Abstract
Many G protein-coupled receptors undergo endocytosis, but the mechanisms involved in endocytic sorting and recycling remain to be fully elucidated. We found that the G protein-coupled calcitonin receptor (CTR) undergoes tonic internalization and accumulates within the cell. Using a fluorescence loss in photobleaching assay, we classified these vesicles functionally as recycling vesicles. In a two-hybrid screening, we found that the actin-binding protein filamin interacted with the C-terminal tail of the CTR. The degradation of the receptor was profoundly increased in the absence of filamin or the CTR-filamin interaction. The absence of filamin was also associated with a marked decrease in recycling of the receptor from the endosomes to the cell surface. In contrast, calcitonin-induced inhibition of spontaneous filamin proteolysis was associated with increased recycling of the receptor to the cell surface and decreased degradation of the CTR, suggesting an important role for filamin in the endocytic sorting and recycling of the internalized CTR.
Highlights
The calcitonin receptor (CTR)1 belongs to subclass B of the G protein-coupled receptors (GPCRs)
Internalization of the receptor was allowed to proceed for different time periods, and the biotin remaining on the cell surface was stripped
The remaining anti-HA antibody bound to the CTR at the surface was labeled with the PE-conjugated secondary antibody, and the cell-associated PE was quantified by fluorescence-activated cell sorter (FACS)
Summary
Reagents and Antibodies—Salmon calcitonin (sCT) was purchased from Peninsula Laboratories, Inc. (Belmont, CA). To measure cell surface expression, a 30-min incubation step was followed by resuspending cells in 100 l of PBS containing 50 g/ml phycoerythrin (PE)-conjugated goat anti-mouse IgG antibody (Molecular Probes). Analysis of Receptor Recycling Using FACS—Cells were prepared as described for the internalization assay, except that cells were incubated for 30 min at 37 °C in the presence of the anti-HA antibody to allow endocytosis of the CTR with the bound antibody. Analysis of Receptor Endocytosis Using Cleavable Biotin—CTRtransfected cells in 10-cm dishes were washed twice with ice-cold PBS and incubated with 0.5 mg/ml sulfo-NHS-S-S-biotin (Pierce) in PBS for 30 min at 4 °C to biotinylate cell surface proteins.
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