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First published online April 8, 2004
Journal of Experimental Biology 207, 1625-1632 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00914

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Dynamin-association with agonist-mediated sequestration of beta-adrenergic receptor in single-cell eukaryote Paramecium

Jolanta Wiejak, Liliana Surmacz and Elzbieta Wyroba^*

Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland

^* Author for correspondence (e-mail: e.wyroba{at}nencki.gov.pl)

Accepted 2 February 2004

Evidence that dynamin is associated with the sequestration of the Paramecium ß₂-adrenergic receptor (ßAR) immunoanalogue is presented. We previously reported a dramatic change in the distribution of ßAR 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 µmol l^–1) ßAR 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 ßAR 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 ß-adrenergic receptor kinase (ßARK): its deduced amino acid sequence shows 51.6% homology in 126 amino acids that overlap with the human ßARK2 (GRK3), and may participate in Paramecium ßAR desensitization.

These results suggest that the molecular machinery for the desensitization/sequestration of the receptor immunorelated to vertebrate ßAR exists in unicellular Paramecium.

Key words: dynamin, sequestration, desensitization, Paramecium, ß₂-adrenergic receptor, GRK, isoproterenol, cloning, confocal imaging, immunological analysis

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