Detection Of The Expression And Activation Of The Endogenous MGlu Receptors By Nanobody-based TR-FRET Sensors

The eight metabotropic glutamate receptors(mGlu Rs or mGlu receptors)belong to the G protein-coupled receptor(GPCR)family in mammals.They are activated by the main excitatory neurotransmitter glutamate(Glu)and involved in tuning synaptic transmission and neuronal excitability.The mGlu receptors are considered promising therapeutic targets for several neurological and psychiatric disorders,including schizophrenia and Parkinson’s disease.Most previous studies assumed that mGlu receptors functioned as homodimers.Although studies have revealed the existence of 16 mGlu heterodimers in vitro,there is no direct proof that they exist in vivo.Furthermore,mGlu homo-and heterodimers distribution in the brain remains elusive.On the other hand,there is no convenient method to detect the activation of endogenous mGlu receptors.This study used the fluorescent-labeled nanobodies combined with time-resolved fluorescence resonance energy(TR-FRET)technology to detect the interaction between mGlu subunits.Based on such a strategy,two kinds of nanobody-based TR-FRET sensors were developed:(i)the conformational change sensors that reveal the activation of receptors upon agonist binding and the detectors that enable the detection of mGlu receptors expressions.Firstly,the properties of nanobodies(DN1,DN10,and FcDN42)targeting mGlu2 and mGlu4 receptors were characterized by the immunofluorescence experiment and the binding assays to determine their specificity and affinity.And the activation of the mGlu2 receptors in the over-expression system or brain tissues was determined in a highthroughput manner by the DN1/DN10 conformational change sensor.And a pharmacological analysis was completed.These data confirmed the effectiveness of the method.Then,the mGlu2-4 heterodimers were observed in the olfactory bulb,prefrontal cortex,striatum,and hippocampus of adult mice by the mGlu2-4 heterodimer detector(FcDN42/DN1),with the highest abundance in the olfactory bulb.Furthermore,the pharmacological fingerprints of mGlu2-4 heterodimers were detected by the mGlu2-4heterodimer conformational change sensor(FcDN42/DN10),indicating that the heterodimer signal in the olfactory bulb comes from the endogenous mGlu2-4 heterodimers,rather than the mGlu2 and mGlu4 homodimers in proximity.These data directly demonstrated the existence of the endogenous mGlu2-4 heterodimers.Finally,the expressions of the mGlu2 homodimers and mGlu4 homodimers in six brain regions were determined by corresponding detectors(DN1/DN1 or FcDN42/FcDN42),respectively,including the olfactory bulb,prefrontal cortex,striatum,hippocampus,midbrain,and cerebellum.By comparing the expression of mGlu2-4 heterodimers and mGlu4 homodimers,found that there were more mGlu2-4 heterodimers than mGlu4 homodimers in most regions outside the cerebellum.These data indicated that mGlu4 mainly existed as heterodimer in these brain regions.In summary,the present study directly demonstrated the existence of the endogenous mGlu2-4 heterodimers and revealed the differential distribution of the mGlu2 homodimers,mGlu4 homodimers,as well as mGlu2-4 heterodimers in the mice brain.These findings provided new clues for addressing the physiological roles of mGlu receptors and developing drugs for mGlu homo-or heterodimers.Furthermore,the data also show that nanobodies are promising TR-FRET probes and that nanobody-based sensors allow for the relative quantification and functional analysis of endogenous membrane proteins from native tissues.