Identification of an epidermal growth factor receptor homologue in trypanosomes

Molecular and Biochemical Parasitology, 36 (1989) 51-60 Elsevier

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Identification of an epidermal growth factor receptor h o m o l o g u e in trypanosomes Geoff Hide, Alex Gray, Catherine M. Harrison and Anorew Tait Wellcome Unit of Molecular Parasitology, Department of Veterinary Parasitology, University of Glasgow, Glasgow, U.K.

(Received 4 January 1989; accepted 13 March 1989)

Considerable advances have been made in our understanding of cell growth regulation in mammalian cells. In particular, studies on transformed and normal cells have highlighted the contribution of growth factor-related control mechanisms in cell growth regulation. We set out to investigate whether host growth factors are involved in the growth regulation of the parasitic protozoan Trypanosoma brucei. We demonstrate that antibodies to the mammalian epidermal growth factor (EGF) receptor bind to the trypanosome T. brucei and, that these antibodies recognise a surface polypeptide of 135 kDa. This polypeptide is one of only two polypeptides in parasite extracts that bind EGF. Furthermore, EGF modifies protein kinase activity and growth rate of trypanosomes in vitro. These results lead to the conclusion that T. brucei has a surface growth factor receptor with considerable homology to the EGF receptor, and raise the possibility that growth factor interactions similar to those found in mammalian cells are involved in cell growth regulation in trypanosomes. Key words: Trypanosome; EGF receptor; Protein kinase; Trypanosome growth

Introduction A n u m b e r o f insect v e c t o r - t r a n s m i t t e d parasitic p r o t o z o a grow within the b l o o d s t r e a m and tissues of their m a m m a l i a n hosts and in the gut and tissues of the insect vector. While it is clear that the i m m u n e system in the m a m m a l i a n host is a m a j o r negative g r o w t h regulator, little is k n o w n of the interactions with o t h e r host- or vector-derived molecules and their role in regulating growth or differentiation of the parasite. T h e African t r y p a n o s o m e T r y p a n o s o m a brucei u n d e r g o e s a series of m o r p h o l o g i c a l l y and metabolically distinct stages [1] during the c o m p l e t i o n of its life cycle t h r o u g h the m a m m a l i a n host and the vector (tsetse fly). T h e factors which trigger these multiple differentiation events r e m a i n largely unCorrespondence address: G. Hide, Wellcome Unit of Molecular Parasitology, Dept. of Veterinary Parasitology, University of Glasgow, Bearsden Road, Glasgow, G61 1QH, U.K. Abbreviations: EGF, epidermal growth factor; LDL, lowdensity lipoprotein; IFA, indirect immunofluorescence assay; SDS, sodium dodecyl sulphate; DSS, discuccimidyl suberate.

k n o w n , although a role for lectins in the events in the insect stages has b e e n established [2,3] and a role for m a m m a l i a n host factors in the slender to s t u m p y differentiation has b e e n suggested [4]. These observations suggest that host-derived molecules have a role in the differentiation and growth of the parasite. In both b l o o d s t r e a m and insect stage t r y p a n o s o m e s grown in culture, there is a r e q u i r e m e n t for foetal bovine serum for growth [5-9]. F u r t h e r m o r e , addition of cells such as fibroblasts, epithelial cells [5,6] or adipocytes [10] can produce changes in growth, implying that m a m m a l i a n host-derived c o m p o n e n t s are involved in determining growth rate. T h e requirem e n t for foetal bovine serum could be explained on a nutritional basis in the light of recent observations d e m o n s t r a t i n g the presence of b o t h transferrin and L D L receptors on the surface of the t r y p a n o s o m e [11]; however, the role of host-derived molecules from co-cultured mammalian cells in growth regulation and differentiation remains unclear. With this b a c k g r o u n d in mind, coupled with preliminary observations that the t r y p a n o s o m e

0166-6851/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

52 genome contained sequences homologous to the src and erbB oncogenes (B.R. Shiels, personal communication), we looked for evidence of the presence of growth factor receptors and growth factor interactions in T. brucei. Our investigation has concentrated on the identification of an E G F receptor homologue and, from the data presented, we propose that a polypeptide with significant homology to the mammalian E G F receptor exists on the trypanosome surface which interacts with EGF. Materials and Methods

Preparation of trypanosomes. Bloodstream trypanosomes were prepared by inoculation of MF1 mice with a cloned T. b. brucei stock (TREU869) and separation from murine blood by anion exchange [12]. This method completely removes contaminating host blood cells. The trypanosomes were then washed twice in phosphate-buffered saline (PBS) pH 8.0, containing 1% glucose (PBSG), Procyclic trypanosomes were grown in culture [13] and the trypanosomes were concentrated by centrifugation and then washed twice in PBSG. Antibodies. Antibodies to the purified E G F receptor were obtained from Stanley Cohen; these are described in [14]. Antibodies made to synthetic peptides representing the external domain of the EGF receptor (2075, 2046, OA-11-852) and the v-erbB oncogene product (2220) were provided by Cambridge Research Biochemicals (CRB). Antibody OA-11-852, commercially available from CRB, has been characterized using A-431 cells and shown to specifically recognise the human E G F receptor (CRB Technical Sheet OA-11-852). Indirect immunofluorescence assay. Indirect immunofluorescence assays (IFAs) were carried out using standard procedures [15] on ethanol-fixed trypanosomes. Immune precipitation. Bloodstream or procyclic trypanosomes were surface-labelled with biotin by incubation for 1 h at room temperature in PBSG containing 0.03% of D-biotin-N-hydroxy-succi-

nimidester (BNHS biotin; ref. 16). After 3 washes in PBSG, the cells were suspended in 1 ml of PBSG and lysed by the addition of Nonidet P40 to 1%. The lysate was then preabsorbed with a 6% solution of fixed Staphylococcus aureus [17] in 50 mM Tris, pH 8.0, 0.15 M NaC1, 5 mM EDTA, 10 mM sodium azide and 1% Triton X100 (NET buffer) for 30 min. The resulting supernatant was incubated overnight at 4°C with the addition of 50 jxl of NET, 20 mg m1-1 bovine serum albumin and 12 ~1 of antiserum diluted to give the desired final concentration (usually 1 in 100 dilution). Washed, fixed S. aureus cells, (from 80 ixl of a 10% suspension in NET buffer) were added to the antibody/antigen mixture and incubated for 60 min at room temperature. The S. aureus cell suspension with the attached antigen/antibody complex was centrifuged, the pellet washed 5 x in NET buffer and then solubilised by first vortexing in 20 txl of 9 M urea, followed by the addition of 10 ~xl of 10% SDS and finally made up to 100 Ixl in SDS sample buffer [18] without mercaptoethanol. The immune precipitates were analysed by polyacrylamide gel electrophoresis and blotting onto nitrocellulose. Polyacrylamide gel electrophoresis and blotting. Polyacrylamide gel electrophoresis was carried out using standard procedures [18] under non-reducing conditions. Either 10% or 6-15% gradient gels were used (see Results). The proteins were transferred to nitrocellulose [19], which was blocked for 1 h in 10 mM Tris, pH 7.2, 0.9% NaC1 (TS) containing 1% Tween 20, washed 3 times with TS containing 0.05% Tween 20, and finally incubated with 125I-labelled streptavidin (10 cpm ml-1 final concentration) in the same buffer. Filters were washed 5 x in TS containing 0.05% Tween and then exposed to X-OMAT film for 1-3 days. Preparation of trypanosomes membranes. Membrane enriched fractions of trypanosomes were prepared as previously described [20]. Kinase assays. Kinase assays were carried out on either 100 txg of a membrane enriched fraction or directly with immune complexes in situ on the S. aureus cells. The assays were carried out, at 0°C, in 10 mM Hepes, pH 7.4, 0.01% Triton X-100,

53 2.5 mM MnC12, 25 m M MgC12, 50 nM sodium vanadate and 5 ixCi of [32p] A T P (specific activity of 3000 Ci mmo1-1) in 50 txM unlabelled A T P for 10 min. The reaction was stopped and the proteins solubilised by the addition of urea, SDS and sample buffer. Phosphorylated proteins were identified by polyacrylamide gel electrophoresis and autoradiography. In some of the experiments E G F was added to the incubation mixture at the appropriate concentrations (see Results).

Epidermal growth factor binding. The identification of the polypeptides which bound E G F was carried out as follows: 65 txg of the membrane enriched fraction from bloodstream trypanosomes were incubated with 100 nM biotinylated E G F for 15 min at 0°C. The biotinylated E G F was then either cross-linked directly, or the membrane preparations were washed 5 × in PBSG before cross-linking was initiated (see Results). The cross-linking was carried out by resuspending the sample in 1 ml of 128 mM NaC1, 5 mM KC1, 1.2 mM CaC12, 1.2 mM MgzSO4, 24 mM Hepes, p H 7.5, and 2 mg ml 1 BSA and crosslinking with 250 nM disuccinimidyl suberate (DSS) for 15 min at 0°C. Following washing and spinning, the preparation was solubilised in urea/SDS and sample buffer and the polypeptides separated by polyacrylamide gel electrophoresis followed by transfer to nitrocellulose. The biotinylated polypeptides were detected by probing the nitrocellulose with 125I-labelled streptavidin and processing the filter as described above.

Measurement of growth rate of cultured trypanosomes. Procyclic trypanosomes were diluted, gradually, into either serum-free SDM79 medium [13] or SDM 79 medium containing 1% foetal calf serum. The growth rate was examined as follows. 40-~1 samples were taken every 3 h from duplicate flasks containing procyclic trypanosomes grown either in the presence or absence of E G F . Cell numbers were determined by counting in a haemocytometer, and a mean of the counts, taken from the duplicate samples, was determined and plotted for the cultures with and without E G F .

Results

Antibodies to the epidermal growth factor receptor bind to trypanosomes. IFAs were carried out to determine whether antibodies to the mammalian E G F receptor reacted with T. brucei. Antibodies were incubated with ethanol-fixed procyclic (insect stage) or bloodstream trypanosomes. The antibodies used in the assays were an antibody raised against the purified mammalian E G F receptor [14] and antibodies recognising the same receptor but raised against synthetic peptides (see Materials and Methods for antibody details) representing the extracellular domain of the E G F receptor (antibodies 2075, 2046 and OA-11-852) and the v-erbB oncogene product (antibody 2220). The results of these experiments are illustrated in Fig. 1, and show that both the antibody raised against the purified receptor (Fig. la) and that raised against the extracellular domain of the receptor (Fig. lc; antibody 2075) react, at a dilution of 1 in 50, with procyclic trypanosomes. Similar positive reactions were observed with bloodstream trypanosomes (data not shown) while a normal rabbit serum control (Fig. lb) showed no reaction. Antibody 2075 was shown to react with A431 cells (Fig. ld), a cell line known to over express the E G F receptor [21]. These results demonstrate that epitopes, recognised by anti-

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Fig. 1. Immunofluorescenceassay of antibody binding to fixed procyclic trypanosomes (a-c) and A431 cells (d). (a) Antibodies to the purified mammalian EGF receptor; (b) control normal rabbit serum at 1 in 100 dilution; (c,d) antibody 2075 at 1 in 100 dilution.

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Fig. 2. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of immunoprecipitates of surface-biotinylated bloodstream trypanosomes after precipitation with: (a) normal control rabbit serum at 1 in 100 dilution; (b) antibody 2075 at 1 in 100 dilution; (c) antibody 2075 at 1 in 300 dilution; (d) antibody 2075 at 1 in 600 dilution. See Materials and Methods for experimental details.

Fig. 3. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of immunoprecipitates of surface-biotinylated trypanosome incubated with antibody 2075. (a) Bloodstream trypanosomes; (b) bloodstream trypanosome in the presence of 50 ~g ml ~ of competing peptide; (c) procyclic trypanosomes.

bodies to both the whole E G F receptor and to a peptide corresponding to the external domain, are present in both bloodstream and procyclic trypanosomes. The antibody reactions observed are specific by virtue of the specific immunoprecipitation of a 135-kDa polypeptide, described in the next section.

tion in the presence of the peptide to which antibody 2075 was raised; the results are shown in Fig. 3. The 135-kDa surface polypeptide precipitated by antibody 2075 (lane a) was successfully competed out by the peptide (lane b). A polypeptide of identical size (135 kDa) was precipitated when using antibodies 2046, OA-11-852 and 2220 (data not shown) or when surface-biotinylated procyclic trypanosome lysates were used (Fig. 3, lane c).

Immunoprecipitation with epidermal growth factor receptor antibodies. Immunoprecipitations were carried out to define the molecules recognised by these antibodies. Viable bloodstream trypanosomes (cloned line TREU869) were surface-biotinylated, and detergent lysates derived from these preparations were incubated with antibody 2075. The immune complexes were precipitated with fixed S. aureus Cells and run on SDS-polyacrylamide gels under non-reducing conditions. Following gel electrophoresis, the immune complexes were transferred to nitrocellulose and probed with ~25I-labelled streptavidin. A single polypeptide of 135 kDa was detected, as shown in Fig. 2, using dilutions of 1 in 100 (Fig. 2b) and 1 in 300 (Fig. 2c). Control normal serum (Fig. 2a) or antibody 2075 at a dilution of 1 in 600 (Fig. 2d) show no detectable polypeptide. The specificity of the immune reaction was further confirmed by carrying out the immune precipita-

Kinase activity associated with the immunoprecipitate. One of the features of the mammalian E G F receptor is its ability to autophosphorylate and to phosphorylate other proteins. The 135-kDa polypeptide was assayed for kinase activity by incubating an immunoprecipitate identical to that described previously (Figs. 2 and 3) in the presence of [32p]ATP under the conditions indicated in Materials and Methods. Analysis of the immunoprecipitate under non-reducing conditions by SDS-polyacrylamide gel electrophoresis, followed by autoradiography, showed a large diffuse radioactive area on the gel (Fig. 4, lane b) which was absent when an anti-Neurospora G D H antibody precipitate was used as a control (Fig. 4, lane a). The latter antibody was shown to react with trypanosomes, by IFA (unpublished observations), and therefore acts as an internal control for any non-specific immune precipitation of polypeptides with kinase activity. We therefore

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Fig. 4. Detection of kinase activity associated with immunoprecipitates of bloodstream trypanosome lysates incubated with antibody. (a) Anti-Neurospora GDH antibody; (b) antibody 2075. The immunoprecipitates were assayed for kinase activity as described in Materials and Methods, and after washing, were electrophoresed on an SDS-polyacrylamidegel under non-reducing conditions.

concluded that the polypeptides specifically immunoprecipitated by antibody 2075 have protein kinase activity; the diffuse nature and high molecular size of the band in Fig. 4, lane b is probably due to a combination of the phosphorylation of the antibody and the non-reducing conditions under which the gels were run. These results suggest that the 135-kDa polypeptide has protein kinase activity, a feature characteristic of the m a m malian E G F receptor.

Effects of epidermal growth factor on protein kinase activity in membrane enriched fractions. M e m b r a n e enriched fractions of bloodstream T. brucei were isolated and phosphorylated proteins characterised by incubation with [32p]-ATP followed by SDS-polyacrylamide gel electrophoresis in the presence and absence of mouse E G F . Polypeptides of 170, 135, 76, 50 and 31 k D a were identified as phosphoproteins (Fig. 5, lane a) and an increase in phosphorylation was observed in the 170, 135, 76 and 50-kDa bands with addition of E G F (Fig. 5, lanes b and c). Equal amounts of protein were loaded in each lane, as shown by the comparable intensity of the 31-kDa band in all lanes. Increases in the intensity of labelling of the other four polypeptides (listed above) with in-

creasing concentrations of E G F are clearly observed. Thus, E G F stimulates an increase in protein kinase activity of specific polypeptides in m e m b r a n e enriched fractions. The 135kDa polypeptide, identified by immune precipitation, may be represented by the 135-kDa band seen in Fig. 5. The other bands which alter in their degree of phosphorylation with changing concentrations of E G F may represent real substrates of the putative receptor, alternative receptors or apparent substrates phosphorylated instead of natural substrates because of the absence of whole cell components. These results clearly demonstrate that the m e m b r a n e s of T. brucei contain protein kinases which can be stimulated by E G F and are therefore, by implication, able to bind E G F .

Binding of epidermal growth factor to trypanosomes. To characterise any m e m b r a n e polypeptides which might bind E G F , trypanosome m e m brane preparations were incubated with biotinylated E G F , followed by cross-linking us-

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~ m m Fig. 5. Phosphoproteins labelled by kinase assays of a bloodstream trypanosome membrane fraction and separated by SDSpolyacrylamide gel electrophoresis. The proteins were labelled by incubation with [32p]ATP in the presence of (a) no EGF; (b) 120 nM EGF; (c) 240 nM EGF.

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45Fig. 6. Binding of epidermal growth factor to polypeptides from a surface membrane enriched fraction of bloodstream trypanosomes. Biotinylated EGF was incubated with membrane preparations and then cross-linked directly (a), or washed 5 times between incubation and cross-linking (b). The polypeptides which had bound EGF were identified (after separation on SDS-polyacrylamide gels and transfer to nitrocellulose) by their binding to ~25I-labelledstreptavidin. A control with no added EGF is also shown (c).

ing DSS either directly or after extensive washing. The polypeptides were separated by SDSpolyacrylamide gel electrophoresis, followed by transfer to nitrocellulose. Any polypeptide which had bound E G F would also be labelled by biotin which was detected by probing of the resultant blot with lz5I-labelled streptavidin. Fig. 6 shows an autoradiograph of such a gel and demonstrates that the E G F binds to two polypeptides, one of 135 k D a and one of 50 kDa. Lane a shows the labelling pattern after the m e m b r a n e s had been cross-linked with the labelled E G F and run on the gel without washing; lane b is identical, except that the membranes were washed five times before cross-linking. Such experiments showed reproducibly the labelling of the 135-kDa and 50k D a polypeptides. The unwashed E G F crosslinked preparations (Fig. 6, lane a) show a number of other labelled peptides which, since they could be r e m o v e d by washing prior to cross-linking, were presumed to represent non-specific binding of E G F . Thus, comparison of lanes a and b in Fig. 6 supports the conclusion that the labelled polypeptides show a specific affinity for E G F , as the other polypeptides are r e m o v e d by washing. It would not be predicted that the cross-

linking of E G F (6 k D a ) would produce an observable change in molecular size at the resolution obtained with these gels. The molecular sizes of the E G F cross-linked polypeptides (135 and 50 k D a ) exclude the possibility that these are derived from the B S A added to the cross-linking reaction; furthermore, the m e m b r a n e preparations were centrifuged and the pelleted m e m b r a n e s washed in buffer, to remove B S A prior to solubilisation and separation by SDS-polyacrylamide gel electrophoresis. Fig. 6, lane c is a control lane where no E G F was present. Thus, we show here that E G F binds to two polypeptides, of 135 and 50 k D a , which are found in the trypanosome m e m b r a n e enriched fraction.

Effect of epidermal growth factor on trypanosome growth rate in vitro. In m a m m a l i a n cell culture systems, E G F has been shown to have an effect on the growth of cells [22]. W e examined the effect of E G F on the growth of trypanosomes. In view of the absence of a bloodstream stage culture system in our laboratory, we examined the effect of E G F on procyclic stage trypanosomes. The procyclics were grown in S D M medium containing either no serum or 1% serum, and the results, shown as growth curves (Fig. 7), represent the m e a n and range of duplicate samples. In the serum-free m e d i u m there was a clear mitogenic effect in the presence of 120 nM E G F (Fig. 7a). The concentration of cells was observed to double over a 6-h period, relative to the cells untreated with E G F . In medium, containing 1% serum, a mitogenic effect was also observed in the presence of E G F (Fig. 7b). This mitogenic effect was consistent over a n u m b e r of experiments (data not shown). Thus, these experiments show that E G F stimulates growth in procyclic trypanosomes. Discussion

E G F was the first factor described which was implicated in the control of m a m m a l i a n cell growth [23]. Subsequently, E G F has been shown to be involved in the growth regulation (both positive and negative) of m a n y cell types [24]. The recent studies on growth regulatory processes in normal and abnormal cell lines have identified a

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Fig. 7. Growth of procyclictrypanosomes in the presence (+) or absence (o) of mouse epidermal growth factor at 120 nM. Growth experiments were carried out in SDM-79 medium containing (a) no foetal calf serum; or (b) 1% foetal calf serum. The cell numbers were measured as described in Materials and Methods. number of other growth factors besides E G F [25], such as platelet-derived growth factor and the transforming growth factors. E G F , the most widely studied growth factor, modifies cell growth rate by binding to a cell surface receptor which triggers, by means of phosphorylation events, changes at the nuclear level which act to alter the rate of cell division. The h u m a n E G F receptor, a t r a n s - m e m b r a n e protein of 170 k D a , has three main domains: an extracellular EGF-binding domain, an intracellular tyrosine kinase domain and a t r a n s - m e m b r a n e domain. In addition to being found in humans, similar receptors have been found in other m a m m a l s such as mice and rats. Recently, E G F receptor homologues have also been identified in Drosophila as proteins of 190 and 100 k D a [26-28]. This conservation of the E G F receptor between m a m m a l s and insects suggests that the E G F receptor plays a fundamental role in a general mechanism of cell growth control. F u r t h e r m o r e , the high degree of conservation of D N A sequences between parts of the E G F receptor and other protein kinases, such as the src gene family [29], suggests that this family of genes has a long evolutionary history, and further emphasises their basic role in cell growth. In this study, we report evidence for a surface polypeptide in t r y p a n o s o m e s which shows a number of properties characteristic of the m a m m a l i a n E G F receptor. W e have demonstrated the exist-

ence of two polypeptides on the m e m b r a n e of the trypanosome which bind E G F and which may therefore represent receptors for E G F . The 135k D a polypeptide can be immune-precipitated by antibodies to the external domain of the h u m a n E G F receptor, has protein kinase activity, and is possibly a structure homologous to the 170-kDa human E G F receptor. The specificity of the immunoprecipitations is confirmed by the absence of any immunoprecipitate when normal serum is used and by the observation that the peptide to which the antibody was raised is capable of competing with the immunoprecipitation of the 135kDa polypeptide. Furthermore, on the basis of the molecular size of this polypeptide, it is clear that antibody 2075 does not cross-react with any of the m a j o r surface proteins of either the bloodstream (variant surface glycoprotein 65-68 k D a or procyclic forms (170, 160, 96, 63 and 58 kDa; ref. 30) of the parasite. The immunoprecipitation experiments, therefore, clearly demonstrate that a surface polypeptide of 135 kDa, common to both the insect and bloodstream stages of the parasite, has an epitope similar or identical to an epitope within the external domain of the E G F receptor. We have also identified a m e m b r a n e polypeptide of identical molecular size which shows EGF-sensitive phosphorylation in the presence of A T P and which is capable of binding E G F . T a k e n together, these results strongly support the conclu-

58 sion that trypanosomes express a 135-kDa surface protein with protein kinase activity, an EGFbinding domain and a domain which has homology to the mammalian E G F receptor. Furthermore, the observation that E G F can stimulate trypanosome growth not only confirms this conclusion, but also suggests that the trypanosome has a cellular growth regulatory pathway. In addition to the 135-kDa E G F binding polypeptide, a second polypeptide of 50 k D a was identified which bound E G F , and showed increased phosphorylation in the presence of E G F , but which was not precipitated by antibody 2075. The relationship of this polypeptide to the 135kDa polypeptide is as yet unclear; it may represent a processing or degradation product, or it may represent a second receptor. The 50-kDa EGF-binding protein was not precipitated with the antisera against the human receptor. The possibility that the polypeptides (135 and 50 kDa represent two distinct receptors is of considerable interest, as a similar observation has been made in D r o s o p h i l a , where proteins of 190 and 100 kDa have been identified as E G F receptor homologues [26,28]. The 100-kDa polypeptide was found to bind a number of EGF-related growth factors, and it has been suggested that this protein might represent an evolutionary precursor to the mammalian growth factor receptors [26,28]. The origins and relationships of the two EGFbinding polypeptides and their relationships to the receptors of higher organisms await the cloning and sequencing of the trypanosome genes for

these putative E G F receptor homologues. Levels of E G F in mammalian body tissues occur at between 1 and 800 ~xg m1-1 [31], or 0.15-130 nM; the concentrations used in our experiments are therefore within the physiological range. The observations that E G F modifies membrane protein kinase activity and stimulates trypanosome growth rate in vitro suggests that a growth factor-dependent cellular growth regulatory pathway is present in the parasite, and that host or vector growth factors may play a role in regulating trypanosome growth. We have shown that the putative receptor is present in both the procyclic (insect) stage and the bloodstream stage. Thus, we cannot discount the possibility that the receptors recognise insect EGF-like growth factors, or that the trypanosomes themselves produce an EGF-like growth factor which acts in both the insect and mammalian life-cycle stages. Considerable further investigation is required to elucidate the biological role of this growth factor receptor and its interaction with growth factors.

Acknowledgements This work was supported by a grant from the Wellcome Trust. We are indebted to Stanley Cohen for gifts of E G F - R antibodies, to Cambridge Research Biochemicals for supplying antibodies to the synthetic peptides, to B.R. Shiels for the preliminary results which led to this work, and to Frank Johnston, Alan McEwan and Alan May for the photography.

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