Functional evidence for biased inhibition of G protein signaling by YM-254890 in human coronary artery endothelial cells

Abstract

G protein-coupled receptor (GPCR) has been the primary therapeutic targets for many diseases. Agonists and antagonists of various GPCR are estimated to occupy approximately 35% of the drug market (1). Extracellular signals perceived by GPCR are transmitted via G proteins and trigger intracellular signaling cascades resulting in a plethora of physiological responses. G proteins usually are categorized into four main classical subfamilies according to their α subunits: Gαq/11, Gαs, Gαi/o, Gα12/13 (2). Main subunits Gαq/11, Gαs, Gαi/o and G12/13 are commonly thought to be coupled with phospholipase C (PLC), adenylyl cyclase activation, adenylyl cyclase inactivation, and other small GTPase families, respectively (3). Gαq/11 activates phospholipase Cβ pathway leading to intracellular Ca 2+ mobilization. Gαs and Gαi/o proteins regulate adenylyl cyclase activation and inhibition, respectively, which control intracellular cAMP level. Thus, G proteins are important signal transducing molecules for various cellular responses. Malfunction of GPCR signaling pathways are involved in many diseases, such as diabetes, cardiovascular diseases, and certain forms of cancers (4).

An experimental strategy to probe Gq contribution to signal transduction in living cells

“…In line with the findings from Peng et al . , who reported YM depression of cAMP production by several G s PCR in human coronary artery endothelial cells ( 65 ), FR and YM diminished cAMP elevation by the Gs-sensitive V2 vasopressin receptor in HEK293 cells ( Fig. 5 B ).…”

“…We and others ( 64 , 124 ) recently reported that Gi-Ca 2+ is also fully blocked when cells are pretreated with FR or YM. FR sensitivity of Gi-βγ-Ca 2+ but not of Gi-mediated cAMP lowering has led some authors to hypothesize that FR and YM might inhibit Gβγ signaling as well ( 64 , 65 ). However, this finding may be subject to alternative interpretation (FR inhibits Gβγ versus Gq is required for Gi-Gβγ-calcium), which is why we previously investigated the molecular mechanism whereby this inhibition occurred ( 124 ).…”

“…Although there is scientific consensus on the Gq inhibition, the absence of potential off-target effects for FR and YM remains to be fully demonstrated. This is all the more important because recent studies surmised FR to also inhibit non-Gq events such as cAMP elevation induced by Gs-coupled GPCRs ( 65 ) or Gβγ signaling originating from GPCR-activated Gαi/o-Gβγ heterotrimers ( 64 ). Conjectures like these indeed question which of the reported effects of FR are attributable to Gq inhibition and which arise from nonspecific interaction with other Gα proteins or, potentially, completely distinct cellular targets.…”

“…Despite prior in-depth characterization of FR and YM ( 22 , 24 , 25 , 26 , 27 , 28 , 60 , 61 , 62 , 63 ), two recent investigations have cast doubt on the selectivity profiles of both inhibitors. Although one study claimed FR to also inhibit Gβγ-mediated Ca 2+ signaling downstream of Gi-coupled GPCRs ( 64 ), a recent report suggested YM to act as a broad-spectrum inhibitor for Gq and Gs proteins, and, additionally, as biased Gi inhibitor ( 65 ). Thus, an important emerging research question is, whether FR and YM exert such nonselective effects by directly inhibiting G proteins other than Gq, G11, and G14 or by inhibiting only its canonical targets in signaling networks that cooperate with other G protein classes to control a given effector system.…”

The bitter end: T2R bitter receptor agonists elevate nuclear calcium and induce apoptosis in non-ciliated airway epithelial cells

“…Thus, Ca 2+ nuc is not due to osmolarity or pH. To confirm GPCR involvement, broad-range G protein inhibitor YM-254890 [27] or phospholipase C (PLC) inhibitor U73122 blocked both Ca 2+ i and Ca 2+ nuc ( Fig. 1G-J ).…”