SUMMARY
Evidence that dynamin is associated with the sequestration of the Paramecium β2-adrenergic receptor (βAR)immunoanalogue is presented. We previously reported a dramatic change in the distribution of βAR analogue in the subcellular fractions upon isoproterenol treatment: it is redistributed from the membraneous to the cytosolic fraction, as revealed by quantitative image analysis of western blots. Here we confirm and extend this observation by laser scanning confocal and immunogold electron microscopy. In the presence of isoproterenol (10μmol l–1) βAR translocated from the cell surface into dynamin-positive vesicles in the cytoplasmic compartment, as observed by dual fluorochrome immunolabeling in a series of the confocal optical sections. Colocalization of βAR and dynamin in the tiny endocytic vesicles was detected by further electron microscopic studies.
Generally receptor sequestration follows its desensitization, which is initiated by receptor phosphorylation by G-protein-coupled receptor kinase. We cloned and sequenced the gene fragment of 407 nucleotides homologous to theβ-adrenergic receptor kinase (βARK): its deduced amino acid sequence shows 51.6% homology in 126 amino acids that overlap with the human βARK2(GRK3), and may participate in Paramecium βAR desensitization.
These results suggest that the molecular machinery for the desensitization/sequestration of the receptor immunorelated to vertebrateβAR exists in unicellular Paramecium.
