Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity.
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.
Pharmacology and Toxicology Section, Institute of Pharmacy, Rheinische Friedrich-Wilhelms-University, Bonn, Germany.
Johannes
J
Antony
Kerstin
K
Kellershohn
Marion
M
Mohr-Andrä
Anna
A
Kebig
Stefanie
S
Prilla
Mathias
M
Muth
Eberhard
E
Heller
Teresa
T
Disingrini
Clelia
C
Dallanoce
Simona
S
Bertoni
Jasmin
J
Schrobang
Christian
C
Tränkle
Evi
E
Kostenis
Arthur
A
Christopoulos
Hans-Dieter
HD
Höltje
Elisabetta
E
Barocelli
Marco
M
De Amici
Ulrike
U
Holzgrabe
Klaus
K
Mohr
D000818:Animals; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000819:agonists / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D019204:GTP-Binding Proteins