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) and 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 internalize 125I-PTHrP(1-34)amide was determined. Most of the C-terminal tail (91 of 127 residues) could be deleted without affecting internalization. However, further truncation removing residues 475-494 resulted in a 50-60% decrease in ligand internalization. A mutant with an internal deletion of these 20 amino acids showed a similar reduction in internalization, confirming the presence of a positive endocytic signal. No additional positive signals were found in the membrane-proximal region of the tail. However, alanine mutagenesis of the membrane-proximal residues 459-461 (EVQ-->AAA) resulted in a mutant PTH/PTHrP receptor displaying a 40% increase in ligand endocytosis, indicating that EVQ functions as a negative signal. Treatment of COS-7 cells with hypertonic 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 membrane-proximal cytoplasmic tail of the PTH/PTHrP receptor and suggest that these signals regulate the ability of the receptor to accumulate in clathrin-coated pits.
Huang
Z
Z
Endocrine Research Unit, Veterans Administration Medical Center, San Francisco, California.
Chen
Y
Y
Nissenson
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D004705:Endocytosis; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000378:metabolism*; D015398:Signal Transduction
D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D019204:GTP-Binding Proteins