Real-time imaging of alpha2 adrenergic receptor subtypes in living cells using green fluorescent protein

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Background and Goals: Despite the increasing knowledge of beta2 adrenergic receptor, the study of agonist-induced intracellular trafficking of alpha2 adrenoceptor has been limited to immunocytochemical analysis. Thus, we examine the real time subtype-specific trafficking of alpha2 adrenoceptors in living cells using a respective fusion protein of green fluorescent protein (GFP) and human alpha2 adrenoceptor subtypes.Material and Methods: Using recombinant DNA techniques, the C terminus of each alpha2A, B, and C adrenoceptor subtype was fused to the N terminus of GFP (alpha2 AR-GFP). The DNA sequence of each alpha2 AR-GFP was confirmed by dideoxy sequencing both strands. For the radiolabeled ligand binding assay, the recombinant constructs cloned into Pcdna3 vector were transfected transiently in COS-1 cells using DEAE dextram method. Satruration binding isotherms were performed by incubating membranes with varying cocentrations of 3 [H]-RX-821002, and nonspecific binding was determined … More by adding 10 Um rauwolscine. Equilibrium dissociation constants were determined from saturation isothems using a nonlinear least-square surve-fitting technique (GraphPDA Software Inc., San Diego, CA). Protein content for the membrane preparations were assayed according to Lowry et al. The recombinant constructs were transfected transiently in HEK293 cells using liposome method for the living cell imaging. We examined dynamic manners of trafficking of alpha2 AR-GFP in response to dexmedetomidne using cooled CCD camera attached to an epifluorescent microscope. The distributon into endosomal compartments were also observed using three-dimensional analysis of time-lapse imagesResults and Discussions: : At steady state, alpha2A and 2B AR subtypes are localized in the plasma membrane, though a significant portion of alpha2C AR subtype is localized in an intracellular pool. We observed that alpha2B AR subtype underwent agonist-induced internalization and seemed to follow the same endosomal pathway within 15 min used by beta2 AR. Agonist treatment of alpha2A and 2C AR subtypes results in some receptor internalization Less