Dynamin-association with agonist-mediated sequestration of beta-adrenergic receptor in single-cell eukaryote Paramecium.
Evidence that dynamin is associated with the sequestration of the Paramecium beta(2)-adrenergic receptor (betaAR) immunoanalogue is presented. We previously reported a dramatic change in the distribution of betaAR 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 (10 micro mol l(-1)) betaAR 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 betaAR 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 the beta-adrenergic receptor kinase (betaARK): its deduced amino acid sequence shows 51.6% homology in 126 amino acids that overlap with the human betaARK2 (GRK3), and may participate in Paramecium betaAR desensitization. These results suggest that the molecular machinery for the desensitization/sequestration of the receptor immunorelated to vertebrate betaAR exists in unicellular PARAMECIUM:
Wiejak
Jolanta
J
Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland.
Surmacz
Liliana
L
Wyroba
Elzbieta
E
D000318:Adrenergic beta-Agonists / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D034281:Dynamins / Q000378:metabolism*; D005456:Fluorescent Dyes; D007150:Immunohistochemistry; D007545:Isoproterenol / Q000378:metabolism*; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010247:Paramecium / Q000378:metabolism* / Q000648:ultrastructure; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D022161:Transport Vesicles / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases
D000318:Adrenergic beta-Agonists; D017931:DNA Primers; D005456:Fluorescent Dyes; D011943:Receptors, Adrenergic, beta; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D034281:Dynamins; D007545:Isoproterenol