Study On The Interaction Between Adenosine Receptor Proteins Based On FRET Technology

Adenosine receptors are a family of membrane proteins with diverse physiological and pathological functions.These receptors have the tendency to form dimers,and dysfunction of these dimers is related to multiple diseases.F（?）rster resonance energy transfer（FRET）is a process by which the excitation of a fluorescent molecule is transferred nonradiatively.The efficiency of energy transfer is related to the distance between donor and receptor,so FRET technology is widely used to detect protein interactions in living cells.Because of the complexity of cell membrane morphology and weak fluorescence signal,FRET quantitative evaluation of membrane protein interactions remains a challenge.In this paper,we improved current FRET technique and used it to study the dynamic interaction of two adenosine receptor subtypes(A₁R and A_2AR).The main research contents include:First,a norvel system constituted with FRET microscopy imaging and spectral detection system was built,which was characterized by the separation of excitation filter and emission filter,expanding the selection range of FRET fluorescent probe.The range of the excitation light was adjusted by the aperture of the system to achieve selectivity detection of the fluorescence characteristics of single cell.Besides,spectrometer and CCD were installed in the system to simultaneously obtain fluorescence spectrum and fluorescence image of single cell.Second,independent emission spectrum separation method（Iem-sp FRET）and acceptor-sensitized 3-cube FRET imaging（E-FRET）methods were combined to measure the high spatio-temporal resolution interactions between A₁R and A_2AR.E-FRET obtained the FRET efficiency value of region of interest（ROI）with the advantage of high spatial resolution.However,due to anthropogenic disturbance,the time for E-FRET to obtain the maximum FRET efficiency value induced by CCPA was 106s later than that obtained by Iem-sp FRET.Because Iem-sp FRET obtained the FRET efficiency value of a single cell as a whole,the deviation of E-FRET results was corrected by the results obtained from Iem-sp FRET.The corrected results were consistent with the results obtained from Iem-sp FRET,indicating that the time resolution of E-FRET in membrane protein research was improved.In addition,the interaction between A₁R and A_2AR was monitored,and conclusions were obtained from the consistency of spectral and imaging analysis.Activation of A₁R or A_2AR led to rapid increases of FRET efficiency value（～12s）,inhibition of A₁R or A_2AR led to slow decreases of FRET efficiency value（～30min）,realizing high spatio-temporal resolution measurement of A₁R and A_2AR interactions.Third,a batch processing program for FRET image of membrane proteins was developed.At present,FRET image processing had some problems,such as time consuming and strong subjectivity.In order to solve the above problems,we proposed a method for batch processing of FRET image,and compared the difference between the method,cell-by-cell processing and spectral method.The results showed that this method could shorten the time of data processing compared with cell-by-cell processing.Change curves of FRET efficiency with time obtained by batch processing were highly coincident with that obtained by Iem-sp FRET,indicating that batch processing improved the accuracy of E-FRET.The combination of FRET image and batch processing was a simple,time-saving and accurate method for studying the interaction of single cell membrane proteins,which had great potential in the field of cell-based FRET efficiency analysis.Fourth,the interaction between A₁R and A_2AR in the process of liver fibrosis was studied by using the method of FRET image and batch processing.Change curves of FRET efficiency value with time were obtained by analyzing 200 cells,which took about100min.The interaction between A₁R and A_2AR in LX-2 cells was slowly attenuated under acetaldehyde stimulation（*P<0.05）,which took about 30min;the weakening effect was even more obvious as the time of acetaldehyde stimulation increases（***P<0.001）.Moreover,LX-2 cells were activated by acetaldehyde.LX-2 cells activation and interactions between A₁R and A_2AR changed observably in time-dependent manner,and the degree of activation and the interaction changed inversely.The method of combining FRET image and batch processing could be applied to the analysis of real samples,and was expected to be applied to the study of more kinds of membrane receptor interactions.This paper was not only of great theoretical significance and scientific value,but also was expected to expand its applications to the dynamic detection and analysis of other membrane receptor protein interactions.In particular,combining FRET imaging with batch processing simplified the data processing process and was promising for the large-scale analysis of protein interactions in living cells.