Novel mechanism for regulation of epidermal growth factor receptor endocytosis revealed by protein kinase A inhibition.
Current models put forward that the epidermal growth factor receptor (EGFR) is efficiently internalized via clathrin-coated pits only in response to ligand-induced activation of its intrinsic tyrosine kinase and is subsequently directed into a lysosomal-proteasomal degradation pathway by mechanisms that include receptor tyrosine phosphorylation and ubiquitylation. Herein, we report a novel mechanism of EGFR internalization that does not require ligand binding, receptor kinase activity, or ubiquitylation and does not direct the receptor into a degradative pathway. Inhibition of basal protein kinase A (PKA) activity by H89 and the cell-permeable substrate peptide Myr-PKI induced internalization of 40-60% unoccupied, inactive EGFR, and its accumulation into early endosomes without affecting endocytosis of transferrin and mu-opioid receptors. This effect was abrogated by interfering with clathrin function. Thus, the predominant distribution of inactive EGFR at the plasma membrane is not simply by default but involves a PKA-dependent restrictive condition resulting in receptor avoidance of endocytosis until it is stimulated by ligand. Furthermore, PKA inhibition may contribute to ligand-induced EGFR endocytosis because epidermal growth factor inhibited 26% of PKA basal activity. On the other hand, H89 did not alter ligand-induced internalization of EGFR but doubled its half-time of down-regulation by retarding its segregation into degradative compartments, seemingly due to a delay in the receptor tyrosine phosphorylation and ubiquitylation. Our results reveal that PKA basal activity controls EGFR function at two levels: 1) residence time of inactive EGFR at the cell surface by a process of "endocytic evasion," modulating the accessibility of receptors to stimuli; and 2) sorting events leading to the down-regulation pathway of ligand-activated EGFR, determining the length of its intracellular signaling. They add a new dimension to the fine-tuning of EGFR function in response to cellular demands and cross talk with other signaling receptors.
Salazar
Gloria
G
Departamento de Inmunología Clínica y Reumatología, Facultad de Medicina. Centro de Regulación Celular y Patología, Pontificia Universidad Católica de Chile, Santiago, Chile.
González
Alfonso
A
D002966:Clathrin / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D010766:Phosphorylation; D010770:Phosphotransferases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011957:Receptors, Opioid / Q000378:metabolism; D013449:Sulfonamides
D002966:Clathrin; D007457:Iodine Radioisotopes; D007546:Isoquinolines; D008024:Ligands; D011957:Receptors, Opioid; D013449:Sulfonamides; D004815:Epidermal Growth Factor; D010770:Phosphotransferases; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide