Biased Signaling Due To Oligomerization Of The G Protein-Coupled Platelet-Activating Factor Receptor

G protein-coupled receptors(GPCRs)located on the cytoplasmic membrane are a superfamily of seven-transmembrane receptors,including more than 800 receptor subtypes.Clinical drugs developed targeting this type of receptors account for over 1/3 of the market.GPCRs can sense different extracellular stimuli including proteins,hormones,small molecules,neurotransmitters,ions,light,phospholipids,etc.and transmit external signals into cells,thereby triggering different intracellular signaling pathways,activating signaling pathways mediated by heterologous polymeric G proteins and β-arrestins.Different agonists acting on the same receptor can cause the receptor bind to different subsets of effectors,thereby altering cellular physiological effects in different ways,a concept known as "biased signaling".More and more researchers are focusing on developing different biased agonists to activate specific signaling pathways,and then more precisely control drug effects to reduce side effects.However,the mechanism of integration and regulation of biased signaling among the downstream pathways of GPCRs is still unclear.In addition,many studies have reported that dimerization and oligomerization of GPCRs exist in natural tissues and play a critical role in physiological and pathological processes.Studies have shown that most of the GPCRs on the cell membrane are mixed in the form of monomers,dimers and oligomers,and there is a dynamic process of mutual conversion between them.It has been reported that GPCRs can modulate the activation efficacy of downstream signaling pathways by changing the ratio of mono/di/oligomers,but it is unclear whether the di/oligomerization of GPCRs can cause downstream biased signaling pathways.This project used the Platelet Activating Factor Receptor(PAFR)as a model to research the effects of GPCRs dimers and oligomers on various downstream pathways.First,using Total Internal Reflection Microscope(TIRFM)single-molecule imaging photobleaching steps analysis technique to confirm that PAFR exists in the form of a mixture of monomers,dimers and oligomers on the cell membrane.And the ratio of its dimers and oligomers showed a dependence on the amount of receptor expression: with the increase of receptor expression density,the proportion of dimers and oligomers increased while the monomeric forms decreased.Secondly,using Bioluminescence Resonance Energy Transfer(BRET)technology,it was found that specific and high interaction occurs between PAFRs.In addition,the addition of competing receptors will weaken the interaction between them,further confirming that the di/oligomerization of the receptors do exist on the cell membrane.Furthermore,through peptide interference and cysteine cross-linking technology to explore the fine mechanism of receptor dimerization,found transmembrane domains TM1,TM4 and TM5 as the interaction interface of receptor dimerization;and when locked TM1 and TM4/5 at the same time,the receptors were found to exist more in oligomeric form.Next,G proteins and β-arrestins pathway of the receptor was tested,and found that when the receptor was stabilized in their dimer or oligomer state,the balance between the downstream signaling pathways of the receptor changed: PAF can biasedly induce the G protein signaling pathway stronger,however,reduces the recruitment of β-arrestin and the related receptor internalization process.Also,β-arrestin plays a role in regulating the phenomenon that PAFR di/oligomerization causes downstream biased signaling.The phenomenon of biased signal integration by such dimerization and oligomerization has also been demonstrated in the natural genetic variation system of PAFR.This study discovered the relationship between GPCRs di/oligomers and the regulation of downstream signaling pathways,and provided a new perspective on the regulatory mechanism of the integration between various signaling pathways of the receptor.