Development And Application Of A G Protein Biosensor In Class C G Protein-Coupled Receptors

G protein-coupled receptors(GPCRs)are one of the largest membrane protein families and are important drug targets.The diverse physiological effects caused by GPCRs are mainly mediated by heterotrimeric G proteins.After binding with extracellular ligand,changes of the conformation of GPCR promote the exchange between GDP and GTP in G?subunit,which is accompanied with changes in the interaction between G?and G??,triggering the intracellular signal transduction.The G?subunit can be divided into four subtypes:G?_i/o,G?_s,G?_q/11 and G?_12/13,each one of which has distinct properties.Therefore,unraveling the activation mechanism of G proteins is an important and direct means to understand the various downstream signaling and intracellular responses mediated by GPCRs.Class C GPCRs mainly include metabotropic glutamate receptors(m Glu Rs),?-aminobutyric acid type B receptors(GABA_BRs),calcium-sensing receptor and taste receptors1.m Glu Rs and GABA_BRs are the main excitatory and inhibitory neurotransmitter receptors in the central nervous system that are closely related to many neurological diseases.Although many studies have found that m Glu Rs and GABA_BRs could activate some G protein subtypes,it is still remained unknown whether other G protein subtypes could also be activated.Meanwhile,the activation of G proteins by these endogenous GPCRs in neurons still remains unclear.Based on the interaction changes after the activation of the G protein heterotrimer,a group of biosensors were developed using bioluminescence resonance energy transfer(BRET)technology to monitor the activation of different G protein subtypes directly in live cells.By detecting the activation of specific G proteins,the biosensors were demonstrated that they could be used to detect the activation of the eight main G protein subtypes:G_i1,G_i2,G_i3,G_{o A},G_{o B},G_q,G_s and G₁₃.Furthermore,the insertion position of the BRET donor on G?proteins and the pairing between G?and G??subunits were optimized to enhance the activation amplitude of the G_q,G_s and G₁₃ biosensors.The activation characteristics of different G protein subtypes by all m Glu Rs(m Glu1 to m Glu8)and GABA_BRs were analyzed with the biosensors.The present results demonstrated that m Glu1 and m Glu5could activate G_q protein,while m Glu2/3/4/6/7/8 and GABA_BRs could activate the members of G_i/o protein family,such as G_i1 and G_{o A},which is in line with previous reports.Meanwhile,m Glu1 and m Glu5 were also found to activate G_i/o proteins,while some m Glu Rs and GABA_BRs could activate G₁₃ and G_q proteins,suggesting that m Glu Rs and GABA_BRs probably exert new functions by coupling new G protein subtypes.Finally,the activation of G_i/o protein by endogenous GABA_BRs was detected in primary cultured neurons.Although the expression level of G_i/obiosensors in the neurons is not high,they had a similar amplitude as in the overexpression system,which further confirmed that the biosensors were powerful.The pharmacological characteristics of GABA_BRs treated with agonists,antagonists and allosteric modulators in neurons were also analyzed with the biosensors.In summary,a group of biosensors were developed to detect the activation of G proteins.Subsequently,they were applied to the two main neurotransmitter GPCRs,and the results showed that m Glu Rs and GABA_BRs could activate several new G protein subtypes.It was further confirmed that the biosensors could be used to detect G protein activation by endogenous GPCR in primary neurons.Existing tools for detecting endogenous GPCR activities are still in shortfall.With high sensitivity,the biosensors may be a new tool to detect the activity of GPCRs,especially for endogenous GPCRs.However,the physiological functions of m Glu Rs and GABA_BRs mediated by new G protein subtypes and the universal application of the biosensors to more endogenous GPCRs remain to be further studied and discussed.