Anomalous binding of epidermal growth factor to A431 cells is due to the effect of high receptor densities and a saturable endocytic system

This study was conducted to determine how extraordinarily high numbers of epidermal growth factor receptors (EGF-R) affected the binding and internalization of EGF in the transformed cell line A431. I found that at low EGF concentrations, the kinetics of binding behaved as a nonsaturable, first-order process showing no evidence of multiple-affinity classes of receptors. However, EGF dissociation rates were strongly dependent on the degree of receptor occupancy in both intact cells and isolated membranes. This occupancy-dependent dissociation appears to be due to diffusion-limited binding. EGF-induced receptor internalization was rapid and first order when the absolute number of occupied receptors was below 4 x 10(3) min-1. However, at higher occupancies the specific internalization rate progressively declined to a final limiting value of 20% normal. The saturation of EGF-R endocytosis was specific since internalization of transferrin receptors was not affected by high concentrations of either transferrin or EGF. Saturation of EGF-R endocytosis probably involves a specific component of the endocytic pathway since fluid phase endocytosis increased coordinately with EGF-R occupancy. I conclude that there are several aspects of EGF-R dynamics on A431 cells are neither similar to the behavior of EGF-R in other cell types nor similar to the reported behavior of other hormone receptors. Although A431 cells have an extraordinary number of EGF-R, they do not seem to have corresponding levels of at least two other crucial cell surface components: one that mediates EGF-induced rapid receptor internalization and one that attenuates EGF-induced membrane responses. These factors, in addition to the presence of diffusion-limited binding at low EGF concentrations, are probably responsible for the appearance of multiple-affinity classes of receptors in this cell type.     

An endosomally localized isoform of Eps15 interacts with Hrs to mediate degradation of epidermal growth factor receptor

Down-regulation of activated and ubiquitinated growth factor (GF) receptors by endocytosis and subsequent lysosomal degradation ensures attenuation of GF signaling. The ubiquitin-binding adaptor protein Eps15 (epidermal growth factor receptor [EGFR] pathway substrate 15) functions in endocytosis of such receptors. Here, we identify an Eps15 isoform, Eps15b, and demonstrate its expression in human cells and conservation across vertebrate species. Although both Eps15 and Eps15b interact with the endosomal sorting protein Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) in vitro, we find that Hrs specifically binds Eps15b in vivo (whereas adaptor protein 2 preferentially interacts with Eps15). Although Eps15 mainly localizes to clathrin-coated pits at the plasma membrane, Eps15b localizes to Hrs-positive microdomains on endosomes. Eps15b overexpression, similarly to Hrs overexpression, inhibits ligand-mediated degradation of EGFR, whereas Eps15 is without effect. Similarly, depletion of Eps15b but not Eps15 delays degradation and promotes recycling of EGFR. These results indicate that Eps15b is an endosomally localized isoform of Eps15 that is present in the Hrs complex via direct Hrs interaction and important for the sorting function of this complex.     

A luminescent assay for real-time measurements of receptor endocytosis in living cells

Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs.     

The Na+/H+ exchanger regulatory factor stabilizes epidermal growth factor receptors at the cell surface

Ligand binding to cell surface receptors initiates both signal transduction and endocytosis. Although signaling may continue within the endocytic compartment, down-regulation is the major mechanism that controls the concentration of cell surface receptors, their ability to receive environmental signals, and the ultimate strength of biological signaling. Internalization, recycling, and trafficking of receptor tyrosine kinases (RTKs) within the endosome compartment are each regulated to control the overall process of down-regulation. We have identified the Na(+)/H(+) exchanger regulatory factor (NHERF) as an important molecular component that stabilizes epidermal growth factor receptors (EGFRs) at the cell surface to restrict receptor down-regulation. The NH(2)-terminal PDZ domain (PDZ 1) of NHERF specifically binds to an internal peptide motif located within the COOH-terminal regulatory domain of EGFR. Expression of NHERF slows the rate of EGF-induced receptor degradation. A point mutation that abolishes the PDZ 1 recognition sequence of EGFR enhances the rate of ligand-induced endocytosis and down-regulation of EGFR. Similarly, expression of a dominant negative mutant of NHERF enhances EGF-induced receptor down-regulation. In contrast to beta-adrenergic receptors where NHERF enhances recycling of internalized receptors, NHERF stabilizes EGFR at the cell surface and slows the rate of endocytosis without affecting recycling. Although the mechanisms differ, for both RTKs and G protein-coupled receptors, the overall effect of NHERF is to enhance the fraction of receptors present at the cell surface.     

Tyrosine 569 in the c-Fms juxtamembrane domain is essential for kinase activity and macrophage colony-stimulating factor-dependent internalization.

The receptor (Fms) for macrophage colony-stimulating factor (M-CSF) is a member of the tyrosine kinase class of growth factor receptors. It maintains survival, stimulates growth, and drives differentiation of the macrophage lineage of hematopoietic cells. Fms accumulates on the cell surface and becomes activated for signal transduction after M-CSF binding and is then internalized via endocytosis for eventual degradation in lysosomes. We have investigated the mechanism of endocytosis as part of the overall signaling process of this receptor and have identified an amino acid segment near the cytoplasmic juxtamembrane region surrounding tyrosine 569 that is important for internalization. Mutation of tyrosine 569 to alanine (Y569A) eliminates ligand-induced rapid endocytosis of receptor molecules. The mutant Fms Y569A also lacks tyrosine kinase activity; however, tyrosine kinase activity is not essential for endocytosis because the kinase inactive receptor Fms K614A does undergo ligand-induced endocytosis, albeit at a reduced rate. Mutation of tyrosine 569 to phenylalanine had no effect on the M-CSF-induced endocytosis of Fms, and a four-amino-acid sequence containing Y-569 could support endocytosis when transferred into the cytoplasmic juxtamembrane region of a glycophorin A construct. These results indicate that tyrosine 569 within the juxtamembrane region of Fms is part of a signal recognition sequence for endocytosis that does not require tyrosine phosphorylation at this site and that this domain also influences the kinase activity of the receptor. These results are consistent with a ligand-dependent step in recognition of the potential cryptic internalization signal.     

Role of the Rab11-associated intracellular pool of receptors formed by constitutive endocytosis of the beta isoform of the thromboxane A2 receptor (TP beta)

Intracellular trafficking pathways of G protein-coupled receptors (GPCRs), following their agonist-induced endocytosis and their consequences on receptor function, are the subject of intense research efforts. However, less is known regarding their constitutive endocytosis. We previously demonstrated that the beta isoform of the thromboxane A(2) receptor (TPbeta) undergoes constitutive and agonist-induced endocytosis. Constitutive endocytosis of GPCRs can lead to the formation of an intracellular pool of receptors from which they can recycle back to the cell surface. In the present report, we show with the help of two TPbeta mutants (TPbeta-Y339A and TPbeta-I343A) specifically deficient in constitutive endocytosis that this intracellular pool of receptors serves to maintain agonist sensitivity over prolonged receptor stimulation in HEK293 cells. Second messenger generation by the TPbeta-Y339A and TPbeta-I343A mutants was drastically reduced compared to the wild-type receptor as suggested by dose-response and time-course experiments of inositol phosphates production following agonist treatment, despite normal coupling between the receptors and the Galpha(q) protein. Moreover, second messenger production after receptor activation was dramatically reduced when cells were pretreated with monensin, a recycling inhibitor. Receptor cell surface expression and endocytosis experiments further revealed that the small GTPase Rab11 protein is a determinant factor in controlling TPbeta recycling back to the cell surface. Co-localization experiments performed by immunofluorescence microscopy indicated that both constitutive and agonist-triggered endocytosis resulted in targeting of TPbeta to the Rab11-positive recycling endosome. Thus, we provide evidence that constitutive endocytosis of TPbeta forms a pool of receptors in the perinuclear recycling endosome from which they recycle to the cell surface, a process involved in preserving receptor sensitivity to agonist stimulation.     

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of beta2-adrenergic receptors

Phosphatidylinositol 3-kinase inhibitors have been shown to affect endocytosis or subsequent intracellular sorting in various receptor systems. Agonist-activated beta(2)-adrenergic receptors undergo desensitization by mechanisms that include the phosphorylation, endocytosis and degradation of receptors. Following endocytosis, most internalized receptors are sorted to the cell surface, but some proportion is sorted to lysosomes for degradation. It is not known what governs the ratio of receptors that recycle versus receptors that undergo degradation. To determine if phosphatidylinositol 3-kinases regulate beta(2)-adrenergic receptor trafficking, HEK293 cells stably expressing these receptors were treated with the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. We then studied agonist-induced receptor endocytosis and postendocytic sorting, including recycling and degradation of the internalized receptors. Both inhibitors amplified the internalization of receptors after exposure to the beta-agonist isoproterenol, which was attributable to the sorting of a significant fraction of receptors to an intracellular compartment from which receptor recycling did not occur. The initial rate of beta(2)-adrenergic receptor endocytosis and the default rate of receptor recycling were not significantly altered. During prolonged exposure to agonist, LY294002 slowed the degradation rate of beta(2)-adrenergic receptors and caused the accumulation of receptors within rab7-positive vesicles. These results suggest that phosphatidylinositol 3-kinase inhibitors (1) cause a misrouting of beta(2)-adrenergic receptors into vesicles that are neither able to efficiently recycle to the surface nor sort to lysosomes, and (2) delays the movement of receptors from late endosomes to lysosomes.     

Internalization and cycling of nerve growth factor in PC12 cells: interconversion of type II (fast) and type I (slow) nerve growth factor receptors

The effects of agents that inhibit receptor-mediated endocytosis on type I (slow or high-affinity) and type II (fast or low-affinity) NGF binding have been examined in rat PC12 cells. Compounds interfering with endocytosis eliminate type I NGF binding; those interfering with acidification of endosomal vesicles cause increased type I binding at the expense of type II binding. Measurement of NGF binding during and after treatment with inhibitors indicates that NGF receptors rapidly cycle from the cell surface into an undefined endocytotic compartment and back to the surface with little degradation of receptor or NGF, consistent with a model in which NGF receptors are rapidly and reversibly endocytosed or sequestered; those receptors free on the surface represent type II NGF receptors, while those in the process of endocytosis represent type I NGF receptors. The type I and type II NGF receptor species can be interconverted by agents that can manipulate the position of the receptor in the internalization cycle.     

The role of endosomal signaling triggered by metastatic growth factors in tumor progression

Within tumor microenvironment, a lot of growth factors such as hepatocyte growth factor and epidermal growth factor may induce similar signal cascade downstream of receptor tyrosine kinase (RTK) and trigger tumor metastasis synergistically. In the past decades, the intimate relationship of RTK-mediated receptor endocytosis with signal transduction was well established. In general, most RTK undergoes clathrin-dependent endocytosis and/or clathrin-independent endocytosis. The internalized receptors may sustain the signaling within early endosome, recycling to plasma membrane for subsequent ligand engagement or sorting to late endosomes/lysosome for receptor degradation. Moreover, receptor endocytosis influences signal transduction in a temporal and spatial manner for periodical and polarized cellular processes such as cell migration. The endosomal signalings triggered by various metastatic factors are quite similar in some critical points, which are essential for triggering cell migration and tumor progression. There are common regulators for receptor endocytosis including dynamin, Rab4, Rab5, Rab11 and Cbl. Moreover, many critical regulators within the RTK signal pathway such as Grb2, p38, PKC and Src were also modulators of endocytosis. In the future, these may constitute a new category of targets for prevention of tumor metastasis.     

Cell surface control of the multiubiquitination and deubiquitination of high-affinity immunoglobulin E receptors.

Multiubiquitination of proteins is a critical step leading to selective degradation for many polypeptides. Therefore, activation-induced multiubiquitination of cell surface receptors, such as the platelet-derived growth factor (PDGF) receptor and the T cell antigen (TCR) receptor, may correspond to a degradation pathway for ligand-receptor complexes. Here we show that the antigen-induced engagement of high-affinity immunoglobulin E receptors (Fc epsilon RI) results in the immediate multiubiquitination of Fc epsilon RI beta and gamma chains. This ubiquitination is independent of receptor phosphorylation and is restricted to activated receptors. Surprisingly, receptor multiubiquitination is immediately reversible when receptors are disengaged. Therefore, multiubiquitination and deubiquitination of Fc epsilon RI receptors is controlled at the cell surface by receptor engagement and disengagement. The rapidity, specificity and, most importantly, the reversibility of the activation-induced receptor multiubiquitination suggest that this process may turn on/off a cell surface receptor signaling function thus far unsuspected.     

Adenovirus RIDbeta subunit contains a tyrosine residue that is critical for RID-mediated receptor internalization and inhibition of Fas- and TRAIL-induced apoptosis

The adenovirus-encoded receptor internalization and degradation (RID) protein (previously named E3-10.4K/14.5K), which is composed of RIDalpha and RIDbeta subunits, down-regulates a number of cell surface receptors in the tumor necrosis factor (TNF) receptor superfamily, namely Fas, TRAIL receptor 1, and TRAIL receptor 2. Down-regulation of these "death" receptors protects adenovirus-infected cells from apoptosis induced by the death receptor ligands Fas ligand and TRAIL. RID also down-regulates certain tyrosine kinase cell surface receptors, especially the epidermal growth factor receptor (EGFR). RID-mediated Fas and EGFR down-regulation occurs via endocytosis of the receptors into endosomes followed by transport to and degradation within lysosomes. However, the molecular interactions underlying this function of RID are unknown. To investigate the molecular determinants of RIDbeta that are involved in receptor down-regulation, mutations within the cytoplasmic tail of RIDbeta were constructed and the mutant proteins were analyzed for their capacity to internalize and degrade Fas and EGFR and to protect cells from death receptor ligand-induced apoptosis. The results demonstrated the critical nature of a tyrosine residue near the RIDbeta C terminus; mutation of this residue to alanine abolished RID function. Mutating the tyrosine to phenylalanine did not abolish the function of RID, arguing that phosphorylation of the tyrosine is not required for function. These data suggest that this tyrosine residue forms part of a tyrosine-based sorting signal (Yxxphi). Additional mutations that target another potential sorting motif and several possible protein-protein interaction motifs had no discernible effect on RID function. It was also demonstrated that mutation of serine 116 to alanine eliminated phosphorylation of RIDbeta but did not affect any of the functions of RID that were examined. These results suggest a model in which the tyrosine-based sorting signal in RID plays a role in RID's ability to down-regulate receptors.     

The role of tyrosine kinase activity in endocytosis, compartmentation, and down-regulation of the epidermal growth factor receptor.

Occupancy-induced down-regulation of cell surface epidermal growth factor (EGF) receptors attenuates signal transduction. To define mechanisms through which down-regulation of this class of growth factor receptors occurs, we have investigated the relative roles of ligand-induced internalization and recycling in this process. Occupied, kinase-active EGF receptors were internalized through a high affinity, saturable endocytic system at rates up to 10-fold faster than empty receptors. In contrast, full length EGF receptors lacking tyrosine kinase activity underwent internalization at a rate independent of occupancy. This "kinase-independent" internalization rate appeared to reflect constitutive receptor internalization since it was similar to the internalization rate of both receptors lacking a cytoplasmic domain and of antibodies bound to empty receptors. EGF internalized by either kinase-active or kinase-inactive receptors was efficiently recycled and was found within endosomes containing recycling transferrin receptors. However, targeting of internalized receptors to lysosomes did not require receptor kinase activity. All receptors that displayed ligand-induced internalization also underwent down-regulation, indicating that the proximal cause of down-regulation is occupancy-induced endocytosis. Tyrosine kinase activity greatly enhances this process by stabilizing receptor association with the endocytic apparatus.     

Phosphorylation and chronic agonist treatment atypically modulate GABAB receptor cell surface stability

GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABA(B) receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABA(B) receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABA(B) receptor agonist, fails to enhance GABA(B) receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABA(B) receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of beta-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABA(B)R2. Our results support a model in which GABA(B)R2 phosphorylation specifically stabilizes surface GABA(B) receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABA(B) receptors.     

Expression of excess receptors and negative feedback control of signal pathways are required for rapid activation and prompt cessation of signal transduction

Over expression of receptor tyrosine kinases is responsible for the development of a wide variety of malignancies. Termination of growth factor signaling is primarily determined by the down regulation of active growth factor/receptor complexes. In recent years, considerable insight has been gained in the endocytosis and degradation of growth factor receptors. A crucial player in this process is the EGFR Protein tyrosine kinase Substrate #15, or Eps15. This protein functions as a scaffolding adaptor protein and is involved both in secretion and endocytosis. Eps15 has been shown to bind to AP-1 and AP-2 complexes, to bind to inositol lipids and to several other proteins involved in the regulation of intracellular trafficking. In addition, Eps15 has been detected in the nucleus of mammalian cells. Activation of growth factor receptors induces tyrosine phosphorylation and mono-ubiquitination of Eps15. The role of these post translational modifications of Eps15 is still a mystery. It is proposed that Eps15 and its family members Eps15R and Eps15b are involved in the regulation of membrane morphology, which is required for intracellular vesicle formation and trafficking.     

Endocytosis of receptor tyrosine kinases is driven by monoubiquitylation,not polyubiquitylation

Growth factors stimulate specific receptor tyrosine kinases, but subsequent receptor endocytosis terminates signaling. The ubiquitin ligase c-Cbl targets epidermal growth factor receptors (EGFRs) to endocytosis by tagging them with multiple ubiquitin molecules. However, the type of ubiquitylation is unknown; whereas polyubiquitin chains signal proteasomal degradation, ubiquitin monomers control other processes. We report that in isolation c-Cbl mediates monoubiquitylation rather than polyubiquitylation of EGFRs. Consistent with the sufficiency of monoubiquitylation, when fused to the tail of EGFR, a single ubiquitin induces receptor endocytosis and degradation in cells. By using receptor and ubiquitin mutants, we infer that c-Cbl attaches a founder monoubiquitin to the kinase domain of EGFR and this is complemented by the conjugation of additional monoubiquitins. Hence, receptor tyrosine kinases are desensitized through conjugation of multiple monoubiquitins, which is distinct from polyubiquitin-dependent proteasomal degradation.     

Modulation of interleukin 2 internalization and interleukin 2-dependent cell growth by antireceptor antibodies

The growth factor interleukin 2 (IL2) binds to and is internalized together with high-affinity surface receptors present on lymphoid cells. This endocytosis thus results in down-regulation of the receptors. However, it is not known if the internalization is relevant to the induction of cell growth. In the present study a rat monoclonal antibody to the P55 chain of the IL2 receptor was used to examine the role of receptor internalization in the IL2-dependent autocrine human tumor T cell line IARC 301. When given alone, this antibody did not inhibit IL2 binding, internalization, or IL2-dependent cell proliferation. However, crosslinking by anti-rat immunoglobulins, which did not affect binding of the growth factor, inhibited both IL2 internalization and cell proliferation. Besides offering a novel means for the specific inhibition of the uptake of IL2 bound to IL2 high-affinity receptors, the results are compatible with the association of this receptor-ligand uptake to the growth stimulation by IL2.     

Ligand-independent degradation of epidermal growth factor receptor involves receptor ubiquitylation and Hgs,an adaptor whose ubiquitin-interacting motif targets ubiquitylation by Nedd4

Ligand-dependent endocytosis of the epidermal growth factor receptor (EGFR) involves recruitment of a ubiquitin ligase, and sorting of ubiquitylated receptors to lysosomal degradation. By studying Hgs, a mammalian homolog of a yeast vacuolar-sorting adaptor, we provide information on the less understood, ligand-independent pathway of receptor endocytosis and degradation. Constitutive endocytosis involves receptor ubiquitylation and translocation to Hgs-containing endosomes. Whereas the lipid-binding motif of Hgs is necessary for receptor endocytosis, the ubiquitin-interacting motif negatively regulates receptor degradation. We demonstrate that the ubiquitin-interacting motif is endowed with two functions: it binds ubiquitylated proteins and it targets self-ubiquitylation by recruiting Nedd4, an ubiquitin ligase previously implicated in endocytosis. Based upon the dual function of the ubiquitin-interacting motif and its wide occurrence in endocytic adaptors, we propose a ubiquitin-interacting motif network that relays ubiquitylated membrane receptors to lysosomal degradation through successive budding events.     

Differential functions of members of the low density lipoprotein receptor family suggested by their distinct endocytosis rates

The low density lipoprotein receptor (LDLR) family is composed of a class of cell surface endocytic receptors that recognize extracellular ligands and internalize them for degradation by lysosomes. In addition to LDLR, mammalian members of this family include the LDLR-related protein (LRP), the very low density lipoprotein receptor (VLDLR), the apolipoprotein E receptor-2 (apoER2), and megalin. Herein we have analyzed the endocytic functions of the cytoplasmic tails of these receptors using LRP minireceptors, its chimeric receptor constructs, and full-length VLDLR and apoER2 stably expressed in LRP-null Chinese hamster ovary cells. We find that the initial endocytosis rates mediated by different cytoplasmic tails are significantly different, with half-times of ligand internalization ranging from less than 30 s to more than 8 min. The tail of LRP mediates the highest rate of endocytosis, whereas those of the VLDLR and apoER2 exhibit least endocytosis function. Compared with the tail of LRP, the tails of the LDLR and megalin display significantly lower levels of endocytosis rates. Ligand degradation analyses strongly support differential endocytosis rates initiated by these receptors. Interestingly apoER2, which has recently been shown to mediate intracellular signal transduction, exhibited the lowest level of ligand degradation efficiency. These results thus suggest that the endocytic functions of members of the LDLR family are distinct and that certain receptors in this family may play their main roles in areas other than receptor-mediated endocytosis.     

The Hrs/STAM complex in the downregulation of receptor tyrosine kinases

Cell surface receptor proteins that have undergone endocytosis are transported to the endosome. From the endosome, ligand-activated receptor tyrosine kinases are further transported to the lysosome for degradation, a process called "receptor downregulation." By contrast, nutrient receptors, such as those for low-density lipoprotein and transferrin, are recycled back to the plasma membrane. Sorting of these two types of receptors occurs at the endosome, where ubiquitination of receptor proteins serves as the sorting signal. Namely, ubiquitinated receptors are incorporated into the lysosomal degradation pathway, whereas those that are not ubiquitinated are returned to the cell surface. Hrs and STAM are proteins that form a complex on the endosomal membrane. Recent studies have shown that the Hrs/STAM complex binds ubiquitin moieties and acts as sorting machinery that recognizes ubiquitinated receptors and transfers them to further sequential lysosomal sorting/trafficking processes.     

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.