Specific Interaction Between Insulin Receptor And GLP-1 Receptor And Its Effect On Signal Transductio

G-protein coupled receptor(GPCR)and receptor tyrosine kinase(RTK)are involved in many physiological and pathological processes.And more and more studies have found that the homomerization and heteromerization of GPCRs and RTKs play an important role in the process of ligand binding and signal transduction.The glucagon-like peptide-1 receptor(GLP-1R)of the GPCR family and the insulin receptor(IR)of the RTK family can transmit signals of insulin release and cell metabolism through different signaling molecules and transduction pathways.In addition,GLP-1R and IR are also co-expressed in many different tissues such as pancreatic β cells,and the signal crosstalk between the two receptors is often reported.Therefore,we hypothesized a possible association of IR and GLP-1R.First of all,the regular fluorescence resonance energy transfer(FRET)was tried,and the interaction between GLP-1R and IR was not confirmed due to the low sensitivity of the detection,but it was confirmed that the two overexpressed receptors were mainly expressed on the cell membrane.Subsequently,the interaction between GLP-1R and IR was found using different constructs for NanoBiT(NanoLuc Binary Technology)in HEK 293ET.The specificity of this interaction was verified through saturation experiments and competition experiments.Similarly,we also detected the interaction between GLP-1R and insulin-like growth factor 1 receptor(IGF-1R),and between glucagon receptor(GCGR)and IR.Using IRδ which lacks almost all the intracellular sequence of δ-chain and mGLP-1R containing tri-mutations at TM4,the region of interaction between GLP-1R and IR was preliminarily determined by detecting whether there was still interaction between different receptor mutants.These results suggest that GLP-1R associates to IR through their transmembrane domains.And the GLP-1R/IR complex is neither affected by functional dimerized form of GLP-1R,nor is it disturbed by receptor agonists or antagonists.In order to fully study the role of the GLP-1R/IR complex in the signaling pathway under ligand stimulation,this study mainly explored the signaling proteins recruitment and signal transduction of IR and GLP-1R.This impaired coupling of IRS-1 to IR under GLP1R/IR complexation was concomitant with its increased coupling to GLP-1R,implying that IRS-1 coupling was dynamically drifted between IR and GLP-1R when they were activated.The GLP-1R/IR complexation diminishes the IRS-1 recruitment to IR without reducing its activation while alleviates the exaggerated degradation of IRS-1 associated to IR.In addition,the GLP-1R/IR complex also facilitates the coupling of GLP-1R to SHC1.Using a process similar to that of IRS-1,the coupling of Gsa to IR was not detected in the GLP-1R/IR complex,but its coupling to IRδ was observed.IR showed a similar inhibition as IRδ in the coupling of Gsa to activated GLP-1R.Further study of cAMP downstream of Gsα in GLP-1R/IR complex showed that GLP-1 induced cAMP generation was inhibited when IR was activated.It was found that GLP-1R>IR complex also inhibited the coupling of Gqα to activated GLP-1 R,similar to Gsα.While GLP-1 R/IR complex did not inhibit the coupling of β-arrestin-2 to GLP-1 R.The differential recruitment of Gsα,Gqα,and β-arrestin-2 which was modulated by the GLP-1 R/IR complex reveal a biased signal transmission of GLP-1 R in the receptor complex.In conclusion,our study found that the specific interaction between GLP-1R and IR which was dependent of transmembrane receptor domains and not responsive to ligand binding.In signaling,the GLP-1 R/IR complex inhibits the coupling of IRS-1 to IR at IR activation,with subsequent IRS-1 degradation suppressed rather than its activation inhibited.In addition,the Gsa recruitment to the activated GLP-1 R was inhibited in the GLP-1 R/IR complex.Its subsequent signaling in cAMP pathway was suppressed by the IR activation.Therefore,the identified GLP-1 R/IR complex recruits their signaling molecules to both receptors,where they are differentially modified and lead to a crosstalk between their signaling.The research of the specific interaction between GLP-1 R and IR and the downstream signal transduction in the complex laid a theoretical foundation for the study of new drug targets.