Structural Insights Into ?2 Adrenergic Receptor Desensitization Upon Binding Of Partial Agonist Or Full Agonist

G protein coupled receptors(GPCRs)regulate a wide variety of physiological functions in response to extracellular stimuli.Repeated or continuous agonist binding leads to heterotrimeric G-protein mediated cAMP signaling that quickly returns to basal levels,which is a remarkably conserved process of GPCR desensitization.This response involves receptor phosphorylation,uncoupling of GPCRs and G proteins,?-arrestin binding to the receptor,receptor endocytosis.Previous studies reported that partial agonists cause less desensitization of GPCR than full agonists.Notably,desensitization of GPCR plays crucial role in modulating receptor activation,drugs designing targeting GPCR.However,structural insights of GPCR desensitization induced by partial or full agonists remained unclear.The ?2-adrenergic receptor(?2AR)is an excellent canonical class A GPCR.A wealth of studies have investigated that the reduced desensitization observed with partial agonists results from a decrease of phosphorylation of third intracellular loop(ICL3)and carboxy-terminal tails(C-tail)of ?2AR.The partial agonist salbutamol,recently was observed to cause slower initial rates of phosphorylation of the ?2AR than the full agonists(e.g.isoprenaline and epinephrine),and significantly reduced desensitization,indicating that partial and full agonists might induce distinct activation conformation of GPCR.In the first part,we affinity purified the ?2-adrenergic receptor and the G protein heterotrimer,and assemble into a complex with the addition of a partial or full agonist.We determined the three-dimensional structures of ?2AR-G?s?? complex bound with partial agonist salbutamol or full agonist isoprenaline,using single-particle cryo-EM.This work is the first to provid structural insights into agonist dependent conformational differences of a GPCR.The two near-atomic structures were determined at 3.26 A and 3.88 A resolution,respectively.The absence of hydrogen bond network and attenuated hydrophobic interactions between salbutamol saligenin group and ?2AR were observed in the salbutamol bound ?2AR,compared with isoprenaline bound ?2AR.This difference may result in the weaker affinity and desensitization of salbutamol.There are two molecular switches termed rotamer toggle switch and ionic lock which attributed to movement of TM6.Compared with full agonist isoprenaline,the partial agonist salbutamol does not trigger the rotamer toggle switch in TM6 but only disrupts the ionic lock between TM3 and TM6,possibly leading to lower efficacy and consequent less desensitization.In the binding interface of the ?2AR-Gs protein,more tight interactions between the second intracellular loop(ICL2),the third intracellular loop(ICL3)and the ?2AR for the sample of the partial agonist salbutamol-bound ?2AR-Gs complex.The induced tight interaction could lead to less accessibility of PKA,attenuated phosphorylation in intracellular loops,and consequent weaker arrestin binding and less desensitization.These findings will definitely enrich our understanding the structural insights of ?2AR desensitization with binding of partial or full agonist,which is very important for pharmacokinetics analysis and GPCR targeting drug developments.In the second part,we used cryo-electron microscopy to analyze the three-dimensional structure of the plant potassium channel KAT1.Plants utilize K+ions to maintain hydrostatic pressure,drive irreversible cell expansion for growth,and facilitate reversible changes in guard cell volume that cause stomatal opening or closure.K+ion uptake and circulation throughout plants rely on potassium channels to facilitate K+ion transfer across the cell membrane.KAT1 is an inwardly rectifying potassium channel from Arabidopsis that mediates K+influx.And it mainly expressed in guard cells and plays a key role in regulating the aperture of stomatal pores on the surface of plant leaves.KAT1 exhibits a reversed voltage dependence,meaning it closes with depolarization and opens with hyperpolarization.The structural mechanism underlying the voltage sensor control of the gate in this class of hyperpolarization-gated ion channels is less studied.We cloned KAT1 and the accessory subunit KAB1 into pFastBac dual vector for expression.After affinity purification and electron microscopy data collection and data processing,we finally obtained the cryo-EM structure of KAT1 with a resolution of 3.2A.However,the auxiliary subunit KAB1 dissociates during the freezing sample preparation.The structure indicates that KAT1 is a homotetrameric potassium channel and contains a short non-helical S4-S5 linker that contributes to a non-domain-swapped topology in each subunit.The voltage-sensitive S4 helix is considered to contribute to the hyperpolarization gating of the channel.This structure greatly advances our knowledge of the gating properties of hyperpolarization-activated ion channels and serves as a guide for structure/function analyses of plant potassium channels.