Intracellular dynamics of sst5 receptors in transfected COS-7 cells: maintenance of cell surface receptors during ligand-induced endocytosis.
Internalization of G protein-coupled receptors is crucial for resensitization of phosphorylation-desensitized receptors, but also for their long term desensitization through sequestration. To elucidate the mechanisms regulating cell surface availability of the somatostatin (SRIF) receptor subtype sst5, we characterized its internalization properties in transfected COS-7 cells using biochemical, confocal microscopic, and electron microscopic techniques. Our results demonstrated rapid and efficient sequestration of specifically bound [125I]Tyr0-D-Trp8-SRIF (up to 45% of bound radioactivity). Combined immunocytochemical detection of sst5 and visualization of a fluorescent SRIF analog by confocal microscopy revealed that whereas the internalized ligand progressively clustered toward the cell center with time, immunoreactive receptors remained predominantly associated with the plasma membrane. The preservation of cell surface receptors was confirmed by binding experiments on whole cells revealing a lack of saturability of [125I]Tyr0-D-Trp8-SRIF binding at 37 C. Binding was rendered saturable by the drug monensin, showing that receptor recycling played a key role in the preservation of cell surface receptors. Electron microscopy demonstrated that in addition to receptor recycling, internalization of receptor-ligand complexes triggered a massive recruitment of sst5 receptor molecules from intracellular stores to the membrane. This combination of recycling and recruitment of spare receptors may protect sst5 from long term down-regulation through sequestration and, therefore, facilitate extended SRIF signaling.
Stroh
T
T
Montreal Neurological Institute, McGill University, Montréal, Québec, Canada.
Jackson
A C
AC
Sarret
P
P
Dal Farra
C
C
Vincent
J P
JP
Kreienkamp
H J
HJ
Mazella
J
J
Beaudet
A
A
D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D004705:Endocytosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D008024:Ligands; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D017481:Receptors, Somatostatin / Q000378:metabolism*; D013004:Somatostatin / Q000031:analogs & derivatives / Q000378:metabolism; D014162:Transfection / Q000235:genetics*
D008024:Ligands; D017481:Receptors, Somatostatin; D013004:Somatostatin; C094573:somatostatin receptor 5