THE CYTOPLASMIC TAIL OF THE G-PROTEIN-COUPLED RECEPTOR FOR PARATHYROID-HORMONE AND PARATHYROID HORMONE-RELATED PROTEIN CONTAINS POSITIVE AND NEGATIVE SIGNALS FOR ENDOCYTOSIS

The present studies were done to evaluate the role of the cytoplasmic tail of the G-protein-coupled receptor for parathyroid hormone (PTH) a nd PTH related protein (PTHrP) in the endocytosis of agonist-occupied receptors. PTH/PTHrP receptor mutants progressively truncated from the C terminus were expressed in COS-7 cells, and their ability to intern alize I-125-PTHrP(1-34) amide was determined. Most of the C terminal t ail (91 of 127 residues) could be deleted without affecting internaliz ation. However, further truncation removing residues 475-494 resulted in a 50-60% decrease in ligand internalization. A mutant with an inter nal deletion of these 20 amino acids showed a similar reduction in int ernalization, confirming the presence of a positive endocytic signal. No additional positive signals were found in the membrane-proximal reg ion of the tail. How ever, alanine mutagenesis of the membrane-proxima l residues 459-461 (EVQ --> AAA) resulted in a mutant PTH/PTHrP recept or displaying a 40% increase in ligand endocytosis, indicating that EV Q functions as a negative signal. Treatment of COS-7 cells with hypert onic sucrose (to disrupt clathrin lattices) markedly suppressed (by > 80%) PTH/PTHrP receptor internalization. These results demonstrate the presence of both positive and negative endocytic signals in the membr ane-proximal cytoplasmic tail of the PTH/PTHrP receptor and suggest th at these signals regulate the ability of the receptor to accumulate in clathrin-coated pits.