Novel Mechanism for Regulation of Epidermal Growth Factor Receptor Endocytosis Revealed by Protein Kinase A Inhibition

Abstract

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 -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. INTRODUCTIONThe mechanisms that regulate the endocytic behavior of epidermal growth factor receptor (EGFR) have been a longstanding subject of intense research as a model system of regulated vesicular protein traffic associated to signaling, cellular demands, and cancer (Trowbridge et al., 1993;Di Fiore and Gill, 1999;Carpenter, 2000;Ceresa and Schmid, 2000;Schlessinger, 2000;Wiley and Burke, 2001). Endocytosis provides a pathway for gradual attenuation or desensitization of receptor signaling and also allows a single receptor to transmit different signals from different locations in the cell before degradation, thus enhancing the range of modulation and response variability (Di Fiore and Gill, 1999;Ceresa and Schmid, 2000;Wiley and Burke, 2001). Defects in internalization and/or degradation pathways, as well as distinct endocytic routing displayed by different EGFR family members, have been associated with cell transformation and oncogenesis (Di Fiore and Gill, 1999;Ceresa and Schm...

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