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• W2000489928 abstract "Members of the epidermal growth factor receptor, or ErbB, family of receptor tyrosine kinases have a single transmembrane (TM) α-helix that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, recent studies with the epidermal growth factor receptor (ErbB1) and the erythropoietin receptor have indicated that interactions between TM α-helices do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimers. In addition, not all of the expected ErbB receptor ligand-induced dimerization events can be recapitulated using isolated extracellular domains, suggesting that other regions of the receptor, such as the TM domain, may contribute to dimerization in vivo. Using an approach for analyzing TM domain interactions in Escherichia colicell membranes, named TOXCAT, we find that the TM domains of ErbB receptors self-associate strongly in the absence of their extracellular domains, with the rank order ErbB4-TM > ErbB1-TM ≈ ErbB2-TM > ErbB3-TM. A limited mutational analysis suggests that dimerization of these TM domains involves one or more GXXXG motifs, which occur frequently in the TM domains of receptor tyrosine kinases and are critical for stabilizing the glycophorin A TM domain dimer. We also analyzed the effect of the valine to glutamic acid mutation in ErbB2 that constitutively activates this receptor. Contrary to our expectations, this mutation reduced rather than increased ErbB2-TM dimerization. Our findings suggest a role for TM domain interactions in ErbB receptor function, possibly in stabilizing inactive ligand-independent receptor dimers that have been observed by several groups. Members of the epidermal growth factor receptor, or ErbB, family of receptor tyrosine kinases have a single transmembrane (TM) α-helix that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, recent studies with the epidermal growth factor receptor (ErbB1) and the erythropoietin receptor have indicated that interactions between TM α-helices do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimers. In addition, not all of the expected ErbB receptor ligand-induced dimerization events can be recapitulated using isolated extracellular domains, suggesting that other regions of the receptor, such as the TM domain, may contribute to dimerization in vivo. Using an approach for analyzing TM domain interactions in Escherichia colicell membranes, named TOXCAT, we find that the TM domains of ErbB receptors self-associate strongly in the absence of their extracellular domains, with the rank order ErbB4-TM > ErbB1-TM ≈ ErbB2-TM > ErbB3-TM. A limited mutational analysis suggests that dimerization of these TM domains involves one or more GXXXG motifs, which occur frequently in the TM domains of receptor tyrosine kinases and are critical for stabilizing the glycophorin A TM domain dimer. We also analyzed the effect of the valine to glutamic acid mutation in ErbB2 that constitutively activates this receptor. Contrary to our expectations, this mutation reduced rather than increased ErbB2-TM dimerization. Our findings suggest a role for TM domain interactions in ErbB receptor function, possibly in stabilizing inactive ligand-independent receptor dimers that have been observed by several groups. The ErbB (or HER) family of growth factor receptor tyrosine kinases has four members: the epidermal growth factor (EGF) 1The abbreviations used are:EGFepidermal growth factorCAMchloramphenicolCATchloramphenicol acetyltransferaseECextracellularEPOerythropoietinErbBproduct of oncogene of avian erythroblastosis virus (receptors in the EGF receptor family)GpAglycophorin AMBPmaltose-binding proteinTMtransmembraneZOIzone of inhibition 1The abbreviations used are:EGFepidermal growth factorCAMchloramphenicolCATchloramphenicol acetyltransferaseECextracellularEPOerythropoietinErbBproduct of oncogene of avian erythroblastosis virus (receptors in the EGF receptor family)GpAglycophorin AMBPmaltose-binding proteinTMtransmembraneZOIzone of inhibition receptor (ErbB1), ErbB2 (also known as HER2 or the Neu oncogene product), ErbB3 (HER3), and ErbB4 (HER4) (1Olayioye M.A. Neve R.M. Lane H.A. Hynes N.E. EMBO J. 2000; 19: 3159-3167Crossref PubMed Google Scholar). Each ErbB receptor has a large extracellular (EC) domain of 600–630 amino acids, a single membrane-spanning α-helix, and an intracellular domain of ∼500 amino acids that contains the tyrosine kinase domain plus regulatory sequences (2Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4581) Google Scholar). It is now quite well established that activation of ErbB receptors involves their ligand-induced (or ligand-stabilized) oligomerization, which in turn leads to receptor trans-phosphorylation and activation within dimers or higher order oligomers (reviewed in Ref. 3Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3484) Google Scholar). In the case of ErbB1, we and others have demonstrated that the isolated EC domain of the receptor dimerizes completely upon EGF binding (4Ferguson K.M. Darling P.J. Mohan M.J. Macatee T.L. Lemmon M.A. EMBO J. 2000; 19: 4632-4643Crossref PubMed Scopus (114) Google Scholar, 5Lemmon M.A., Bu, Z. Ladbury J.E. Zhou M. Pinchasi D. Lax I. Engelman D.M. Schlessinger J. EMBO J. 1997; 16: 281-294Crossref PubMed Scopus (302) Google Scholar, 6Brown P.M. Debanne M.T. Grothe S. Bergsma D. Caron M. Kay C. O'Connor-McCourt M.D. Eur. J. Biochem. 1994; 225: 223-233Crossref PubMed Scopus (56) Google Scholar, 7Hurwitz D.R. Emanuel S.L. Nathan M.H. Sarver N. Ullrich A. Felder S. Lax I. Schlessinger J. J. Biol. Chem. 1991; 266: 22035-22043Abstract Full Text PDF PubMed Google Scholar, 8Lax I. Mitra A.K. Ravera C. Hurwitz D.R. Rubinstein M. Ullrich A. Stroud R.M. Schlessinger J. J. Biol. Chem. 1991; 266: 13828-13833Abstract Full Text PDF PubMed Google Scholar, 9Elleman T.C. Domagala T. McKern N.M. Nerrie M. Lonnqvist B. Adams T.E. Lewis J. Lovrecz G.O. Hoyne P.A. Richards K.M. Howlett G.J. Rothacker J. Jorissen R.N. Lou M. Garrett T.P. Burgess A.W. Nice E.C. Ward C.W. Biochemistry. 2001; 40: 8930-8939Crossref PubMed Scopus (80) Google Scholar, 10Odaka M. Kohda D. Lax I. Schlessinger J. Inagaki F. J. Biochem. (Tokyo). 1997; 122: 116-121Crossref PubMed Scopus (45) Google Scholar). Similarly, the isolated EC domain of ErbB4 oligomerizes strongly when it binds to its growth factor ligand neuregulin 1-β1 (4Ferguson K.M. Darling P.J. Mohan M.J. Macatee T.L. Lemmon M.A. EMBO J. 2000; 19: 4632-4643Crossref PubMed Scopus (114) Google Scholar). These findings have led to the argument that ErbB receptor activation results directly and solely from ligand-induced oligomerization of EC domains (3Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3484) Google Scholar, 11Lemmon M.A. Schlessinger J. Trends Biochem. Sci. 1994; 19: 459-463Abstract Full Text PDF PubMed Scopus (432) Google Scholar, 12Heldin C.-H. Cell. 1995; 80: 213-223Abstract Full Text PDF PubMed Scopus (1427) Google Scholar). In this view, the transmembrane (TM) and intracellular domains of receptor molecules need not contribute directly to receptor oligomerization, but are instead driven together by EC domain interactions in the process of receptor activation.By contrast with this model, Tanner and Kyte (13Tanner K.G. Kyte J. J. Biol. Chem. 1999; 274: 35985-35990Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) have argued that interactions between TM domains do contribute significantly to ErbB1 dimerization. In fact, they go so far as to argue from their data that ErbB1 TM domain interactions provide the primary driving force for receptor dimerization. According to their proposal the unliganded ErbB1 EC domain sterically inhibits TM domain-mediated dimerization of the receptor. Activation of the receptor by EGF is then proposed to occur when ligand binding induces conformational changes in the EC domain that relieve this inhibition and permit TM domain-mediated ErbB1 homodimerization (13Tanner K.G. Kyte J. J. Biol. Chem. 1999; 274: 35985-35990Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). This model was prompted by the finding of Tanner and Kyte (13Tanner K.G. Kyte J. J. Biol. Chem. 1999; 274: 35985-35990Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) that EGF-induced dimerization is far more efficient for a fragment of ErbB1 that contains both the EC domain and the TM domain (when studied in detergent solution) than it is for the EC domain alone. Several other pieces of data also support a role for TM domains in dimerization of bitopic cell-surface receptors. For example, a specific Val → Glu mutation within the TM domain of the Neu oncogene product (ErbB2) is well known to induce ErbB2 dimerization and activation (14Bargmann C.I. Hung M.C. Weinberg R.A. Cell. 1986; 45: 649-657Abstract Full Text PDF PubMed Scopus (808) Google Scholar, 15Weiner D.B. Liu J. Cohen J.A. Williams W.V. Greene M.I. Nature. 1989; 339: 230-231Crossref PubMed Scopus (357) Google Scholar). Although precisely how the Val → Glu mutation activates the receptor is not clear, it has been proposed to stabilize interactions between TM α-helices directly (16Sternberg M.J. Gullick W.J. Nature. 1989; 339: 587Crossref PubMed Scopus (104) Google Scholar). Tzahar et al. (17Tzahar E. Pinkas-Kramarski R. Moyer J.D. Klapper L.N. Alroy I. Levkowitz G. Shelly M. Henis S. Eisenstein M. Ratzkin B.J. Sela M. Andrews G.C. Yarden Y. EMBO J. 1997; 16: 4938-4950Crossref PubMed Scopus (210) Google Scholar) have also suggested a role for TM domain interactions in ligand-induced ErbB receptor heteromerization. Finally, it was recently shown that the single TM domain from the erythropoietin (EPO) receptor forms oligomers (18Gurezka R. Laage R. Brosig B. Langosch D. J. Biol. Chem. 1999; 274: 9265-9270Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 19Kubatzky K.F. Ruan W. Gurezka R. Cohen J. Ketteler R. Watowich S.S. Neumann D. Langosch D. Klingmuller U. Curr. Biol. 2001; 11: 110-115Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) and that this self-association may play a role in EPO receptor signaling across the membrane (19Kubatzky K.F. Ruan W. Gurezka R. Cohen J. Ketteler R. Watowich S.S. Neumann D. Langosch D. Klingmuller U. Curr. Biol. 2001; 11: 110-115Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 20Constantinescu S.N. Keren T. Socolovsky M. Nam H. Henis Y.I. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4379-4384Crossref PubMed Scopus (213) Google Scholar).Although recent findings by our laboratory and others argue that EC domains are often sufficient to drive receptor dimerization, this may not always be the case. For example, neuregulin-induced homodimerization of the isolated ErbB3 EC domain cannot be detectedin vitro (4Ferguson K.M. Darling P.J. Mohan M.J. Macatee T.L. Lemmon M.A. EMBO J. 2000; 19: 4632-4643Crossref PubMed Scopus (114) Google Scholar, 21Horan T. Wen J. Arakawa T. Liu N. Brankow D., Hu, S. Ratzkin B. Philo J.S. J. Biol. Chem. 1995; 270: 24604-24608Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Furthermore, the ligand-induced heteromeric association of ErbB receptors detected in vivocannot be recapitulated using isolated EC domains in vitro(4Ferguson K.M. Darling P.J. Mohan M.J. Macatee T.L. Lemmon M.A. EMBO J. 2000; 19: 4632-4643Crossref PubMed Scopus (114) Google Scholar, 21Horan T. Wen J. Arakawa T. Liu N. Brankow D., Hu, S. Ratzkin B. Philo J.S. J. Biol. Chem. 1995; 270: 24604-24608Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Motivated by these observations, together with the reports of receptor TM domain self-association outlined above, we have initiated studies of TM domain interactions in the ErbB receptor family. Using TOXCAT, a system recently developed by the Engelman laboratory for analyzing TM domain interactions in the inner membrane ofEscherichia coli (22Russ W.P. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 863-868Crossref PubMed Scopus (322) Google Scholar), we find that the TM domains from ErbB receptors homo-oligomerize efficiently. Identification of mutations that disrupt association of ErbB receptor TM domains suggests that the interactions responsible are similar to those that mediate dimerization of the glycophorin A TM α-helix (22Russ W.P. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 863-868Crossref PubMed Scopus (322) Google Scholar, 23Lemmon M.A. Flanagan J.M. Treutlein H.R. Zhang J. Engelman D.M. Biochemistry. 1992; 29: 12719-12725Crossref Scopus (463) Google Scholar, 24Lemmon M.A. Treutlein H.R. Adams P.D. Brünger A.T. Engelman D.M. Nat. Struct. Biol. 1994; 1: 157-163Crossref PubMed Scopus (295) Google Scholar). Surprisingly, we also find that the Neu mutation destabilizes rather than stabilizes dimerization of the ErbB2 TM domain. Our results support a role for TM domains in stabilizing (possibly ligand-independent) dimerization of ErbB receptors and suggest that the Neu mutation may achieve its effects by altering the nature, rather than extent, of ErbB2 TM domain dimerization.RESULTSTo investigate the ability of ErbB receptor TM domains to self-associate in the plasma membrane of E. coli, we employed the TOXCAT system developed by Russ and Engelman (22Russ W.P. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 863-868Crossref PubMed Scopus (322) Google Scholar). This system exploits two properties of the ToxR protein from Vibrio cholerae, namely that it is a specific transcriptional activator that requires dimerization to induce expression from its target promoters and that it is an integral membrane protein with modular structure (26Kolmar H. Hennecke F. Gotze K. Janzer B. Vogt B. Mayer F. Fritz H.J. EMBO J. 1995; 14: 3895-3904Crossref PubMed Scopus (63) Google Scholar). Fusion of the cytoplasmic domain of ToxR (ToxR′) to the strongly dimerizing TM domain from glycophorin A (GpA) (27Bormann B.J. Knowles W.J. Marchesi V.T. J. Biol. Chem. 1989; 264: 4033-4037Abstract Full Text PDF PubMed Google Scholar, 28MacKenzie K.R. Prestegard J.H. Engelman D.M. Science. 1997; 276: 131-133Crossref PubMed Scopus (868) Google Scholar) allows ToxR′ to activate transcription from the ctx promoter (22Russ W.P. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 863-868Crossref PubMed Scopus (322) Google Scholar,29Langosch D. Brosig B. Kolmar H. Fritz H.J. J. Mol. Biol. 1996; 263: 525-530Crossref PubMed Scopus (217) Google Scholar). This finding has been exploited in several studies of TM domain interactions (18Gurezka R. Laage R. Brosig B. Langosch D. J. Biol. Chem. 1999; 274: 9265-9270Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 19Kubatzky K.F. Ruan W. Gurezka R. Cohen J. Ketteler R. Watowich S.S. Neumann D. Langosch D. Klingmuller U. Curr. Biol. 2001; 11: 110-115Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 30Russ W.P. Engelman D.M. J. Mol. Biol. 2000; 296: 911-919Crossref PubMed Scopus (775) Google Scholar, 31Zhou F.X. Cocco M.J. Russ W.P. Brunger A.T. Engelman D.M. Nat. Struct. Biol. 2000; 7: 154-160Crossref PubMed Scopus (356) Google Scholar, 32Zhou F.X. Merianos H.J. Brunger A.T. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2250-2255Crossref PubMed Scopus (316) Google Scholar). The TM domain of interest is fused between a NH2-terminal cytoplasmic ToxR′ transcriptional activation domain and a periplasmic (assuming correct membrane insertion) MBP domain. Correct membrane insertion of the ToxR′ (TM)MBP chimera can be determined by assessing both its ability to complement an MBP deficiency in mutant E. coli and its accessibility to protease digestion in spheroplasts. TM domain dimerization is assessed in the TOXCAT system by monitoring expression of CAT, which is under control of the ToxR-responsive ctxpromoter on a reporter plasmid.ErbB Receptor TM Domains DimerizeBy assessing the ability of ToxR′ chimerae to activate CAT reporter expression in E. coli we found that each ErbB receptor TM domain homodimerizes significantly, even when compared with the strongly dimerizing GpA TM domain (GpA-TM). The ErbB receptor TM domains homodimerize in the rank order ErbB4-TM > ErbB1-TM ≈ ErbB2-TM > ErbB3-TM. CAT expression in E. coli can be monitored using a disc diffusion assay (see “Experimental Procedures”), in which resistance to CAM around a disc impregnated with this antibiotic is assessed. As shown graphically in Fig. 2 (see also Ref. 22Russ W.P. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 863-868Crossref PubMed Scopus (322) Google Scholar), only a small ZOI surrounds a CAM-impregnated disc placed on a plate ofE. coli expressing a dimerizing ToxR′ chimera (ToxR′(GpA-TM)MBP, for example), since these cells are CAM-resistant. The ZOI is much larger when the bacteria express a nondimerizing ToxR′ chimera; either with no TM domain (No TM) or with a nondimerizing TM domain (the G83I mutant of GpA-TM (23Lemmon M.A. Flanagan J.M. Treutlein H.R. Zhang J. Engelman D.M. Biochemistry. 1992; 29: 12719-12725Crossref Scopus (463) Google Scholar)), since activation of CAT expression is reduced. As shown in Fig. 2, expression of ToxR′ chimerae containing the ErbB1, ErbB2, or ErbB4 TM domains results in ZOI’s similar in size to that seen for GpA-TM, indicating strong TM domain dimerization. A chimera containing the ErbB3 TM domain also appears to confer some CAM resistance, but significantly less than for the other ErbB TM domains or GpA-TM. The ZOI measured for the ErbB3 chimera is only slightly smaller than with the G83I mutant of GpA-TM in which dimerization is severely disrupted (23Lemmon M.A. Flanagan J.M. Treutlein H.R. Zhang J. Engelman D.M. Biochemistry. 1992; 29: 12719-12725Crossref Scopus (463) Google Scholar).For a more quantitative comparison of TM domain dimerization we next assayed CAT activity directly in lysates of E. coliexpressing the different chimerae, by measuring the transfer of3H acetyl groups to biotinylated CAM (see “Experimental Procedures”). As shown in Fig. 3, expression of the ToxR′(GpA-TM)MBP chimera resulted in a high level of CAT activity in E. coli lysates, which was reduced over 12-fold by the G83I mutation that disrupts GpA-TM dimerization. ToxR′ chimerae containing the ErbB1 or ErbB2 TM domains induced approximately half the CAT activity seen for GpA-TM: the ErbB3 TM domain induced slightly less (approximately 40%) and the ErbB4 TM domain more (approximately 70%) activity.Figure 3Quantitative CAT assays of E. coliexpressing ToxR′(ErbB-TM) MBP chimerae. CAT assays were performed on normalized quantities of lysates from E. colithat expressed chimerae with the noted TM domains (see “Experimental Procedures”). At least three independent experiments were performed (in triplicate) for each TM domain, and means are plotted (±S.D.). CAT activities are expressed as the percentage of that seen with the GpA-TM chimera in parallel experiments. Background correction was also applied according to the number of counts (less than 3–5% of the value obtained with GpA-TM) measured in a negative control (pccKAN, with no TM domain).View Large Image Figure ViewerDownload (PPT)In control experiments, we demonstrated by Western blotting that expression levels of the ToxR′ chimerae containing ErbB receptor TM domains were essentially identical (Fig.4 A). To demonstrate that the chimerae are correctly integrated into the membrane, each was shown to complement a malE deletion in E. coli, allowing a strain that does not express MBP (MM39) to grow with maltose as its sole carbon source (Fig. 4 B). This can only occur if the MBP moiety of the chimera is present in the periplasm and so provides evidence that the chimera inserts correctly in the plasma membrane: with MBP in the periplasm, ToxR′ sequences in the cytoplasm and the TM domain spanning the inner membrane. Correct membrane integration was further supported by that fact that each chimera was accessible to partial proteinase K digestion in spheroplasts (but not in whole cells), while cytoplasmic proteins were not (representative data are shown in Fig. 5). In these experiments, nearly all of the expressed chimera was accessible to proteinase K in the spheroplast preparation, suggesting that almost all has acquired the correct transmembrane orientation.Figure 4Mutation of ErbB TM domains does not affect expression or membrane insertion of ToxR' chimerae. A, lysates from E. coli expressing the marked chimerae were normalized for protein content, run on 7.5% SDS-PAGE gels, and immunoblotted with an antibody to the COOH-terminal MBP domain. All chimerae tested expressed at essentially identical levels, except for the ErbB4 N2/C mutant (which actually showed enhanced apparent dimerization). Results for all mutants that were defective in oligomerization are shown, to confirm that loss of CAT transcription did not simply result from a reduction in the quantity of the transcriptional activator. B, MM39 (malE-deficient) E. coli expressing the indicated chimera were streaked on an M9 minimal media plate with maltose as the sole carbon source. Only cells in which MBP is periplasmic (and therefore in which the chimera is correctly inserted into the membrane) can survive with maltose as sole carbon source. This was true for all TM domain chimerae used in this study, indicating proper membrane integration. As controls, growth of cells expressing periplasmic MBP from pMAL-p2 is shown, as is the lack of growth of cells expressing only cytoplasmic MBP from pMAL-c2 (see “Experimental Procedures”).View Large Image Figure ViewerDownload (PPT)Figure 5ToxR(ErbB-TM) MBP chimerae are susceptible to protease digestion in spheroplasts but not in whole cells. The accessibility of ToxR′/MBP chimerae to proteinase K digestion in spheroplasts (S) and whole cells (W) was compared. Data from these control experiments are shown here for chimerae containing GpA-TM (A), ErbB2-TM mutated in motifs 2N and 2C (B), wild-type ErbB2-TM (C), and ErbB2-TM containing the Neu Val → Glu mutation (D). Similar data were obtained for all other chimerae discussed in this paper. In each panel, the left-hand gel is a Western blot probed with an anti-MBP antibody, while the right-hand gelis stained with Coomassie Blue to monitor the extent to which proteinase K has proteolyzed E. coli proteins in general. The intact ToxR′(TM)MBP fusion is denoted at the left of each anti-MBP Western blot with an arrow, and MBP that has been liberated from the chimera by proteolysis is denoted with anasterisk. In whole cells (W) there is no cleavage of the chimerae whether the cells are left untreated (U), treated with proteinase K (P) or treated with 1% Nonidet P-40 plus proteinase K (DP). The Coomassie-stained gel on the right of each panel shows no general cleavage ofE. coli proteins. By contrast, while chimerae are not cleaved in spheroplasts (S) that are left untreated (U), substantial cleavage is seen when proteinase K is added (P), despite a lack of general proteolysis in the Coomassie-stained gels. This indicates that the ToxR′(TM)MBP chimerae are accessible to added proteinase K in spheroplasts but not in intactE. coli. Detergent solubilization of spheroplasts followed by proteinase K treatment (labeled DP) results in substantially more general proteolysis of E. coli proteins, but no significant increase in ToxR′(TM)MBP chimera cleavage. This indicates that nearly all of the chimeric protein is accessible to proteinase K in the spheroplast preparation. These results support the data presented in Fig. 4 that the ToxR′(TM)MBP chimerae are correctly integrated into the E. coli inner membrane, with MBP in the periplasm. Molecular mass markers in the Coomassie-stained gels are (from the highest seen) are 201, 130, 94, 48, and 36 kDa (close to the bottom of each gel).View Large Image Figure ViewerDownload (PPT)Presence of Potential Dimerization Motifs in ErbB Receptor TM DomainsThree primary modes have been described for the strong noncovalent self-association of individual TM domains. In one, a heptad motif of leucines is thought to mediate oligomerization, presumably through side chain packing interactions similar to those seen in leucine zippers (18Gurezka R. Laage R. Brosig B. Langosch D. J. Biol. Chem. 1999; 274: 9265-9270Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The ErbB receptor TM domains contain no such heptad motifs, arguing against the relevance of this type of interaction for our studies. A second mode involves stabilization of TM domain oligomers by intramembraneous hydrogen bonds between polar side chains (32Zhou F.X. Merianos H.J. Brunger A.T. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2250-2255Crossref PubMed Scopus (316) Google Scholar, 33Gratkowski H. Lear J.D. DeGrado W.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 880-885Crossref PubMed Scopus (286) Google Scholar). The ErbB receptor TM domains contain no strongly polar side chains, allowing us to dismiss this as a possibility. The third mode, likely to be of most relevance for ErbB receptor TM domains, involves the so called GXXXG motif (30Russ W.P. Engelman D.M. J. Mol. Biol. 2000; 296: 911-919Crossref PubMed Scopus (775) Google Scholar). The GXXXG motif is a central component of the dimerization interface for the GpA TM domain (28MacKenzie K.R. Prestegard J.H. Engelman D.M. Science. 1997; 276: 131-133Crossref PubMed Scopus (868) Google Scholar) and also appears to be critical for self-association of the M13 coat protein transmembrane segment (34Wang C. Deber C.M. J. Biol. Chem. 2000; 275: 16155-16159Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Furthermore, nearly all dimerizing sequences isolated from a TM domain library in a TOXCAT-based screen contained a sequence related to the GXXXG motif (30Russ W.P. Engelman D.M. J. Mol. Biol. 2000; 296: 911-919Crossref PubMed Scopus (775) Google Scholar). Structural studies of the GpA-TM dimer have shown that the GXXXG motif increases the total area of the helix-helix interface by allowing close proximity of adjacent straight helices when they cross in a right-handed sense (28MacKenzie K.R. Prestegard J.H. Engelman D.M. Science. 1997; 276: 131-133Crossref PubMed Scopus (868) Google Scholar). In addition, the motif provides a flat surface against which side chains of other interfacial residues may pack.Inspection of the sequences for the ErbB receptor TM domains (Fig. 1) shows several elements that closely resemble the GXXXG motifs found by Russ and Engelman (30Russ W.P. Engelman D.M. J. Mol. Biol. 2000; 296: 911-919Crossref PubMed Scopus (775) Google Scholar) to drive TM domain dimerization. These motifs are listed in Table I. Sternberg and Gullick (35Sternberg M.J. Gullick W.J. Protein Eng. 1990; 3: 245-248Crossref PubMed Scopus (129) Google Scholar) previously pointed out the frequent occurrence of this general pentapeptide motif (P0-P1-P2-P3-P4) in the TM domains of receptor tyrosine kinases, in which the first position (P0) is occupied by a residue with a small side chain (G/A/S/T/P), position P4 is occupied by alanine or glycine, and position P3 is most often occupied by a residue with a large aliphatic side chain. The TM domains from ErbB1, ErbB2, and ErbB4 all possess at least two such potential motifs, one toward the amino terminus and one toward the carboxyl terminus of the domain (separated by ∼3 turns of α-helix). Each motif is designated with the receptor name and respective terminus in Table I (for example, motif 1C is the ErbB1 COOH-terminal motif with sequence AXXXG). In the ErbB1 and ErbB4 TM domains there are two possible NH2-terminal motifs that overlap. These are denoted by the addition of a subscripted “1” or “2” to the motif name. For example, the ErbB1 NH2-terminal TXXXG pattern is motif 1N1, and the overlapping GXXXA pattern is motif 1N2 (see Table I). The ErbB3 TM domain is exceptional in having only one possible motif, a TXXXG sequence (3N), that is positioned similarly to the GXXXG motif in GpA-TM (Fig. 1).Table IPotential GXXXG-based dimerization motifs in the TM domains of ErbB receptorsMotifTM domainResidue 1Residue 2Residue 3Residue 4Residue 5Role in dimerization1N1 (TXXXG)ErbB1Thr648Gly649Met650Val651Gly652No1N2 (GXXXA)ErbB1Gly649Met650Val651Gly652Ala653No1C (AXXXG)ErbB1Ala661Leu662Gly663Ile664Gly665Yes2N (SXXXG)ErbB2Ser656Ala657Val658Val659Gly660Yes2C (GXXXG)ErbB2Gly668Val669Val670Phe671Gly672Yes3N (TXXXG)ErbB3Thr647Val648Ile649Ala650Gly651No4N1 (AXXXG)ErbB4Ala655Gly656Val657Ile658Gly659ND1-aND, not determined.4N2 (GXXXG)ErbB4Gly656Val657Ile658Gly659Gly660Yes4C (GXXXA)ErbB4Gly668Leu669Thr670Phe671Ala672No↑Mutated position1-a ND, not determined. Open table in a new tab Mutations in Potential Dimerization Motifs Disrupt Dimerization of ErbB1, ErbB2, and ErbB4, but not ErbB3 TM DomainsTo assess the importance of each GXXXG motif in dimerization of ErbB receptor TM domains, the residue corresponding to the second glycine (equivalent to Gly83 in GpA-TM) was mutated to valine (see Table I), and dimerization of the mutated TM domain was assessed using TOXCAT. In previous studies of GpA-TM dimerization, all substitutions at Gly83 were found to be strongly disruptive (23Lemmon M.A. Flanagan J.M. Treutlein H.R. Zhang J. Engelman D.M. Biochemistry. 1992; 29: 12719-12725Crossref Scopus (463) Google Scholar). In the ErbB1, ErbB2, and ErbB4 TM domains, an equiva" @default.
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• W2000489928 title "The Single Transmembrane Domains of ErbB Receptors Self-associate in Cell Membranes" @default.
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