Internalization of V2-vasopressin receptors in LLC-PK1-cells: evidence for receptor-mediated endocytosis.

The mechanism of internalization of the vasopressin-receptor (V 2 -subtype) of LLC-PK 1 -cells, a pig renal tubular cell line, is unknown. We studied internalization utilizing a novel, highly specific vasopressin analogue (( 125 I)-[8-p(OH)-phenylpropionyl]-LVP 2000 Ci/mmol). Scatchard analysis performed with membranes of LLC-PK 1 -cells revealed a K d of 0.8 +/− 0.2 nM and a B max of 366 +/− 41 fmol/mg of protein. Degradation of the ligand was excluded by RP-HPLCanalysis. Internalization was proven by the acid-wash technique, quantitative light-microscopic autoradiography and electron microscopy. The ligand was internalized in a time- and temperature-dependent manner. At 4°C, no uptake was found; at 22°C, after 30 min of incubation, more than 50% of the radioligand was found inside the cell. Electron microscopy demonstrated that plasma-membrane bound vasopressin receptors are internalized by receptor-mediated endocytosis via coated pits.