Ligand-induced caveolae-mediated internalization of A1 adenosine receptors: morphological evidence of endosomal sorting and receptor recycling.
The involvement of caveolae in the internalization of A(1) adenosine receptors (A(1)R) and the receptor sorting and recycling was studied in the smooth muscle cell line DDT(1)MF-2, by binding assays, by confocal microscopy, and at the structural level. The use of cholera toxin-binding subunit adsorbed to gold as a specific probe for labeling the ganglioside GM(1) and immunoelectron microscopy techniques showed that agonist stimulation produced a clustering and sequestration of adenosine receptors in caveolae. Furthermore, pull-down experiments showed there to be a direct interaction between the C-terminal domain of A(1)R and caveolin-1. Addition of exogenous adenosine deaminase (ADA), a protein that binds to A(1)R and acts as a receptor activity modifying protein (RAMP) stimulated R-PIA-induced A(1) receptor internalization. Finally, the sorting and recycling of A(1)R/ADA complexes was analyzed. Detailed electron microscopy revealed that A(1)R/ADA complexes internalize together through caveolae, are differentially sorted in endosomes, and are recycled back to the cell surface by different groups of recycling endosomes. These results give insight into the spatiotemporal regulation and traffic of A(1)R and RAMPs.
Escriche
Marisol
M
Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University of Barcelona, 08028, Barcelona, Spain.
Burgueño
Javier
J
Ciruela
Francisco
F
Canela
Enric I
EI
Mallol
Josefa
J
Enrich
Carlos
C
Lluís
Carmen
C
Franco
Rafael
R
D000243:Adenosine Deaminase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000378:metabolism; D006224:Cricetinae; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism / Q000648:ultrastructure; D017979:Gold Colloid / Q000378:metabolism; D008024:Ligands; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology*; D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D051242:Caveolin 1; D022461:Caveolins; D017979:Gold Colloid; D008024:Ligands; D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1; D011993:Recombinant Fusion Proteins; D002772:Cholera Toxin; D000243:Adenosine Deaminase; D002118:Calcium