Recycling of a secretory granule membrane protein after stimulated secretion.
Recycling of a secretory granule membrane protein, dopamine-beta-hydroxylase, was examined in primary cultures of bovine adrenal chromaffin cells. Cells were stimulated to secrete in the presence of antibodies directed against the luminal domain of dopamine-beta-hydroxylase. The location of the antibodies after various times of reincubation and after a second secretory stimulus was assessed using immunofluorescence microscopy. Stimulation led to the exposure of dopamine-beta-hydroxylase at the plasma membrane, which could be detected by a polyclonal antibody in living and fixed cells. The plasma membrane dopamine-beta-hydroxylase, either alone or complexed with antibody, was rapidly internalized after removal of the secretagogue. Internalized protein-antibody complex remained stable for at least 24 hours of reculture. Twenty four hours after stimulation the cells with internalized antibody could respond to further stimulation and some of the antibody was re-exposed at the plasma membrane. These findings were confirmed using FACS analysis. This suggests that the antibody-protein complex had returned to secretory granules that could respond to further secretagogue stimulation.
Hurtley
S M
SM
Department of Biochemistry, University of Edinburgh Medical School, UK.
D000311:Adrenal Glands / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001464:Barium / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D002838:Chromaffin System / Q000187:drug effects / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004299:Dopamine beta-Hydroxylase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D008565:Membrane Proteins / Q000378:metabolism*
D000906:Antibodies; D008565:Membrane Proteins; D001464:Barium; D004299:Dopamine beta-Hydroxylase