GIT1 mediates Src-dependent activation of phospholipase Cgamma by angiotensin II and epidermal growth factor

Critical events for vasoconstrictor and growth factor signal transduction include stimulation of phospholipase Cgamma (PLCgamma) and elevation of intracellular calcium. c-Src has been proposed as a common mediator for these signals activated by both G protein-coupled receptors (GPCRs) and tyrosine kinase-coupled receptors (TKRs). Here we show that the GPCR kinase-interacting protein-1 (GIT1) is a substrate for c-Src that undergoes tyrosine phosphorylation in response to angiotensin II (AngII) and EGF in vascular smooth muscle and 293 cells. GIT1 associates with PLCgamma via the PLCgamma Src homology 2 and 3 domains constitutively, and the interaction is unaltered by AngII and EGF. GIT1 interaction with PLCgamma is required for PLCgamma activation based on inhibition of tyrosine phosphorylation and calcium mobilization after GIT1 knockdown with antisense GIT1 oligonucleotides. GIT1 interacts with PLCgamma via a novel Spa homology domain (SHD) and a coiled-coil domain. Deletion mutation analysis showed that GIT1(SHD) is required for AngII- and EGF-mediated PLCgamma activation (measured by phosphorylation of Tyr783 and inositol 1,4,5-trisphosphate formation). We propose that GIT1 is a novel regulator of PLCgamma function that mediates PLCgamma activation by c-Src and integrates signal transduction by GPCRs and TKRs.     

Retinoids suppress epidermal growth factor-associated cell proliferation by inhibiting epidermal growth factor receptor-dependent ERK1/2 activation.

Human papillomavirus (HPV) is an important etiological agent in the genesis of cervical cancer. HPV-positive cervical tumors and human papillomavirus-positive cell lines display increased epidermal growth factor receptor (EGFR) expression, which is associated with increased cell proliferation. ECE16-1 cells are an HPV-immortalized human ectocervical epithelial cell line that is a model of HPV-associated cervical neoplasia and displays elevated EGFR levels. In the present study, we evaluated the effects of receptor-selective retinoid ligands on EGFR-associated signal transduction. We show that retinoic acid receptor (RAR)-selective ligands reduce EGFR level and the magnitude and duration of EGFR activation in EGF-stimulated cells. These effects are reversed by cotreatment with an RAR antagonist. To identify the mechanism, we examined the effects of retinoid treatments on EGF-dependent signaling. Stimulation with EGF causes a biphasic activation of the ERK1/2 MAPK. The first peak of activation is present at 20 min, and the second is present at 36 h. This activation subsequently leads to an increase in the cyclin D1 level and increased cell proliferation. Simultaneous treatment with EGF and a RAR-selective retinoid inhibits both phases of ERK1/2 activation, completely eliminates the cyclin D1 induction, and suppresses EGF-dependent cell proliferation. This effect is specific as retinoid treatment does not alter the level or activity of other EGFR-regulated kinases, including AKT and the MAPKs p38 and JNK. Retinoid X receptor-selective ligands, in contrast, did not regulate these responses. These results suggest that RAR ligand-associated down-regulation of EGFR activity reduces cell proliferation by reducing the magnitude and duration of EGF-dependent ERK1/2 activation.     

ADP-ribosylation factor 6 regulates mu-opioid receptor trafficking and signaling via activation of phospholipase D2

Endocytosis of the mu-opioid receptor (MOPr) has been shown to play a protective role against the development of tolerance to opioid drugs by facilitating receptor reactivation and recycling. It has been further demonstrated, that the opioid-mediated and ADP-ribosylation factor (ARF)-dependent activation of phospholipase D2 (PLD2) is a prerequisite for MOPr endocytosis. In this study, we investigated which particular ARF protein is involved in opioid-mediated PLD2 activation and what are the mechanisms of ARF function in MOPr trafficking and signaling. By coexpressing the MOPr and dominant negative or constitutively active ARF mutants in human embryonic kidney (HEK) 293 cells and primary cultured cortical neurons as well as by using siRNA technology, we identified the ARF6 protein to be involved in the regulation of MOPr endocytosis. We also found that expression of an effector domain mutant of ARF6, which is incapable of activating PLD, blocked agonist-induced endocytosis suggesting that ARF6 function in MOPr trafficking is PLD2-mediated. Analogously, opioid-mediated activation of PLD2 is blocked in the presence of dominant negative ARF6 mutants. Finally, we also showed that ARF6 protein influences the recycling/reactivation of internalized MOPr and thus modulates agonist-induced MOPr desensitization. Together, these results provide evidence that ARF6 protein regulates MOPr trafficking and signaling via PLD2 activation and hence affects the development of opioid receptor desensitization and tolerance.     

The small GTPase ADP-ribosylation factor 6 negatively regulates dendritic spine formation

Actin cytoskeletal reorganization and membrane trafficking are important for spine morphogenesis. Here we investigated whether the small GTPase, ADP-ribosylation factor 6 (ARF6), which regulates actin dynamics and peripheral vesicular trafficking, is involved in the regulation of spine formation. The developmental expression pattern of ARF6 in mouse hippocampus was similar to that of the post-synaptic density protein-95, and these molecules colocalized in mouse hippocampal neurons. Overexpression of a constitutively active ARF6 mutant in cultured hippocampal neurons decreased the spine density, whereas a dominant-negative ARF6 mutant increased the density. These results demonstrate a novel function for ARF6 as a key regulator of spine formation.     

ADP-ribosylation factor-dependent phospholipase D2 activation is required for agonist-induced mu-opioid receptor endocytosis

Agonist exposure of many G protein-coupled receptors induces a rapid receptor phosphorylation and uncoupling from G proteins. Resensitization of these desensitized receptors requires endocytosis and subsequent dephosphorylation. Using a yeast two-hybrid screen, the rat mu-opioid receptor (MOR1, also termed MOP) was found to be associated with phospholipase D2 (PLD2), a phospholipid-specific phosphodiesterase located in the plasma membrane, which has been implicated in the formation of endocytotic vesicles. Coimmunoprecipitation experiments in HEK293 cells coexpressing MOR1 and PLD2 confirmed that MOR1 constitutively interacts with PLD2. Treatment with the mu receptor agonist DAMGO ([d-Ala(2), Me Phe(4), Glyol(5)]enkephalin) led to an increase in PLD2 activity, whereas morphine, which does not induce MOR1 receptor internalization, failed to induce PLD2 activation. The DAMGO-mediated PLD2 activation was inhibited by brefeldin A, an inhibitor of ADP-ribosylation factor (ARF) but not by the protein kinase C (PKC) inhibitor calphostin C indicating that opioid receptor-mediated activation of PLD2 is ARF- but not PKC-dependent. Furthermore, heterologous stimulation of PLD2 by phorbol ester led to an accelerated internalization of the mu-opioid receptor after both DAMGO and morphine exposure. Conversely the inhibition of PLD2-mediated phosphatidic acid formation by 1-butanol or overexpression of a negative mutant of PLD2 prevented agonist-mediated endocytosis of MOR1. Together, these data suggest that PLD2 play a key role in the regulation of agonist-induced endocytosis of the mu-opioid receptor.     

Regulation of epidermal growth factor receptor traffic by the small GTPase rhoB.

Members of the Rho family of small GTPases control cell adhesion and motility through dynamic regulation of the actin cytoskeleton. Although twelve family members have been identified, only three of these - RhoA, Rac and Cdc42 - have been studied in detail. RhoA regulates the formation of focal adhesions and the bundling of actin filaments into stress fibres. It is also involved in other cell signalling pathways including the regulation of gene expression and the generation of lipid second messengers [1] [2]. RhoA is very closely related to two other small GTPases about which much less is known: RhoB and RhoC (which are approximately 83% identical). Perhaps the most intriguing of these is RhoB. RhoA is largely cytosolic but translocates to the plasma membrane on activation. RhoB, however, is entirely localised to the cytosolic face of endocytic vesicles [3] [4]. This suggests a potential role for RhoB in regulating endocytic traffic; however, no evidence has been presented to support this. RhoA has been shown to act at the plasma membrane to regulate the clathrin-mediated internalisation of transferrin receptor [5] and of the muscarinic acetylcholine receptor [6]. We have recently demonstrated that RhoB binds the RhoA effector, PRK1 and targets it to the endosomal compartment [7]. We show here that RhoB acts through PRK1 to regulate the kinetics of epidermal growth factor receptor traffic.     

The ADP-ribosylation factor (ARF)-related GTPase ARF-related protein binds to the ARF-specific guanine nucleotide exchange factor cytohesin and inhibits the ARF-dependent activation of phospholipase D

ADP-ribosylation factor-related protein (ARP) is a membrane-associated GTPase with remote similarity to the family of ADP-ribosylation factors (ARF). In a yeast two-hybrid screen designed to identify proteins interacting with ARP, we isolated a partial cDNA of the ARF-specific guanine nucleotide exchange factor mSec7-1/cytohesin encoding its N terminus and most of the Sec7 domain (codons 1-200). ARP and ARP-Q79L (GTPase-negative ARP) exhibited a higher affinity to mSec7-1-(1-200) than ARP-T31N (nucleotide exchange-defective ARP) in the two-hybrid assay. Similarly, full-length [35S]mSec7-1/cytohesin was specifically adsorbed to glutathione-Sepharose loaded with glutathione S-transferase (GST)-ARP-Q79L, GST-ARP, or GST-ARP-T31N, the latter exhibiting the lowest binding affinity. Overexpression of ARP-Q79L, but not of ARP-T31N, in COS-7 cells reduced the fluorescence from co-expressed green fluorescent protein fused with mSec7-1/cytohesin or mSec7-2/ARNO in plasma membranes as detected by deconvolution microscopy. Recombinant ARP and ARP-Q79L, but not ARP-T31N, inhibited the phospholipase D (PLD) activity stimulated by mSec7-2/ARNO and ARF in a system of isolated membranes. Furthermore, transfection of HEK-293 cells with ARP or ARP-Q79L, but not ARP-T31N, inhibited the muscarinic acetylcholine receptor-3 induced PLD stimulation and translocation of ARF from cytosol to membranes. These data suggest that the GTP-bound form of ARP specifically binds mSec7-1/cytohesin, and that ARP may be involved in a pathway inhibiting the ARF-controlled activity of PLD.     

The direct interaction of phospholipase C-gamma 1 with phospholipase D2 is important for epidermal growth factor signaling

The epidermal growth factor (EGF) receptor has an important role in cellular proliferation, and the enzymatic activity of phospholipase C (PLC)-gamma1 is regarded to be critical for EGF-induced mitogenesis. In this study, we report for the first time a phospholipase complex composed of PLC-gamma1 and phospholipase D2 (PLD2). PLC-gamma1 is co-immunoprecipitated with PLD2 in COS-7 cells. The results of in vitro binding analysis and co-immunoprecipitation analysis in COS-7 cells show that the Src homology (SH) 3 domain of PLC-gamma1 binds to the proline-rich motif within the Phox homology (PX) domain of PLD2. The interaction between PLC-gamma1 and PLD2 is EGF stimulation-dependent and potentiates EGF-induced inositol 1,4,5-trisphosphate (IP(3)) formation and Ca(2+) increase. Mutating Pro-145 and Pro-148 within the PX domain of PLD2 to leucines disrupts the interaction between PLC-gamma1 and PLD2 and fails to potentiate EGF-induced IP(3) formation and Ca(2+) increase. However, neither PLD2 wild type nor PLD2 mutant affects the EGF-induced tyrosine phosphorylation of PLC-gamma1. These findings suggest that, upon EGF stimulation, PLC-gamma1 directly interacts with PLD2 and this interaction is important for PLC-gamma1 activity.     

Matrix hyaluronan alters epidermal growth factor receptor-dependent cell morphology

EGFR, a critical regulator of oncogenic signaling during cancer progression, is capable of integrating multireceptor signaling pathways that promote metastasis. EGFR is subject to regulatory cues from the extracellular matrix (ECM), of which hyaluronan (HA) is a major component. In mammary tumors, HA is deposited in the ECM where it functions in biomechanical support and modulates intracellular signaling. We utilized a 3D collagen system in which HA is either polymerized in collagen matrix or provided soluble in the media (sHA). Here we report that collagen-embedded HA (eHA) inhibits EGFR activation, filopodia formation, and cell spreading on a collagen matrix. These findings demonstrate a novel role for eHA as a protective molecule when encountered in the collagen matrix during cancer progression.     

ADP-ribosylation factor-dependent phospholipase D activation by the M3 muscarinic receptor

G protein-coupled receptors can potentially activate phospholipase D (PLD) by a number of routes. We show here that the native M3 muscarinic receptor in 1321N1 cells and an epitope-tagged M3 receptor expressed in COS7 cells substantially utilize an ADP-ribosylation factor (ARF)-dependent route of PLD activation. This pathway is activated at the plasma membrane but appears to be largely independent of G, phospholipase C, Ca2+ q/11, protein kinase C, tyrosine kinases, and phosphatidyl inositol 3-kinase. We report instead that it involves physical association of ARF with the M3 receptor as demonstrated by co-immunoprecipitation and by in vitro interaction with a glutathione S-transferase fusion protein of the receptor's third intracellular loop domain. Experiments with mutant constructs of ARF1/6 and PLD1/2 indicate that the M3 receptor displays a major ARF1-dependent route of PLD1 activation with an additional ARF6-dependent pathway to PLD1 or PLD2. Examples of other G protein-coupled receptors assessed in comparison display alternative pathways of protein kinase C- or ARF6-dependent activation of PLD2.     

Maximal beta3-adrenergic regulation of lipolysis involves Src and epidermal growth factor receptor-dependent ERK1/2 activation

Catecholamine-stimulated lipolysis is primarily a beta-adrenergic and cAMP-dependent event. In previous studies we established that the beta(3)-adrenergic receptor (beta(3)AR) in adipocytes utilizes a unique mechanism to stimulate extracellular signal-regulated kinases 1 and 2 (ERK) by direct recruitment and activation of Src kinase. Therefore, we investigated the role of the ERK pathway in adipocyte metabolism and found that the beta(3)AR agonist CL316,243 regulates lipolysis through both cAMP-dependent protein kinase (PKA) and ERK. Inhibition of PKA activity completely eliminated lipolysis at low (subnanomolar) CL316,243 concentrations and by 75-80% at higher nanomolar concentrations. The remaining 20-25% of PKA-independent lipolysis, as well as ERK activation, was abolished by inhibiting the activity of either Src (PP2 or small interfering RNA), epidermal growth factor receptor (EGFR with AG1478 or small interfering RNA), or mitogen-activated protein kinase kinase 1 or 2 (MKK1/2 with PD098059). PD098059 inhibited lipolysis by 53% in mice as well. Finally, the effect of estradiol, a reported acute activator of ERK and lipolysis, was also totally prevented by PP2, AG1478, and PD098059. These results suggest that ERK activation by beta(3)AR depends upon Src and epidermal growth factor receptor kinase activities and is responsible for the PKA-independent portion of the lipolytic response. Together these results illustrate the distinct and complementary roles for PKA and ERK in catecholamine-stimulated lipolysis.     

Assessment of receptor-dependent activation of phosphatidylcholine hydrolysis by both phospholipase D and phospholipase C.

Enhancement of cellular phospholipase D (PLD)-1 and phospholipase C (PLC)-mediated hydrolysis of endogenous phosphatidylcholine (PC) during receptor-mediated cell activation has received increasing attention inasmuch as both enzymes can result in the formation of 1,2-diacylglycerol (DAG). The activities of PLD and PLC were examined in purified mast cells by quantitating the mass of the water-soluble hydrolysis products choline and phosphorylcholine, respectively. Using an assay based on choline kinase-mediated phosphorylation of choline that is capable of measuring choline and phosphorylcholine in the low picomole range, we quantitated the masses of both cell-associated and extracellular choline and phosphorylcholine. Activating mast cells by crosslinking its immunoglobulin E receptor (Fc epsilon-RI) resulted in an increase in cellular choline from 13.1 +/- 1.2 pmol/10(6) mast cells (mean +/- SE in unstimulated cells) to levels 5- to 10-fold higher, peaking 20 s after stimulation and rapidly returning toward baseline. The increase in cellular choline mass paralleled the increase in labeled phosphatidic acid accumulation detected in stimulated cells prelabeled with [3H]palmitic acid and preceded the increase in labeled DAG. Although intracellular phosphorylcholine levels were approximately 15-fold greater than choline in unstimulated cells (182 +/- 19 pmol/10(6) mast cells), stimulation resulted in a significant fall in phosphorylcholine levels shortly after stimulation. Pulse chase experiments demonstrated that the receptor-dependent increase in intracellular choline and the fall in phosphorylcholine were not due to hydrolysis of intracellular phosphorylcholine and suggested a receptor-dependent increase in PC resynthesis. When the extracellular medium was examined for the presence of water-soluble products of PC hydrolysis, receptor-dependent increases in the mass of both choline and phosphorylcholine were observed. Labeling studies demonstrated that these extracellular increases were not the result of leakage of these compounds from the cytosol. Taken together, these data lend support for a quantitatively greater role for receptor-mediated PC-PLD compared with PC-PLC during activation of mast cells.     

Secretory phospholipase A(2) inhibits epidermal growth factor-induced receptor activation

Secretory phospholipase A(2) (sPLA(2)) plays important roles in mediating various cellular processes, including cell proliferation, differentiation, apoptosis, and inflammatory response. In this study, we demonstrated that a basic sPLA(2) inhibits epidermal growth factor (EGF)-induced EGF receptor activation, as determined by autophosphorylation of EGF receptor, EGF-activated phospholipase D (PLD) activity, and phospholipase C-gamma(1) (PLC-gamma(1)) tyrosine phosphorylation in a human epidermoid carcinoma cell line, A-431. Treatment of cells with exogenous neutral sphingomyelinase (SMase) or a cell permeable ceramide analog, C(2)-ceramide, also caused similar inhibitory effects on EGF-induced activation of EGF receptor, tyrosine phosphorylation of PLC-gamma(1), and the activation of PLD. sPLA(2)-induced inhibition of EGF receptor was associated with arachidonic acid release, which was followed by an increase in intracellular ceramide formation. Both sPLA(2) and exogenous C(2)-ceramide are able to inhibit the proliferation of A-431. The data presented indicate for the first time that sPLA(2) downregulates the EGF receptor-mediated intracellular signal transduction that may be mediated by arachidonic acid and/or ceramide.     

Effects of substitution of threonine 654 of the epidermal growth factor receptor on epidermal growth factor-mediated activation of phospholipase C

Addition of epidermal growth factor (EGF) to many cell types activates phospholipase C resulting in increased levels of diacylglycerol and intracellular Ca2+ which may lead to activation of protein kinase C. EGF treatment of cells can also lead to phosphorylation of the EGF receptor at threonine 654 (a protein kinase C phosphorylation site) which appears to attenuate some aspects of receptor signaling. Thus, a feedback loop involving the EGF receptor, phospholipase C, and protein kinase C may regulate EGF receptor function. In this report, the role of phosphorylation of threonine 654 of the EGF receptor in regulation of EGF-stimulated activation of phospholipase C was investigated. NIH-3T3 cells expressing the normal human EGF receptor or expressing EGF receptor in which an alanine residue had been substituted at residue 654 of the receptor were used. Addition of EGF to cells expressing wild-type receptor induced a rapid, but transient, increase in phosphorylation of threonine 654. EGF addition also caused the rapid accumulation of inositol phosphates in these cells. EGF-stimulated accumulation of inositol phosphates was significantly higher in cells expressing Ala-654 receptors compared to control cells. Treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulated phosphorylation of threonine 654 to a greater degree than EGF, completely inhibited EGF-dependent inositol phosphate accumulation in cells expressing wild-type receptor, but caused only a 20-30% inhibition in Ala-654 expressing cells. EGF stimulated phosphorylation of phospholipase C-gamma on serine and tyrosine residues in cells expressing wild-type of Ala-654 receptors. However, TPA treatment of cells inhibited EGF-induced tyrosine phosphorylation of phospholipase C-gamma only in cells expressing wild-type receptors. Similarly, TPA inhibited tyrosine-specific autophosphorylation of the EGF receptor and tyrosine phosphorylation of several other proteins in wild-type receptor cells, but not in Ala-654 cells. TPA treatment abolished high affinity binding of EGF to cells expressing wild-type receptors, while decreasing the number of high affinity binding sites 20-30% in Ala-654 cells. These data suggest that phosphorylation of threonine 654 can regulate early events in EGF receptor signal transduction such as phosphoinositide turnover, probably through a feedback mechanism involving protein kinase C. Subsequent dephosphorylation of threonine 654 could reactivate the EGF receptor for participation in later signaling events.     

MICAL-like1 mediates epidermal growth factor receptor endocytosis

Small GTPase Rabs are required for membrane protein sorting/delivery to precise membrane domains. Rab13 regulates epithelial tight junction assembly and polarized membrane transport. Here we report that Molecule Interacting with CasL (MICAL)-like1 (MICAL-L1) interacts with GTP-Rab13 and shares a similar domain organization with MICAL. MICAL-L1 has a calponin homology (CH), LIM, proline rich and coiled-coil domains. It is associated with late endosomes. Time-lapse video microscopy shows that green fluorescent protein-Rab7 and mcherry-MICAL-L1 are present within vesicles that move rapidly in the cytoplasm. Depletion of MICAL-L1 by short hairpin RNA does not alter the distribution of a late endosome/lysosome-associated protein but affects the trafficking of epidermal growth factor receptor (EGFR). Overexpression of MICAL-L1 leads to the accumulation of EGFR in the late endosomal compartment. In contrast, knocking down MICAL-L1 results in the distribution of internalized EGFR in vesicles spread throughout the cytoplasm and promotes its degradation. Our data suggest that the N-terminal CH domain associates with the C-terminal Rab13 binding domain (RBD) of MICAL-L1. The binding of Rab13 to RBD disrupts the CH/RBD interaction, and may induce a conformational change in MICAL-L1, promoting its activation. Our results provide novel insights into the MICAL-L1/Rab protein complex that can regulate EGFR trafficking at late endocytic pathways.     

Nerve growth factor-dependent activation of the small GTPase Rin

The Rit and Rin proteins comprise a distinct and evolutionarily conserved subfamily of Ras-related small GTPases. Although we have defined a role for Rit-mediated signal transduction in the regulation of cell proliferation and transformation, the function of Rin remains largely unknown. Because we demonstrate that Rin is developmentally regulated and expressed in adult neurons, we examined its role in neuronal signaling. In this study, we show that stimulation of PC6 cells with either epidermal growth factor or nerve growth factor (NGF) results in rapid activation of Rin. This activation correlates with the onset of Ras activation, and dominant-negative Ras completely inhibits Rin activation induced by NGF. Further examination of Ras-mediated Rin activation suggests that this process is dependent upon neuronally expressed regulatory factors. Expression of mutationally activated H-Ras fails to activate Rin in non-neuronal cells, but results in potent stimulation of Rin-GTP levels in a variety of neuronal cell lines. Furthermore, although constitutively activated Rin does not induce neurite outgrowth on its own, both NGF-induced and oncogenic Ras-induced neurite outgrowth were inhibited by the expression of dominant-negative Rin. Together, these studies indicate that Rin activation is a direct downstream effect of growth factor-dependent signaling in neuronal cells and suggest that Rin may function to transduce signals within the mature nervous system.     

Transcytosis of epidermal growth factor. The epidermal growth factor receptor mediates uptake but not transcytosis

Madin-Darby canine kidney (MDCK) cells polarize and generate distinct apical and basolateral membrane domains when grown on permeable filter supports. Under these conditions, they transcytose fluid-phase markers. Recently, receptor-mediated transcytosis of epidermal growth factor (EGF) across MDCK cells has been reported (Maratos-Flier, E., Kao, C.-Y. Y., Verdin, E. M., and King, G. L. (1987) J. Cell Biol. 105, 1595-1601). We examined the role of the EGF receptor in this process. Transcytosis of EGF occurred only in the basolateral-to-apical direction, was time-dependent, and inhibited by the addition of unlabeled EGF in a concentration-dependent manner. In contrast to previous work, we found that only about 5% of basolaterally bound EGF was transported to the apical chamber. The half-time of transport was 90 min. A mutant cell line of MDCK, MDCKII-RCAr, was used to study the expression of the EGF receptor. Cell surface glycoproteins of these mutant cells can be efficiently labeled with [3H]galactose by exogalactosylation. The EGF receptor was found to be expressed only on the basolateral surface. Addition of EGF to the basolateral medium resulted in rapid internalization and degradation of the receptor. Testing directly for transcytosis of basolateral glycoproteins, we detected several proteins transported across the cell. The EGF receptor, however, was not among this group of proteins. Taking these results together, we suggest the following model. Internalization of EGF on the basolateral surface is mediated by the EGF receptor. EGF dissociates from the receptor in an endocytic compartment. A fraction of the EGF is then diverted nonselectively to the transcytotic pathway, as found for other fluid-phase markers previously (Bomsel, M., Prydz, K., Parton, R. G., Gruenberg, J., and Simons, K. (1989) J. Cell Biol. 109, 3243-3258.