COMMUNICATION
High-affinity Binding of Epidermal Growth Factor (EGF) to EGF Receptor Is Disrupted by Overexpression of Mutant Dynamin (K44A)*

Tove RingerikeDagger , Espen StangDagger , Lene E. JohannessenDagger , Dagny Sandnes§, Finn Olav Levyparallel , and Inger Helene MadshusDagger **

From the Dagger  Institute of Pathology,  MSD Cardiovascular Research Center, and parallel  Institute for Surgical Research, University of Oslo, The National Hospital, N-0027 Oslo and the § Department of Pharmacology, University of Oslo, P. O. Box 1057, N-0316 Oslo, Norway

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results & Discussion
References

Activation of the epidermal growth factor receptor (EGFR) kinase was analyzed in cells conditionally defective for clathrin-dependent endocytosis by overexpression of mutant dynamin (K44A). EGF-induced autophosphorylation of the EGFR on ice was strongly reduced in cells overexpressing mutant dynamin, and consistently, binding analyses showed that high-affinity EGFRs were lost. In the absence of mutant dynamin the cells displayed both high- and low-affinity EGFR. At 4 °C EGF-EGFR localized mainly outside coated pits regardless of expression of mutant dynamin. However, also low-affinity EGFR efficiently moved to coated pits upon incubating cells at 37 °C. Thus, expression of mutant dynamin disrupts high-affinity binding of EGF, but not ligand-induced recruitment of EGFR to clathrin-coated pits.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results & Discussion
References

Binding of epidermal growth factor (EGF)1 to the EGF receptor (EGFR) effects recruitment of EGFR to clathrin-coated pits, and eventually activated EGFR is delivered to lysosomes and degraded (for review, see Refs. 1 and 2). Binding of EGF furthermore results in autophosphorylation of the EGFR, and several authors have described tyrosine-phosphorylated EGFR contained in endosomes (for review, see Ref. 3). The adaptor proteins growth factor receptor-binding protein 2 (Grb2) and SH2-domain-containing alpha 2 collagen-related protein (Shc), as well as Son of Sevenless protein (SOS) (all involved in activation of Ras) were shown to be enriched in the endosomal fraction of EGF-treated hepatocytes (4). Based on these observations, several authors have suggested that endocytosed EGFR is actively engaged in signal transduction.

The GTPase dynamin is required for clathrin-coated vesicle formation (for review, see Refs. 5 and 6), and a conditional defect in endocytosis is imposed by the regulated expression of the K44A mutant form of dynamin (7). HeLa cells expressing the K44A dynamin mutant are conditionally and specifically defective in clathrin-mediated endocytosis (7), and it was reported recently that EGF-induced signaling was altered under these conditions (8). This raises the possibility that trafficking might regulate specificity of signaling. However, dynamin is a protein reported to interact with a number of cellular proteins, among them proteins directly involved in signal transduction (for review, see Ref. 9). We therefore set out to investigate whether the altered signaling observed upon overexpression of mutant dynamin was indeed caused by inhibition of clathrin-dependent endocytosis directly or whether overexpression of mutant dynamin per se affected signaling. To our surprise we found that upon overexpression of mutant dynamin high-affinity EGF binding was disrupted. Overexpression of mutant dynamin therefore directly affects signaling by EGF.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

Materials-- Human recombinant EGF was from Bachem Feinchemikalien AG, Budendorf, Switzerland. All chemicals were from Sigma unless otherwise indicated.

Cells-- HeLa cells transfected with a plasmid encoding mutant (K44A) or wild-type dynamin 1, where the promoter is negatively controlled by tetracycline (7), were generously provided by Dr. Sandra L. Schmid, The Scripps Research Institute. The cells were grown in Costar 3275 flasks (Costar Corp., Cambridge, MA), and the medium used was Dulbecco's modified Eagle's medium (BioWhittaker, Walkersville, MD) containing 400 µg/ml geneticin (Life Technologies, Inc., Paisley, UK), 200 ng/ml puromycin, 2 mM L-glutamine (BioWhittaker), and 1 × penicillin/streptomycin/fungizone mixture (BioWhittaker) supplemented with 10% (v/v) fetal bovine serum (BioWhittaker). Cells were seeded at a density of 15,000 cells/cm2 in disposable microtiter plates and grown for 24 h at 37 °C in the presence (uninduced cells) or absence (induced cells) of 1 µg/ml tetracycline. For the next 24 h, cells were serum-starved by incubation in the same medium with only 0.5% fetal bovine serum.

Western Blotting-- Cells in 12-well microtiter plates were incubated as indicated in legends to figures and subsequently subjected to Western blot analysis. The cells were lysed in lysis buffer (10 mM Tris-HCl (pH 6.8), 5 mM EDTA, 50 mM NaF, 30 mM sodium pyrophosphate, 2% (w/v) sodium dodecyl sulfate (SDS) (Bio-Rad), 1% (v/v) beta -mercaptoethanol, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 4% (v/v) glycerol, and 0.005% (w/v) bromphenol blue) on ice for 10 min, heated at 95 °C for 10 min, and centrifuged at 20 000 × g for 15 min before the supernatant fraction was subjected to SDS-polyacrylamide gel electrophoresis, and the proteins were electrotransferred to nitrocellulose (NitroBind, Micron Separations Inc., Westborough, MA). Antibodies used were sheep anti-EGFR (Life Technologies, Inc.), peroxidase-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA), mouse anti-phosphotyrosine (Upstate Biotechnology, Lake Placid, NY), and peroxidase-conjugated anti-sheep IgG (Jackson ImmunoResearch Laboratories Inc.). The reactive proteins were detected using an enhanced chemiluminescence (ECL) method (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Scatchard Plot-- Binding of 125I-EGF (Amersham Pharmacia Biotech) to whole cells was measured by incubating cells in 24-well microtiter plates for 3 h on ice in 50 µl of minimal essential medium without sodium bicarbonate (Life Technologies, Inc.) with 0.1% bovine serum albumin (BSA) and increasing concentrations of 125I-EGF in the absence (total binding) or presence (nonspecific binding) of unlabeled EGF (200 nM). The incubation medium was carefully removed and transferred to tubes for gamma  counting. The cells were washed three times with ice-cold phosphate-buffered saline and hydrolyzed in 0.3 ml of 1 M NaOH. The hydrolyzed cells were transferred to tubes for gamma  counting. The specific binding data (total binding minus nonspecific binding) were analyzed by nonlinear curve fitting to equations describing one (Y = Bmax × X/(Kd + X)) or two (Y = Bmax1 × X/(Kd1 + X) Bmax2 × X/(Kd2 + X)) binding sites using Prism 2.01 (GraphPad Software). For the two-site model, the data were weighted by 1/Y2. The observed data and the theoretical curves were plotted according to Scatchard (10) using Sigma-Plot scientific graphing software (SPSS, Jandel Scientific, GmbH, Erkrath, Germany).

Immunoelectron Microscopy-- Cells were grown in 25-cm2 flasks. To label endosomes with an electron-dense marker, cells were incubated with BSA-coated 5-10 nm colloidal gold (11) in the medium for 30 min at 37 °C. Following removal of the BSA-gold-containing medium cells were chilled on ice and incubated with 10-8 M EGF on ice for 30 min. At the end of the incubation period the cells were washed with ice-cold phosphate-buffered saline, fixed, and processed for cryosections and immunogold labeling (12) either immediately or after chase in BSA-gold- and EGF-free medium at 37 °C. The sections were labeled using sheep anti-EGFR antibodies (Life Technologies, Inc.) followed by goat anti-sheep IgG-coated 18 nm colloidal gold (Jackson ImmunoResearch Laboratories Inc.).

Determination of IP3 Mass-- Medium was removed from cells grown in six-well disposable plates, and 600 µl of 0.4 M perchloric acid was added. The cells were scraped from the plates with a rubber policeman, and the contents of each well was transferred to glass tubes. The samples were left on ice for 30 min before centrifugation and neutralization of the supernatant with 4 M KOH, 1 M Tris, 60 mM EDTA, in the presence of Universal Indicator. The IP3 content of the neutralized supernatant was determined by a competitive radioligand binding assay, using a binding protein prepared from bovine adrenal glands (13). Results are presented as mean ± S.E. of three experiments, each performed with three to six wells.

    RESULTS AND DISCUSSION
Top
Abstract
Introduction
Procedures
Results & Discussion
References

EGF-dependent EGFR Kinase Activity in the Absence of Endocytosis-- As it was reported that EGF-induced signaling was altered by arrest of clathrin-dependent endocytosis (8), we studied EGF-dependent EGFR kinase activity in the absence of endocytosis. We chose to compare the same transfected cell line with or without overexpression of mutant (K44A) dynamin 1 to avoid the risk of clone variations. EGF was added to transfected HeLa cells with or without tetracycline (without or with overexpression of K44A dynamin) on ice, because endocytosis does not occur on ice. We found that cells overexpressing K44A dynamin contained more EGFR than cells not overexpressing K44A dynamin (Fig. 1A), but surprisingly we found that the EGF-induced EGFR autophosphorylation in cells overexpressing K44A dynamin was strongly reduced (Fig. 1B). Immunoprecipitation experiments demonstrated that the phosphorylated protein of molecular mass 170 kDa was indeed the EGFR (data not shown). The specific reduction in EGFR autophosphorylation on ice in cells overexpressing K44A dynamin is stronger than apparent from Fig. 1B, considering the increased amount of EGFR in cells overexpressing K44A dynamin (Fig. 1A).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1.   EGF-dependent tyrosine phosphorylation of EGFR on ice is inhibited in HeLa cells overexpressing mutant dynamin (K44A). Serum-starved cells grown in the presence or absence of tetracycline as described under "Experimental Procedures" were treated with or without 3.5 × 10-9 M EGF for 10 min on ice, lysed, and subjected to Western blotting as described under "Experimental Procedures." A, EGF-treated uninduced cells (lane 1) and EGF-treated cells overexpressing K44A dynamin (lane 2) were Western blotted with an antibody to EGFR. B, uninduced cells (lanes 1 and 2) and cells overexpressing K44A dynamin (lanes 3 and 4) incubated without (lanes 1 and 3) or with (lanes 2 and 4) EGF were Western blotted with an antibody to phosphotyrosine.

Analysis of EGF Binding in HeLa Cells with or without Overexpression of K44A Dynamin-- We measured binding of radiolabeled EGF to EGFR in cells with or without tetracycline and analyzed the binding data by nonlinear curve fitting and Scatchard analyses. As shown in Fig. 2A, the data obtained from cells grown in the presence of tetracycline gave a curvilinear Scatchard plot, demonstrating the presence of two affinity classes of receptors. The high-affinity class of the EGFR (4.2 ± 1.4% of total EGFR) had an apparent Kd of 8 ± 3 pM, and the low-affinity class of EGFR had an apparent Kd of 400 ± 190 pM. Analysis of EGF binding in cells overexpressing K44A dynamin, however, revealed a linear Scatchard plot (Fig. 2B), indicating the presence of only one receptor class. This class of receptors had an apparent Kd of 700 ± 140 pM, not significantly different from the low affinity class observed in wild-type cells. Overexpression of wild-type dynamin did not reduce the level of the high affinity class of the EGFR (data not shown).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2.   Scatchard analysis of binding of 125I-EGF to uninduced HeLa cells (A) and to HeLa cells overexpressing K44A dynamin (B). The cells were serum-starved and grown in the presence (A) or absence (B) of tetracycline. Binding of different dilutions of 125I-EGF in the absence or presence of unlabeled EGF to whole cells was measured and Scatchard analysis done as described under "Experimental Procedures." The figure shows one representative experiment of three, yielding similar results. Pooled results (mean ± S.D., n = 3): A, KdH = 8 ± 3 pM, BmaxH = 3,000 ± 1000 sites/cell (4.2 ± 1.4%), KdL = 400 ± 190 pM, BmaxL = 68,000 ± 16,000 sites/cell. B, Kd = 700 ± 140 pM, Bmax = 88,000 ± 20,000 sites/cell.

Subcellular Localization of EGFR with or without Overexpression of K44A Dynamin-- Immunoelectron microscopy studies (9) showed that in the absence of tetracycline the cells contained more EGFR at the plasma membrane than did uninduced cells (data not shown), consistent with the results in Fig. 1A and in Fig. 2. When EGF was added to cells on ice, the EGFR localized to smooth, uncoated, uninvaginated regions of the plasma membrane regardless of whether K44A dynamin was expressed or not (Fig. 3, A, F, and G). Only rarely did the EGFR localize to coated pits. Chasing at 37 °C, however, caused a rapid relocalization of the EGFR. After 10 min at 37 °C the receptor in uninduced cells had moved into coated pits (Fig. 3B) and been internalized into coated vesicles Fig. 3C and BSA-gold containing early endosomes (Fig. 3D) as well as later endosomes with the morphology of multivesicular bodies (Fig. 3E). Some labeling, probably representing newly synthesized EGFR, was observed in the Golgi region of the cells (Fig. 3D). Expression of K44A dynamin prevents the formation of clathrin-coated vesicles, but not the initial formation of clathrin-coated pits (7). In cells overexpressing K44A dynamin no labeling was found in endosomes, instead the EGFR localized to the blind bulb-shaped end of long, narrow tubular invaginations of the plasma membrane (Fig. 3, H and I). At the end of the invaginations, where the EGFR localized, there was a clear cytoplasmic coat previously shown to consist of clathrin (14). Some labeling was also found in what looked like clathrin-coated vesicles (Fig. 3, J and K). However, we interpret these structures to be clathrin-coated pits, because the plane of sectioning will not always allow visualization of the narrow, tubular connection with the plasma membrane. These data clearly show that even the low-affinity class of EGFR is efficiently recruited to clathrin-coated pits. The observation that in contrast to in uninduced cells the EGFR in cells overexpressing K44A dynamin could not be detected in endosomes is consistent with the reported inhibition of EGFR endocytosis in these cells (7) and with our observation of efficient arrest of clathrin-dependent endocytosis as measured with radiolabeled transferrin (data not shown). The general impression, when comparing cells with or without overexpression of K44A dynamin, was that the number of coated structures was highly increased in cells overexpressing K44A dynamin. In addition to their long, tubular neck these coated pits often showed a more irregular shape compared with coated pits/vesicles seen in wild-type cells. Coated structures were often found in groups, and often two or more coated structures seemed interconnected, seen as figure eight-shaped patterns (Fig. 3, I and K). More precise morphological studies are required to elucidate the reason for this, but our observations indicate that more than one coated pit can exist at the end of each tubule.


View larger version (126K):
[in this window]
[in a new window]
 
Fig. 3.   Immunocytochemical localization of EGFR. HeLa cells grown in the presence of tetracycline (A-E) or in the absence of tetracycline (F-K) had endocytosed 5-10 nm BSA-gold before incubation with EGF on ice. Following binding of 10-8 M EGF the cells were either fixed immediately (A, F, and G) or chased for 10 min in EGF-free prewarmed medium (B-E and H-K). Immunocytochemical labeling, as described under "Experimental Procedures," showed that immediately after binding of EGF on ice the EGFR (large arrowheads) localized to smooth regions of the plasma membrane both in the presence of tetracycline (A) and in the absence of tetracycline (F and G). Note the atypical morphology of the coated pits with long tubular plasma membrane connections (small arrowheads) in K44A dynamin expressing cells (F-I). After 10-min chase at 37 °C the EGFR in uninduced cells had moved into coated pits (cp) (B) and been internalized into coated vesicles (C) and BSA-gold containing early endosomes (e.e.) (D) as well as multivesicular bodies (mvb) (E). Some labeling was observed in the Golgi region (g) of the cells (D). In cells expressing K44A dynamin the EGFR localized to the coated end of the long tubular invaginations (H and I). Some labeling was also found in what look like clathrin coated vesicles (J and K); note the irregular morphology of the coated pits, vesicles shown (outlined) in I and K. The large discontinuity in the outlining indicates the possible position of the tubular plasma membrane connection. Bars represent 100 nm.

Activity of Phospholipase C gamma  (PLCgamma ) in Uninduced Cells and in Cells Overexpressing K44A Dynamin-- A possible way whereby the specific kinase activity as well as the affinity of the EGFR could be negatively modulated is by phosphorylation on Thr654 in the cytoplasmic part of the EGFR, a substrate of protein kinase C (PKC) (15). PKC can be activated by diacylglycerol, produced by activation of PLCgamma . As PLCgamma in the absence of EGF was reportedly more tyrosine-phosphorylated in cells overexpressing K44A dynamin than in uninduced cells (8), we determined IP3 mass in HeLa cells with or without overexpression of K44A dynamin. The IP3 mass in the presence of tetracycline was 34.1 ± 2.8 pmol/mg of protein, and in the absence of tetracycline the IP3 mass was 35.0 ± 0.5 pmol/mg of protein. The hyperphosphorylation of PLCgamma did therefore not result in increased IP3 production, thus excluding the possibility that the hyperphosphorylation of PLCgamma caused increased formation of diacylglycerol and thereby increased activation of PKC. We cannot exclude the possibility that other phospholipases are activated in cells overexpressing K44A dynamin.

Our data show that HeLa cells overexpressing K44A dynamin have lost high affinity binding of EGF to the EGFR. Because it has been reported that high-affinity receptors are required and sufficient for all EGF-induced responses (16, 17), some of the previously reported changes in EGF-dependent signaling in cells overexpressing mutant dynamin (8) can be explained by the lack of high-affinity EGF-binding. The molecular background for the high- and low-affinity state of the EGFR is still unclear. The observation that activation of PKC by phorbol esters converted high-affinity EGFR to low-affinity EGFR (18) indicated that the intracellular part of the EGFR regulates its affinity for EGF. Cells expressing EGFR lacking the entire intracellular domain or the C-terminal 63 amino acids possessed only low-affinity receptors (19, 20). Deletion of the major autophosphorylation sites (21) or PKC phosphorylation sites (22) did not alter the affinity of the receptor, neither did inactivation of the tyrosine kinase by the introduction of a point mutation (19, 20) or by an insertion of 4 amino acids (23, 24). High-affinity EGFR have been demonstrated to be cytoskeleton-associated, and the EGFR binds specifically to actin (25-29). However, it was recently demonstrated that removal of the actin-binding site did not affect the affinity of EGFR for EGF (30). Instead, another domain of the intracellular part of the receptor located within the kinase domain was found to regulate the affinity for EGF, and the possibility of an interaction between the intracellular part of the EGFR and an affinity-modulating protein was suggested recently (30). A potential affinity-modulating protein might behave differently in cells overexpressing wild-type and mutant (K44A) dynamin, because overexpression of wild-type dynamin did not reduce high-affinity EGF-EGFR interaction (data not shown).

We could not detect direct interaction between overexpressed mutant or wild-type dynamin and the EGFR (data not shown). A possible explanation is therefore that mutant dynamin affects the EGFR affinity by sequestration of an affinity-modulating molecule. Such a molecule could be one of a variety of macromolecules demonstrated to bind dynamin, including microtubules (31), acidic phospholipids (32), and several SH3 (Src homology domain-3) domain-containing intracellular signal-transducing proteins (9).

Vieira et al. (8) observed that ligand-induced EGFR signaling is attenuated in cells overexpressing K44A dynamin. They explained this finding by the arrested endocytosis of the ligand-bound EGFR under these conditions, thus implying that the EGFR must traffic to endosomes for proper signaling to occur. Although confirming the finding that ligand-induced signaling is attenuated in cells overexpressing K44A dynamin, we have demonstrated that the EGF-induced kinase activity is strongly reduced in cells overexpressing K44A dynamin compared with in uninduced cells, even when no endocytosis occurs in either cell line. The reason for this is the fact that overexpression of mutant dynamin per se affects the affinity state of the EGFR. Thus, in conclusion, other factors than the arrested endocytosis might explain the reduced EGFR signaling in cells overexpressing K44A dynamin.

    ACKNOWLEDGEMENT

We thank Sandra L. Schmid for kindly providing HeLa cells transfected with wild-type or mutant (K44A) dynamin.

    FOOTNOTES

* This work was supported by The Norwegian Cancer Society, The National Council for Science and the Humanities, Medinnova, Nordic Insulin Foundation Committee, The Anders Jahre's Foundation for the Promotion of Science, Blix Legacy and Bruuns Legacy.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Institute of Pathology, The National Hospital, N-0027 Oslo, Norway. Tel.: 47-22868609; Fax: 47-22112261; E-mail: i.h.madshus{at}labmed.uio.no.

1 The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; BSA, bovine serum albumin; PLCgamma , phospholipase C gamma ; PKC, protein kinase C; IP3, inositol 1,4,5-trisphosphate.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

  1. Haigler, H. T., McKanna, J. A., and Cohen, S. (1979) J. Cell Biol. 81, 382-395[Abstract]
  2. Sorkin, A., and Waters, C. M. (1993) Bioessays 15, 375-382[Medline] [Order article via Infotrieve]
  3. Bergeron, J. J. M., Di Guglielmo, G. M., Baass, P. C., Authier, F., and Posner, B. I. (1995) Biosci. Rep. 15, 411-418[Medline] [Order article via Infotrieve]
  4. Di Guglielmo, G. M., Baass, P. C., Ou, W.-J., Posner, B. I., and Bergeron, J. J. M. (1994) EMBO J. 13, 4269-4277[Abstract]
  5. Liu, J.-P., and Robinson, P. J. (1995) Endocr. Rev. 16, 590-607[Medline] [Order article via Infotrieve]
  6. Warnock, D. E., and Schmid, S. L. (1996) Bioessays 18, 885-893[Medline] [Order article via Infotrieve]
  7. Damke, H., Baba, T., Warnock, D. E., and Schmid, S. L. (1994) J. Cell Biol. 127, 915-934[Abstract]
  8. Vieira, A. V., Lamaze, C., and Schmid, S. L. (1996) Science 274, 2086-2089[Abstract/Free Full Text]
  9. McClure, S. J., and Robinson, P. J. (1996) Mol. Membr. Biol. 13, 189-215[Medline] [Order article via Infotrieve]
  10. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51, 660-672
  11. Slot, J. W., and Geuze, H. J. (1985) Eur. J. Cell Biol. 38, 87-93[Medline] [Order article via Infotrieve]
  12. Griffiths, G., McDowall, A., Bach, R., and Dubochet, J. (1984) J. Ultrastruct. Res. 89, 65-78[Medline] [Order article via Infotrieve]
  13. Palmer, S., Hughes, K. T., Lee, D. Y., and Wakelam, M. J. (1989) Cell Signalling 1, 147-156[Medline] [Order article via Infotrieve]
  14. Takei, K., McPherson, P. S., Schmid, S. L., and De Camilli, P. (1995) Nature 374, 186-190[Medline] [Order article via Infotrieve]
  15. Hunter, T., Ling, N., and Cooper, J. A. (1984) Nature 311, 480-483[Medline] [Order article via Infotrieve]
  16. Defize, L. H. K., Boonstra, J., Meisenhelder, J., Kruijer, W., Tertoolen, L. G. J., Tilly, B. C., Hunter, T., van Bergen en Henegouwen, P. M. P., Moolenaar, W. H., and de Laat, S. W. (1989) J. Cell Biol. 109, 2495-2507[Abstract]
  17. Bellot, F., Moolenaar, W., Kris, R., Mirakhur, B., Verlaan, I., Ullrich, A., Schlessinger, J., and Felder, S. (1990) J. Cell Biol. 110, 491-502[Abstract]
  18. King, A. C., and Cuatrecasas, P. (1982) J. Biol. Chem. 257, 3053-3060[Free Full Text]
  19. Livneh, E., Prywes, R., Kashles, O., Reiss, N., Sasson, I., Mory, Y., Ullrich, A., and Schlessinger, J. (1986) J. Biol. Chem. 261, 12490-12497[Abstract/Free Full Text]
  20. Prywes, R., Livneh, E., Ullrich, A., and Schlessinger, J. (1986) EMBO J. 5, 2179-2190[Abstract]
  21. Decker, S. J. (1993) J. Biol. Chem. 268, 9176-9179[Abstract/Free Full Text]
  22. Countaway, J. L., McQuilkin, P., Girones, N., and Davis, R. J. (1990) J. Biol. Chem. 265, 3407-3416[Abstract/Free Full Text]
  23. Honegger, A. M., Schmidt, A., Ullrich, A., and Schlessinger, J. (1990) J. Cell Biol. 110, 1541-1548[Abstract]
  24. van Belzen, N, Spaargaren, M., Verkleij, A. J., and Boonstra, J. (1990) J. Cell. Physiol. 145, 365-375[Medline] [Order article via Infotrieve]
  25. Landreth, G. E., Williams, L. K., and Rieser, G. D. (1985) J. Cell Biol 101, 1341-1350[Abstract]
  26. Wiegant, F. A. C, Blok, F. J., Defize, L. H. K., Linnemans, W. A. M., Verkleij, A. J., and Boonstra, J. (1986) J. Cell Biol. 103, 87-94[Abstract]
  27. Roy, L. M., Gittinger, C. K., and Landreth, G. E. (1989) J. Cell. Physiol. 140, 295-304[Medline] [Order article via Infotrieve]
  28. van Bergen en Henegouwen, P. M. P., Defize, L. H. K., de Kroon, J., van Damme, H., Verkleij, A. J., and Boonstra, J. (1989) J. Cell. Biochem. 39, 455-465[Medline] [Order article via Infotrieve]
  29. van Bergen en Henegouwen, P. M. P., den Hartigh, J. C., Romeyn, P., Verkleij, A. J., and Boonstra, J. (1992) Exp. Cell Res 199, 90-97[Medline] [Order article via Infotrieve]
  30. van der Heyden, M. A. G., Nievers, M., Verkleij, A. J., Boonstra, J., and van Bergen en Henegouwen, P. M. P. (1997) FEBS Lett. 410, 265-268[CrossRef][Medline] [Order article via Infotrieve]
  31. Herskovits, J. S., Shpetner, H. S., Burgess, C. C., and Vallee, R. B. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11468-11472[Abstract]
  32. Tuma, P. L., Stachniak, M. C., and Collins, C. A. (1993) J. Biol. Chem. 268, 17240-17246[Abstract/Free Full Text]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.