Ligand internalization and recycling by human recombinant somatostatin type 4 (h sst4) receptors expressed in CHO‐K1 cells

There is controversy as to whether somatostatin sst4 receptors internalize. In this study, CHO‐K1 cells expressing human sst4 receptor (CHOsst4 cells) cells internalized [125I]‐[11Tyr]‐SRIF in a time‐dependent manner, reaching a steady state at 60 min (1.4±0.1×104 molecules internalized per cell). Internalization was blocked by hypertonic sucrose (0.5 M), ATP depletion or by decreasing the temperature to 4°C. Internalization of [125I]‐[11Tyr]‐SRIF was also inhibited (pIC50 values) by increasing concentrations of SRIF (7.74), L‐362855 (6.27) and NNC‐296100 (6.50) with pIC50 values approximately 10 fold lower than those obtained for inhibition of [125I]‐[11Tyr]‐SRIF binding to membrane homogenates. Internalized ligand recycled rapidly to the extracellular media (t1/2 3.9±0.7 min) with only 6.8±0.6% of internalized radioactivity remaining in the cell after 45 min. Confocal microscopy of permeabilized, HA‐epitope tagged CHOsst4 cells labelled with a Cy‐3 conjugated antibody revealed little internal immunostaining after SRIF (1 μM) treatment, consistent with the small proportion of receptors (3.5%) estimated to be internalized by radioimmunoassay. In summary, CHOsst4 cells internalized [125I]‐[11Tyr]‐SRIF in a clathrin‐ and ATP‐dependent manner with subsequent rapid recycling to the extracellular medium. Rapid receptor recycling and the consequent low proportion of receptors internalized at any one time may explain the inability to visualize internalized receptors by confocal microscopy. It seems unlikely therefore that the marked receptor desensitization observed in CHOsst4 cells following SRIF treatment can be accounted for by a decrease in cell surface receptor expression. British Journal of Pharmacology (2001) 132, 1102–1110; doi:10.1038/sj.bjp.0703896