subtype of muscarinic acetylcholine receptors has the highest sensitivity for allosteric modulators and the M 5 subtype the lowest. The M 2 /M 5 selectivity of some structurally diverse allosteric agents is known to be completely explained by M 2 177 Tyr and M 2 423 Thr in receptors whose orthosteric site is occupied by the conventional ligand N-methylscopolamine (NMS Trp by alanine revealed a pronounced contribution of these epitopes to subtype independent baseline affinity in NMSbound and NMS-free receptors for all agents except diallylcaracurine V. In a few instances, this tryptophan also influenced cooperativity and subtype selectivity. Docking simulations using a three-dimensional M 2 receptor model revealed that the aromatic rings of M 2 177 Tyr and M 2 422 Trp, in a concerted action, might fix one of the aromatic moieties of alkane-bisammonio compounds between them. Thus, M 2 422 Trp and the spatially adjacent M 2 177 Tyr, as well as M 2 423 Thr, form a cluster of amino acids within the allosteric binding cleft that is pivotal for both M 2 /M 5 subtype selectivity and baseline affinity of allosteric agents.All five subtypes of muscarinic acetylcholine receptors contain an allosteric site apart from the orthosteric site that is addressed by acetylcholine and conventional muscarinic agonists and antagonists. Binding of an allosteric modulator allows formation of ternary complexes consisting of the allosteric agent, the orthosteric ligand, and the receptor protein. Through ternary complex formation, allosteric agents may evoke particular actions that cannot be induced by orthosteric ligands alone and that may have therapeutic potential. For instance, allosteric modulators may increase the binding of orthosteric agonists or antagonists (positive cooperativity) or they may inhibit orthosteric ligand binding (negative cooperativity). In either case, the magnitude of the cooperativity will define an intrinsic limit on the magnitude of the positive or negative effect, in marked contrast to the unconstrained action of orthosteric agonists and antagonists. It is also possible for allosteric modulators to leave orthosteric ligand binding unchanged (neutral cooperativity) while nevertheless changing the kinetics of binding (Ellis, 1997;Christopoulos and Kenakin, 2002;Krejčí et al., 2004;Soudijn et al., 2004;Birdsall and Lazareno, 2005;Wess, 2005). Finally, in addition to modulating orthosteric ligand binding properties, allosteric agents also may modulate agonist induced intrinsic efficacy (Zahn et al., 2002). A better understanding of the molecular topology and mechanisms of allosteric