Involvement of caveolin in ligand-induced recruitment and internalization of A(1) adenosine receptor and adenosine deaminase in an epithelial cell line.
Chronic exposure of A(1) adenosine receptors (A(1)R) to A(1)R agonists leads to activation, phosphorylation, desensitization, and internalization to intracellular compartments of the receptor. Desensitization and internalization of A(1)R is modulated by adenosine deaminase (ADA), an enzyme that regulates the extracellular concentration of adenosine. ADA interacts with A(1)R on the cell surface of the smooth muscle cell line DDT1 MF-2, and both proteins are internalized following agonist stimulation of the receptor. The mechanism involved in A(1)R and ADA internalization upon agonist exposure is poorly understood in epithelial cells. In this report, we show that A(1)R and ADA interact in LLC-PK(1) epithelial cells. Exposure of LLC-PK(1) cells to A(1)R agonists induces aggregation of A(1)R and ADA on the cell surface and their translocation to intracellular compartments. Biochemical and cell biology assays were used to characterize the intracellular vesicles containing both proteins after agonist treatment. A(1)R and ADA colocalized together with the rafts marker protein caveolin. Filipin, a sterol-binding agent that disrupts rafts (small microdomains of the plasma membrane), was able to inhibit A(1)R internalization. In contrast, acid treatment of the cells, which disrupts internalization via clathrin-coated vesicles, did not inhibit agonist-stimulated A(1)R internalization. We demonstrated that A(1)R agonist N(6)-(R)-phenylisopropyl adenosine promotes the translocation of A(1)R into low-density gradient fractions containing caveolin. Furthermore, a direct interaction of the C-terminal domain of A(1)R with caveolin-1 was demonstrated by pull down experiments. These results indicate that A(1)R and ADA form a stable complex in the cell surface of LLC-PK(1) cells and that agonist-induced internalization of the A(1) adenosine receptor and ADA is mediated by clathrin-independent endocytosis.
Ginés
S
S
Departament de Bioquímica i Biologia Molecular, Facultat de Química. Universitat de Barcelona, Spain.
Ciruela
F
F
Burgueño
J
J
Casadó
V
V
Canela
E I
EI
Mallol
J
J
Lluís
C
C
Franco
R
R
D000243:Adenosine Deaminase / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D051242:Caveolin 1; D022461:Caveolins / Q000502:physiology*; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism; D004847:Epithelial Cells / Q000201:enzymology / Q000378:metabolism* / Q000502:physiology; D008024:Ligands; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D013552:Swine
D051242:Caveolin 1; D022461:Caveolins; D002966:Clathrin; D008024:Ligands; D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1; D000243:Adenosine Deaminase