The Function Of Seven Transmembrane Domains In CaSR

The calcium-sensing receptor?CaSR?is one member of Class C G Protein Coupled Receptors?GPCRs?.CaSR is widely distributed in the human body and participates in many physiological and pathological processes.It mainly regulates the Ca²⁺homeostasis by monitoring the concentration of extracellular Ca²⁺and a variety of stimulations.CaSR is a homodimer,and each monomer includes an N-terminal extracellular domain?ECD?,seven transmembrane domains?7TM?and an intracellular C-terminus?C-tail?.The ECD of CaSR is the major region for various ligands binding,among which Ca²⁺is the main CaSR agonist.The CaSR binding sites of allosteric molecules are located in 7TM.Previous studies have shown that the 7TM of CaSR may have binding sites for natural ligands.This phenomenon that multiple natural ligands bind in the 7TM is rarely seen in Class C GPCRs.For Class A GPCRs,since they have no ECD,their ligand binding site is located at 7TM.It can be seen that CaSR mutants lacking the ECD exhibited similar Class A GPCRs characteristics.According to statistics,wild-type GPCRs with constitutive activity are mainly concentrated in Class A GPCRs.It has been shown that the constitutively active Class C receptor mGluR5a can still constitutively activate G proteins in the absence of ECD.It revealed that the structural basis of constitutive activity is the7TM.In addition,a large number of constitutively active mutations found in GPCRs are also mainly concentrated in the 7TM.In addition,These promote that CaSR mutants lacking the ECD may have constitutive activity.It is known that most Class C GPCRs including wild-type CaSR have no constitutive activity,and there has been no report about the constitutive activity of mutants lacking the ECD in most Class C GPCRs.Whether the 7TM of CaSR has constitutive activity and its role in CaSR sensing extracellular signals and regulating downstream signaling needs to be further elucidated.We first constructed?CaSR and?CaSR?truncation mutants with mGluR5 signal peptide which most part of ECD is deleted.It was confirmed that the two truncated mutants normally express and locate in HEK293 cells in a similar manner as wild-type CaSR by ELISA and immunofluorescence methods.When the mutants were stimulated alone with Ca²⁺,Mg²⁺or spermine without PAM by analyzing the intracellular calcium signal and IP1 production,we found that both mutants responded to extracellular Ca²⁺stimulation,?CaSR?had a certain response to spermine.The results strongly confirm the presence of Ca²⁺binding site in the 7TM of CaSR.In order to explore whether the two truncated mutants have constitutive activity,the accumulation of IP1 and the activation of basal ERK1/2 were detected.It was found that both WT and mutants could not constitutively promote IP1 accumulation and activate G_q signal.However,compared with WT,both mutants could significantly promote basal ERK1/2 activation,and the activation could be regulated by allosteric modulator.It has been reported that CaSR promotes ERK1/2 activation through multiple G protein-dependent or independent signaling pathways.To investigate the signaling mechanism of the constitutive activation of ERK1/2 by mutants,we introduced two mutations F801A and L797A in?CaSR?which abolished the coupling of receptors with G_q and G_i/o.Both mutations have no effect on the constitutive activation of ERK1/2 by?CaSR?.This suggested that basal ERK1/2 may be caused by CaSR 7TM in a basied signaling pathway independent of G_q and G_i/o.In summary,this study found that CaSR mutants without ECD can be activated by extracellular Ca²⁺alone independently of PAM.This confirmed the presence of Ca²⁺binding site in the 7TM of CaSR.This is the first time to demonstrate that CaSR mutants lacking the ECD can constitutively activate ERK1/2,which may be mediated through G_q and G_i/o protein-independent signaling pathways.The study provides a solid basis for exploring the role of 7TM in CaSR mediated signaling transduction,and is able to deepen the current understanding of CaSR structure and activation mechanism.At the same time,it provides new ideas for the study of the structure and function of Class C non-constitutive active GPCRs.