title: Azaspiracid-1 inhibits endocytosis of plasma membrane proteins in epithelial cells.
abstract: The effect of azaspiracid-1 (AZA-1) on the plasma membrane proteins E-cadherin, Na(+)/K(+)-ATPase, and prolactin receptor (R(prl)) has been investigated in MCF-7 cells. Cell treatment for 24 h with 1nM AZA-1 induced the accumulation of a proteolytic fragment of E-cadherin and significant increases in the levels of Na(+)/K(+)-ATPase and R(prl) at the level of membranous structures. The effect induced by AZA-1 was mimicked by latrunculin A, suggesting that the toxin might act by blocking the endocytosis of plasma membrane proteins. The exposure of intact cells to a biotinylation reagent that does not permeate the plasma membrane provided data showing that AZA-1 treatment of MCF-7 cells doubled the levels of total protein located on the cell surface. The exposure of intact cells to exogenous proteases (trypsin and proteinase K) showed that AZA-1 treatment of MCF-7 cells modifies the availability of the three membrane protein markers to proteolytic attacks, providing evidence that significant portions of the protein pools are located in structures that are not exposed to the cell surface after the treatment with AZA-1. Distinct subcellular locations of the membrane protein markers in MCF-7 cells exposed to AZA-1 were confirmed by immunofluorescence microscopy. Direct evidence that AZA-1 inhibits endocytosis was obtained by showing that AZA-1 blocked the intracellular transfer of E-cadherin-bound antibody in MCF-7 cells. The effects of AZA-1 on the E-cadherin system were confirmed in Caco-2 and Madin Darby canine kidney epithelial cell lines. We conclude that AZA-1 inhibits endocytosis of plasma membrane proteins in epithelial cells.
authors.affiliation: Dipartimento di Scienze Biomediche, Università di Modena e Reggio Emilia, Modena, Italy.
authors.forename: Mirella
authors.initials: M
authors.lastname: Bellocci
authors.forename: Gian Luca
authors.initials: GL
authors.lastname: Sala
authors.forename: Federica
authors.initials: F
authors.lastname: Callegari
authors.forename: Gian Paolo
authors.initials: GP
authors.lastname: Rossini
mesh_terms: D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000633:toxicity; D015820:Cadherins / Q000378:metabolism; D045744:Cell Line, Tumor; D002738:Chloroquine / Q000633:toxicity; D004705:Endocytosis / Q000187:drug effects*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008387:Marine Toxins / Q000633:toxicity*; D008565:Membrane Proteins / Q000378:metabolism*; D013141:Spiro Compounds / Q000633:toxicity*; D013347:Subcellular Fractions / Q000378:metabolism; D053778:Thiazolidines / Q000633:toxicity; D000095702:Polyether Toxins
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D015820:Cadherins; D002738:Chloroquine; D008387:Marine Toxins; D008565:Membrane Proteins; D013141:Spiro Compounds; D053778:Thiazolidines; C406592:azaspiracid; D000095702:Polyether Toxins; C037067:latrunculin A