title: Role of the proline-rich domain of dynamin-2 and its interactions with Src homology 3 domains during endocytosis of the AT1 angiotensin receptor.
abstract: In nonneural tissues, the dynamin-2 isoform participates in the formation of clathrin-coated vesicles during receptor endocytosis. In this study, the mechanism of dynamin-2 action was explored during endocytosis of the G protein-coupled AT1A angiotensin receptor expressed in Chinese hamster ovary cells. Dynamin-2 molecules with mutant pleckstrin homology domains or deleted proline-rich domains (PRD) exerted dominant negative inhibition on the endocytosis of radiolabeled angiotensin II. However, only the PRD mutation interfered with the localization of the dynamin-2 molecule to clathrin-coated pits and reduced the inhibitory effect of the GTPase-deficient K44A mutant dynamin-2. Green fluorescent protein-tagged Src homology 3 (SH3) domains of endophilin I and amphiphysin II, two major binding partners of dynamins, also inhibited AT1A receptor-mediated endocytosis of angiotensin II. These effects were partially or fully, respectively, restored by the overexpression of dynamin-2. Transient overexpression of these SH3 domains also reduced the localization of dynamin-2 to clathrin-coated pits. These data indicate that, similar to the recruitment of dynamin-1 during the recycling of synaptic vesicles, interaction of the dynamin-2 PRD with SH3 domains of proteins such as the amphiphysins and endophilins is essential for AT1A receptor endocytosis. This mechanism could be of general importance in dynamin-dependent endocytosis of other G protein-coupled receptors in nonneural tissues.
authors.affiliation: Department of Physiology, Semmelweis University, Faculty of Medicine, H-1088 Budapest, Hungary.
authors.forename: Márta
authors.initials: M
authors.lastname: Szaszák
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Gáborik
authors.forename: Gábor
authors.initials: G
authors.lastname: Turu
authors.forename: Peter S
authors.initials: PS
authors.lastname: McPherson
authors.forename: Adrian J L
authors.initials: AJ
authors.lastname: Clark
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Catt
authors.forename: László
authors.initials: L
authors.lastname: Hunyady
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D034284:Dynamin I; D034281:Dynamins; D004705:Endocytosis; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000502:physiology*; D017353:Gene Deletion; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011392:Proline / Q000737:chemistry*; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D051381:Rats; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D018909:src Homology Domains
chemical_list: D018076:DNA, Complementary; D008164:Luminescent Proteins; D020033:Protein Isoforms; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D049452:Green Fluorescent Proteins; D011392:Proline; D034284:Dynamin I; D020558:GTP Phosphohydrolases; D034281:Dynamins