Intracellular dynamics of sst(5) receptors in transfected COS-7 cells: Maintenance of cell surface receptors during ligand-induced endocytosis

Internalization of G protein-coupled receptors is crucial for resensitizati on of phosphorylation-desensitized receptors, but also for their long term desensitization through sequestration. To elucidate the mechanisms regulati ng cell surface availability of the somatostatin (SRIF) receptor subtype ss t,, we characterized its internalization properties in transfected COS-7 ce lls using biochemical, confocal microscopic, and electron microscopic techn iques. Our results demonstrated rapid and efficient sequestration of specif ically bound [I-125]Tyr(0)-D-Trp(8)-SRIF (up to 45% of bound radioactivity) . Combined immunocytochemical detection of sst, and visualization of a fluo rescent SRIF analog by confocal microscopy revealed that whereas the intern alized ligand progressively clustered toward the cell center with time, imm unoreactive receptors remained predominantly associated with the plasma mem brane. The preservation of cell surface receptors was confirmed by binding experiments on whole cells revealing a lack of saturability of [I-125]Tyr(0 )-D-Trp(8)-SRIF binding at 37 C. Binding was rendered saturable by the drug monensin, showing that receptor recycling played a key role in the preserv ation of cell surface receptors. Electron microscopy demonstrated that in a ddition to receptor recycling, internalization of receptor-ligand complexes triggered a massive recruitment of sst, receptor molecules from intracellu lar stores to the membrane. This combination of recycling and recruitment o f spare receptors may protect sst(5) from long term downregulation through sequestration and, therefore, facilitate extended SRIF signaling.