Abstract
The recognition that individual GPCRs can activate multiple signaling pathways has raised the possibility of developing drugs selectively targeting therapeutically relevant ones. This requires tools to determine which G proteins and βarrestins are activated by a given receptor. Here, we present a set of BRET sensors monitoring the activation of the 12 G protein subtypes based on the translocation of their effectors to the plasma membrane (EMTA). Unlike most of the existing detection systems, EMTA does not require modification of receptors or G proteins (except for Gs). EMTA was found to be suitable for the detection of constitutive activity, inverse agonism, biased signaling and polypharmacology. Profiling of 100 therapeutically relevant human GPCRs resulted in 1,500 pathway-specific concentration-response curves and revealed a great diversity of coupling profiles ranging from exquisite selectivity to broad promiscuity. Overall, this work describes unique resources for studying the complexities underlying GPCR signaling and pharmacology.
Competing Interest Statement
AM, BB, CN, FG and SM were employees of Domain Therapeutics North America during part or all of this research. EF and J-FF are employees and shareholders of Pfizer. SS and XL are employees and are part of the management of Domain Therapeutics. MB is the president of Domain Therapeutics scientific advisory board. BB, CLG, HK, MH, VL, MB have filed patent applications related to the biosensors used in this work and the technology has been licensed to Domain Therapeutics. CA, ASH, SS-O, MC, MH and DEG have no competing interests to declare.
Footnotes
↵† These authors contributed equally.
‡ These authors contributed equally.
§ Institute for Research in Immunology and Cancer (IRIC), Université de Montréal; Montréal, Québec, H3T 1J4, Canada.
The recognition that individual GPCR can engage multiple signaling pathways has raised the possibility of developing drugs selectively targeting therapeutically relevant ones. This requires tools to determine which G proteins and βarrestins are engaged by a given receptor. Here, we present a set of BRET sensors monitoring the activation of the 12 G protein subtypes based on the translocation of their effectors to the plasma membrane (EMTA). Unlike most of the existing detection systems, EMTA does not require modification of receptors or G proteins (except for Gs). EMTA was found to be suitable for the detection of constitutive activity, inverse agonism, biased signaling and polypharmacology. Profiling of 100 therapeutically relevant human GPCRs resulted in 1,500 pathway-specific concentration-response curves and revealed a great diversity of coupling profiles ranging from exquisite selectivity to broad promiscuity. Overall, this work describes unique resources for studying the complexities underlying GPCR signaling and pharmacology.