]']8
Biochimiea el Biophysica Acre, 1134(1992) 178~181 © 1~2 ElsevierSciencePublishersB,V÷All rightsreserved0167-4889/92/$0b.00
IBBAMCR10285
Rapid Report
The iigand binding domain of the epidermal growth factor receptor is not required for receptor dimerization M a d a n M . K w a t r a a,b D a r e l [ D . B i g n e r ~,d a n d J o n a t h a n A. C o h n b~ De47artments of ~ Pmholug~, ~ Medicine and c Cell Biology and the LtPreus~ laboratory for Bruin Tumor ReseQrch. Duke Unicers~ty Medical Center and VA Medical CenTer, Durham, NC (USA)
(Received 13 November1991) Key words: Epidermal8-owlh [aclor receptor; ReceptordimerJzati0.
r n examine the role of the ligand binding domain of epidermal growth factor receptor in its dimerization, we studied the dimerization of a truncated form of the receptor that resembles v-erbB in that it tacks a ligand binding domain. Receptor dimerizalion was determined by sedimentation analysis on sucrose density gradients at different concentrations of Triton X-t00, At high concentrations of Triton X-t00 (0,2%), the truncated receptor occurred as a monomer and displayed low basal autophqsphoryiation. By contrast, at low concentrations of Triton X-t00 (0.ill%), it eaisted as a dimer aad exhibited high basal al~tonhosphmytation. The ability of the truncated receptor to dimerize indicates that the ligand binding domain of the epidermal growth factor templar is nol required for receptor dimcrization.
Ligand binding, receptor dimcrization, increased tyrosine kinasr activity and receptor autophosphorylation are key ¢¢¢nts during signal transduction by the epidermal growth factor (EGF) receptor. As recently reviewed [1], much evidence indicates that activation of the receptor kinase is preceded by ligand.dependent receptor dimerizatian/aggregation. Specifically, EGF induces dimerization of the receptor in isolated membranes, in purified receptor preparations and in living cells; and the dimerized form of the receptor exhibits enhanced tyrosine kinase activity [2-4]. While it is unknown how EGF binding promotes receptor dimerization, it seems likely that this event results from an effect of ligand binding on the receptor's monomer-dimar equilibrium. Evidence also e~ists indicating that activation of the EGF receptor kinase can occur in the absence of receptor dimerization [5]. Furthermore, it has been shown that EGF recept,or undergoes ligand-independent dimerization at low concentrations of Triton X-]00 (0.01%) [6]. Even though these findings indicate that iigand binding is not required for EGF receptor dimerization and activation, it is unknown whether the Jigand binding domain of the receptor is necessary for these functions. Studies using the v-erbB oncoprotein provide indirect evidence related to this question, This
Correspondence:M,M. Kwalra, Box 3821, Duke Uaive~siq,Medical Center, Durham. NC 27710, USA.
oncoprotein has a high homology with the intraceliular domain of the EGF receptor, but lacks the recvptor's ligand binding domain. The finding that v-erbB oleoprotein does not dimerize [7] suggests that dimerization of the EGF receptor can only occur with an intact ligand binding domain. Recent studies in this labor_~tory have characterized a truncated f~rm of the EOF receptor that occurs in the human gfiorna, D-245 MG [8]. The eDNA sequence of this truncated receptor indicates that it is identical to the intact receptor, e ~ e p t that the truncated form lacks the first 542 amino acids of the intact form [9]. Thus, the N-terminal of the truncated receptor corresponds to the amino acid in position 543 of the intact receptor. The truncated form has an apparent M r of 100000 by ~ d i u m dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and exh~its no ligand binding [10], consistent with predictions based on the eDNA sequence. Nonetheless, it resembles the intact receptor with respect to tyrosinc kinase activity [11]. Thus, this truncated EGF tempter resembles the v - e r b B oncoprotein in that it b, eks a ligand binding domain, The two proteins differ, however, in that only the truncated receptor is identical to the intact receptor in its intraceilular domain. In this study we have used this truncated receptor to test, in a more direct fashion, the hypothesis that the ligand binding domain of the EGF receptor is required for its dimerization. Fig. 1 shows the ligand-independent autophosphorylation of partially purified EGF receptors from A431
179 EGFP.eCel:,e,~. Ima¢l T.,~r,~l
265~ -
kDa+
45
tom-
TrltenX-tN[~) 0,24.0"1. 0.20.al Fig. 1. Autoradiogram showing the effect of Triton X-I[]U on autophosphory|ation of intact and Iruncatcd EGF receptors. EGF receptnrs were partially purified from A431 ('intact+)and from D+24.5MG h~tman glioma ('tmucgted'3 xenogra|ts grown in nude mice as described [18,19]. The ?uranus were minced and were waslled and homogell.izt,d ia buffer A (0.25 M sucrose/20 mM Hopes/5 mM ED3"A/5 mM EOTA/] mM dithinthreitol (pH 7.4)) containing the following proleinaS¢ inbibitors: 0.l mM phenylmethylsul[onyl fluOn,,Ic, ] mM iodoscctsmide, III t.~g/m| ~ybean trypsin inhJbilor, 1 pg/m'l aprotlnin, t ,~M peps!aria, and 2 p.~./ml !eupeptin. "The
]'.omo4enale was centrifuged al 40ll00x g [or 3U'rain and the rcsuJting pellet was ~e~pendad in buffer B (20 mM Hopes (pH 7AL with Ihe above proteinase inhibitoPs) at L to 5 m4 prolcin Per m| and stored at -70+C. EGF receptors were solubUized and partially purif'ted rting wheat germ agglulioin (WOA) arfinily chromatography as described [I 1,20]. The truncated receptor could be purified on the leclio column, as it retains three potential N-linked gl~cosylztinn sites in ils short extracellnlar amino terminal domain [9]. The autophnsphoryladon ~ea¢tion was p¢.-fonned in a '~0-p,Ivolume conraining 20 .'aM Hepe~, ]0 mM M4CI2, 2 mM MnCl2, 0.2% or 0.01%
intact E G F receptor dimerizes at low concentrations of Triton X+I00 [6] and that the dlrneric form of the receptor exhibits it.creased kinasc aetivjty (Ref. 4; however, see Ref. 12). ]~ecaus~ the truncated receptor resembled the intact receptor with respect to the effect of reduced detergent concentration on autophosphorylation (Fig. 1), it s e e m e d likely that the truncated receptor would also undergr) dimerization, D i m e r i z a d o n of the truncated a n d intact receptors waS studied by s e d i m e n t a t i o n analysis on st,+rose d e w sJty gradients. For these studies, the receptors were autoph0sphorylated by incubation with [7- 32 P]ATP under phosphorylating conditions a n d were l o a d e d o n sucrose density gradients. A s shown in Fig. 2, the s e d i m e n t a t i o n properties of the atttophosphorylated intect receptor were strikingly d e p e n d e n t o n t h e detergent concentration. A t 0.2% T d t o n X-100, the receptor seal!monied with a ~ d i r n e n l a t i o n coefficient close to 7 S, which corresponds to the monna!eric form of the r e c e p t ~ [12]. By contrast., a! 0.01% Triton X-100, the receptor sod!merited more rapidly with a sedimentation coefficicm n e a r 12 S, consistent with the formation of receptor dimers [I2]. These findings indicate that the a u t o p h o s p h m y l a t e d receptor dim=rizcs at reduced levels of detergent, consistent with earlier studies of the intact receptor in which tyros!no kinase activity was used to d e t e c t the protein [6]. Additional studies c o m p a r e d the s e d i m e n t a t i o n p r o p e r t i e s of the
6.8 S T
11.3 S T 0 . 2 0 % Triton X - 1 0 0
Triton X-I00, l to 2 P8 partially purified receptor, and 1 ,uM
[~-32P]ATP+ 10to 20 cpm/fmol [21}+Following incubation at 4+C for 3 rain, the reaction w"~ terminated by adding 25 ~1 4% SDS, 20% glycerol, and 125 mM "Iris (pH 6.8). Phosphowtated receptors were analyzed by SDS, PAGE (7% g¢}s) and atlioradiography [22]. Nacetylgiucosamin¢, cain!USe,human placental alkaline phnsphatase, ~oteioase inhibitors, and WGA were obtained from Sigma Chemical Co., St. Louis, Me. Triton X-i00 was obtained from Pierce (Rockford, IL). and [T--~2P]ATP(60~0 Ci/mrnol) was Obtained from New England Nnclear/Dupont (Boston, MA). WGA-Seph~resc was prepared by coupling WGA Io cyanogen bromide-activated Sepharose 6MB (Pharma¢ia. Vineland, NJ).
cells
Biochimiea el Biophysica Acre, 1134(1992) 178~181 © 1~2 ElsevierSciencePublishersB,V÷All rightsreserved0167-4889/92/$0b.00
IBBAMCR10285
Rapid Report
The iigand binding domain of the epidermal growth factor receptor is not required for receptor dimerization M a d a n M . K w a t r a a,b D a r e l [ D . B i g n e r ~,d a n d J o n a t h a n A. C o h n b~ De47artments of ~ Pmholug~, ~ Medicine and c Cell Biology and the LtPreus~ laboratory for Bruin Tumor ReseQrch. Duke Unicers~ty Medical Center and VA Medical CenTer, Durham, NC (USA)
(Received 13 November1991) Key words: Epidermal8-owlh [aclor receptor; ReceptordimerJzati0.
r n examine the role of the ligand binding domain of epidermal growth factor receptor in its dimerization, we studied the dimerization of a truncated form of the receptor that resembles v-erbB in that it tacks a ligand binding domain. Receptor dimerizalion was determined by sedimentation analysis on sucrose density gradients at different concentrations of Triton X-t00, At high concentrations of Triton X-t00 (0,2%), the truncated receptor occurred as a monomer and displayed low basal autophqsphoryiation. By contrast, at low concentrations of Triton X-t00 (0.ill%), it eaisted as a dimer aad exhibited high basal al~tonhosphmytation. The ability of the truncated receptor to dimerize indicates that the ligand binding domain of the epidermal growth factor templar is nol required for receptor dimcrization.
Ligand binding, receptor dimcrization, increased tyrosine kinasr activity and receptor autophosphorylation are key ¢¢¢nts during signal transduction by the epidermal growth factor (EGF) receptor. As recently reviewed [1], much evidence indicates that activation of the receptor kinase is preceded by ligand.dependent receptor dimerizatian/aggregation. Specifically, EGF induces dimerization of the receptor in isolated membranes, in purified receptor preparations and in living cells; and the dimerized form of the receptor exhibits enhanced tyrosine kinase activity [2-4]. While it is unknown how EGF binding promotes receptor dimerization, it seems likely that this event results from an effect of ligand binding on the receptor's monomer-dimar equilibrium. Evidence also e~ists indicating that activation of the EGF receptor kinase can occur in the absence of receptor dimerization [5]. Furthermore, it has been shown that EGF recept,or undergoes ligand-independent dimerization at low concentrations of Triton X-]00 (0.01%) [6]. Even though these findings indicate that iigand binding is not required for EGF receptor dimerization and activation, it is unknown whether the Jigand binding domain of the receptor is necessary for these functions. Studies using the v-erbB oncoprotein provide indirect evidence related to this question, This
Correspondence:M,M. Kwalra, Box 3821, Duke Uaive~siq,Medical Center, Durham. NC 27710, USA.
oncoprotein has a high homology with the intraceliular domain of the EGF receptor, but lacks the recvptor's ligand binding domain. The finding that v-erbB oleoprotein does not dimerize [7] suggests that dimerization of the EGF receptor can only occur with an intact ligand binding domain. Recent studies in this labor_~tory have characterized a truncated f~rm of the EOF receptor that occurs in the human gfiorna, D-245 MG [8]. The eDNA sequence of this truncated receptor indicates that it is identical to the intact receptor, e ~ e p t that the truncated form lacks the first 542 amino acids of the intact form [9]. Thus, the N-terminal of the truncated receptor corresponds to the amino acid in position 543 of the intact receptor. The truncated form has an apparent M r of 100000 by ~ d i u m dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and exh~its no ligand binding [10], consistent with predictions based on the eDNA sequence. Nonetheless, it resembles the intact receptor with respect to tyrosinc kinase activity [11]. Thus, this truncated EGF tempter resembles the v - e r b B oncoprotein in that it b, eks a ligand binding domain, The two proteins differ, however, in that only the truncated receptor is identical to the intact receptor in its intraceilular domain. In this study we have used this truncated receptor to test, in a more direct fashion, the hypothesis that the ligand binding domain of the EGF receptor is required for its dimerization. Fig. 1 shows the ligand-independent autophosphorylation of partially purified EGF receptors from A431
179 EGFP.eCel:,e,~. Ima¢l T.,~r,~l
265~ -
kDa+
45
tom-
TrltenX-tN[~) 0,24.0"1. 0.20.al Fig. 1. Autoradiogram showing the effect of Triton X-I[]U on autophosphory|ation of intact and Iruncatcd EGF receptors. EGF receptnrs were partially purified from A431 ('intact+)and from D+24.5MG h~tman glioma ('tmucgted'3 xenogra|ts grown in nude mice as described [18,19]. The ?uranus were minced and were waslled and homogell.izt,d ia buffer A (0.25 M sucrose/20 mM Hopes/5 mM ED3"A/5 mM EOTA/] mM dithinthreitol (pH 7.4)) containing the following proleinaS¢ inbibitors: 0.l mM phenylmethylsul[onyl fluOn,,Ic, ] mM iodoscctsmide, III t.~g/m| ~ybean trypsin inhJbilor, 1 pg/m'l aprotlnin, t ,~M peps!aria, and 2 p.~./ml !eupeptin. "The
]'.omo4enale was centrifuged al 40ll00x g [or 3U'rain and the rcsuJting pellet was ~e~pendad in buffer B (20 mM Hopes (pH 7AL with Ihe above proteinase inhibitoPs) at L to 5 m4 prolcin Per m| and stored at -70+C. EGF receptors were solubUized and partially purif'ted rting wheat germ agglulioin (WOA) arfinily chromatography as described [I 1,20]. The truncated receptor could be purified on the leclio column, as it retains three potential N-linked gl~cosylztinn sites in ils short extracellnlar amino terminal domain [9]. The autophnsphoryladon ~ea¢tion was p¢.-fonned in a '~0-p,Ivolume conraining 20 .'aM Hepe~, ]0 mM M4CI2, 2 mM MnCl2, 0.2% or 0.01%
intact E G F receptor dimerizes at low concentrations of Triton X+I00 [6] and that the dlrneric form of the receptor exhibits it.creased kinasc aetivjty (Ref. 4; however, see Ref. 12). ]~ecaus~ the truncated receptor resembled the intact receptor with respect to the effect of reduced detergent concentration on autophosphorylation (Fig. 1), it s e e m e d likely that the truncated receptor would also undergr) dimerization, D i m e r i z a d o n of the truncated a n d intact receptors waS studied by s e d i m e n t a t i o n analysis on st,+rose d e w sJty gradients. For these studies, the receptors were autoph0sphorylated by incubation with [7- 32 P]ATP under phosphorylating conditions a n d were l o a d e d o n sucrose density gradients. A s shown in Fig. 2, the s e d i m e n t a t i o n properties of the atttophosphorylated intect receptor were strikingly d e p e n d e n t o n t h e detergent concentration. A t 0.2% T d t o n X-100, the receptor seal!monied with a ~ d i r n e n l a t i o n coefficient close to 7 S, which corresponds to the monna!eric form of the r e c e p t ~ [12]. By contrast., a! 0.01% Triton X-100, the receptor sod!merited more rapidly with a sedimentation coefficicm n e a r 12 S, consistent with the formation of receptor dimers [I2]. These findings indicate that the a u t o p h o s p h m y l a t e d receptor dim=rizcs at reduced levels of detergent, consistent with earlier studies of the intact receptor in which tyros!no kinase activity was used to d e t e c t the protein [6]. Additional studies c o m p a r e d the s e d i m e n t a t i o n p r o p e r t i e s of the
6.8 S T
11.3 S T 0 . 2 0 % Triton X - 1 0 0
Triton X-I00, l to 2 P8 partially purified receptor, and 1 ,uM
[~-32P]ATP+ 10to 20 cpm/fmol [21}+Following incubation at 4+C for 3 rain, the reaction w"~ terminated by adding 25 ~1 4% SDS, 20% glycerol, and 125 mM "Iris (pH 6.8). Phosphowtated receptors were analyzed by SDS, PAGE (7% g¢}s) and atlioradiography [22]. Nacetylgiucosamin¢, cain!USe,human placental alkaline phnsphatase, ~oteioase inhibitors, and WGA were obtained from Sigma Chemical Co., St. Louis, Me. Triton X-i00 was obtained from Pierce (Rockford, IL). and [T--~2P]ATP(60~0 Ci/mrnol) was Obtained from New England Nnclear/Dupont (Boston, MA). WGA-Seph~resc was prepared by coupling WGA Io cyanogen bromide-activated Sepharose 6MB (Pharma¢ia. Vineland, NJ).
cells
