Active Conformation of the Erythropoietin Receptor

In the absence of erythropoietin (Epo) cell surface Epo receptors (EpoR) are dimeric; dimerization is mediated mainly by the transmembrane domain. Binding of Epo changes the orientation of the two receptor subunits. This conformational change is transmitted through the juxtamembrane and transmembrane domains, leading to activation of JAK2 kinase and induction of proliferation and survival signals. To define the active EpoR conformation(s) we screened libraries of EpoRs with random mutations in the transmembrane domain and identified several point mutations that activate the EpoR in the absence of ligand, including changes of either of the first two transmembrane domain residues (Leu226 and Ile227) to cysteine. Following this discovery, we performed cysteine-scanning mutagenesis in the EpoR juxtamembrane and transmembrane domains. Many mutants formed disulfide-linked receptor dimers, but only EpoR dimers linked by cysteines at positions 223, 226, or 227 activated EpoR signal transduction pathways and supported proliferation of Ba/F3 cells in the absence of cytokines. These data suggest that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane and the connected cytosolic domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR, constitutively activating downstream signaling. Based on our data and the previously determined structure of Epo bound to a dimer of the EpoR extracellular domain, we present a model of the active and inactive conformations of the Epo receptor. In the absence of erythropoietin (Epo) cell surface Epo receptors (EpoR) are dimeric; dimerization is mediated mainly by the transmembrane domain. Binding of Epo changes the orientation of the two receptor subunits. This conformational change is transmitted through the juxtamembrane and transmembrane domains, leading to activation of JAK2 kinase and induction of proliferation and survival signals. To define the active EpoR conformation(s) we screened libraries of EpoRs with random mutations in the transmembrane domain and identified several point mutations that activate the EpoR in the absence of ligand, including changes of either of the first two transmembrane domain residues (Leu226 and Ile227) to cysteine. Following this discovery, we performed cysteine-scanning mutagenesis in the EpoR juxtamembrane and transmembrane domains. Many mutants formed disulfide-linked receptor dimers, but only EpoR dimers linked by cysteines at positions 223, 226, or 227 activated EpoR signal transduction pathways and supported proliferation of Ba/F3 cells in the absence of cytokines. These data suggest that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane and the connected cytosolic domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR, constitutively activating downstream signaling. Based on our data and the previously determined structure of Epo bound to a dimer of the EpoR extracellular domain, we present a model of the active and inactive conformations of the Epo receptor. The cytokine erythropoietin (Epo) 2The abbreviations used are: Epo, erythropoietin; EpoR, erythropoietin receptor; EBP, Epo-binding protein; EMP, Epo mimetic peptide; JAK, Janus kinase; STAT, signal transducers and activators of transcription; IL, interleukin; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorter; FBS, fetal bovine serum; ER, endoplasmic reticulum; Endo H, endoglycosidase H; HA, hemagglutinin; JH, Janus kinase homology; IRES, internal ribosomal entry site. is the primary regulator of mammalian erythropoiesis. The erythropoietin receptor (EpoR), a member of the cytokine receptor family, is comprised of an extracellular ligand binding domain, a single transmembrane domain, and an intracellular domain (1D'Andrea A.D. Fasman G.D. Lodish H.F. Cell. 1989; 58: 1023-1024Abstract Full Text PDF PubMed Scopus (151) Google Scholar). Epo binding triggers trans-phosphorylation and activation of the Janus family protein tyrosine kinase JAK2 that is appended to the intracellular domain of EpoR. Activated JAK2 then phosphorylates tyrosine residues in EpoR, creating docking sites for intracellular signaling proteins such as STAT5, phosphatidylinositol 3′-kinase, and SHP1. These events lead to the activation of several signal transduction pathways and specific gene expression, resulting in the survival, proliferation, and differentiation of erythroid progenitors (2Constantinescu S.N. Ghaffari S. Lodish H.F. Trends Endocrinol. Metab. 1999; 10: 18-23Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Without Epo or EpoR, definitive erythropoiesis cannot occur (3Wu H. Liu X. Jaenisch R. Lodish H.F. Cell. 1995; 83: 59-67Abstract Full Text PDF PubMed Scopus (861) Google Scholar). Previous studies on a constitutively activated EpoR mutant, R129C, revealed that the activated EpoR is a dimer (4Watowich S.S. Yoshimura A. Longmore G.D. Hilton D.J. Yoshimura Y. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2140-2144Crossref PubMed Scopus (273) Google Scholar). Recent evidence indicates that surface EpoR exists as a dimer in the absence of Epo, and that Epo binding likely activates the EpoR through defined conformational changes (5Constantinescu 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 (215) Google Scholar, 6Seubert N. Royer Y. Staerk J. Kubatzky K.F. Moucadel V. Krishnakumar S. Smith S.O. Constantinescu S.N. Mol. Cell. 2003; 12: 1239-1250Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The EpoR extracellular segment consists of two fibronectin III domains, D1 and D2. The crystal structure of two EpoR extracellular ligand binding domains (Epo-binding protein, EBP) bound to one Epo molecule showed that the D1 domains of the two dimerized EpoRs are positioned at a 120° angle when viewed perpendicular to the membrane plane (7Syed R.S. Reid S.W. Li C. Cheetham J.C. Aoki K.H. Liu B. Zhan H. Osslund T.D. Chirino A.J. J. J. S. D.J. J. PubMed Scopus Google Scholar). the crystal structure of the EpoR extracellular domains bound to a Epo mimetic a orientation with a angle the two D1 domains PubMed Scopus Google Scholar). This that is one conformation that activate EpoR and that a 120° angle the two receptors for signaling (7Syed R.S. Reid S.W. Li C. Cheetham J.C. Aoki K.H. Liu B. Zhan H. Osslund T.D. Chirino A.J. J. J. S. D.J. J. PubMed Scopus Google Scholar). that Epo activates EpoR by the two EpoR in a dimeric the active of one JAK2 kinase to the tyrosine in the activation of the this the JAK2 downstream signaling EpoR dimerization in the absence of ligand is mediated mainly by transmembrane domain (5Constantinescu 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 (215) Google and the EpoR transmembrane domain is a that dimer R. R. B. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). dimerization by mutations in the transmembrane domain EpoR signaling in mammalian cells R. R. B. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, K.F. R. J. R. S.S. U. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The EpoR cytosolic juxtamembrane domain a and that is for JAK2 activation Constantinescu S.N. Lodish H.F. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). in or the transmembrane segment and this that the transmembrane and cytosolic juxtamembrane domains of EpoR a and S.N. Nam H. Lodish H.F. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). This structure for the dimeric EpoR in an inactive conformation and receptor the EpoR transmembrane and juxtamembrane in the EpoRs in active and inactive and of the EpoR transmembrane domain for the of EpoR as is structure of cytokine receptor transmembrane or intracellular domain, and we of the appended are by cytokine To this the EpoR transmembrane domain to a segment the and active and inactive of EpoR transmembrane dimer by the dimer through in the transmembrane domain of the N. Royer Y. Staerk J. Kubatzky K.F. Moucadel V. Krishnakumar S. Smith S.O. Constantinescu S.N. Mol. Cell. 2003; 12: 1239-1250Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). In the dimeric by the domains likely the activated by the Epo mimetic the active conformation by Epo binding N. Royer Y. Staerk J. Kubatzky K.F. Moucadel V. Krishnakumar S. Smith S.O. Constantinescu S.N. Mol. Cell. 2003; 12: 1239-1250Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). we a to the active of the EpoR. the EpoR transmembrane and juxtamembrane domains a that in an inactive in the absence of ligand, mutations in this the conformational changes that the Epo binding on the EpoR, the of the intracellular domains and the JAK2 and leading to activation of libraries of EpoRs with single and mutations in transmembrane domains, we identified several mutations that activate EpoR in the absence of Many in the transmembrane domain, but several changes of either of the first two transmembrane domain residues (Leu226 and Ile227) to cysteine. then performed cysteine-scanning mutagenesis of the EpoR juxtamembrane extracellular and transmembrane domains. Many mutants disulfide-linked dimers, but only EpoR dimers linked by at positions 223, 226, or 227 activate of the EpoR signal transduction pathways we and proliferation of Ba/F3 cells in the absence of cytokines. These suggest that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR. of disulfide-linked EpoR transmembrane domains, in either an inactive or an active as defined by the conformational changes in a dimeric EpoR activates JAK2 and downstream signaling EpoR in a to X. Constantinescu S.N. Y. Lodish H.F. PubMed Scopus Google Scholar). mutations to sites the EpoR transmembrane and juxtamembrane and for EpoR sites and or and by with two or random in The random the of the transmembrane and juxtamembrane the and libraries EpoRs a random to random Ba/F3 cells with To in one the that only of Ba/F3 cells with fluorescent protein cells by These cells then in fetal bovine and of The of revealed by with and of the for and cells or EpoRs and in with FBS, at and with a To proliferation, Ba/F3 cells or EpoRs in with and a of of Epo to in of to to the and the of by a and cells or EpoRs in and with and These then with JAK2 or as by protein then in and the proteins with the by in on a and to These with specific for by with and the To the for JAK2 and STAT5, in with and for at then in with and The then with specific for JAK2 or as of EpoR by Epo Epo with Ba/F3 cells or EpoRs a first for and cells with These cells then in with of for at in and by cells through a of Ba/F3 cells used as a to Epo determined by the model for a of binding sites to data a of ligand and the of receptors on the cell surface and the of cells Lodish H.F. J. Full Text Full Text PDF PubMed Scopus Google Scholar). Endo of the cells or EpoRs in with at in the cell in and by with endoglycosidase at for The then on to and with in the of EpoR in of transmembrane and juxtamembrane domains in receptor dimerization and activation (5Constantinescu 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 (215) Google Scholar, S.N. Nam H. Lodish H.F. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). that certain mutations in this the activated dimeric of EpoR, the survival and proliferation of erythroid progenitors in the absence of activating mutations in the transmembrane and juxtamembrane domain identified in cytokine receptors M. Y. S. A. T. PubMed Google Scholar, M. S. H. PubMed Scopus Google Scholar, A. A. PubMed Scopus Google but to in EpoR. To a of EpoR we a mutagenesis we used with two or random in to the for the EpoR transmembrane and juxtamembrane domains in the X. Constantinescu S.N. Y. Lodish H.F. PubMed Scopus Google In libraries EpoRs and used to Ba/F3 Ba/F3 cells EpoRs by FACS, and Ba/F3 in the absence of Epo and EpoR These EpoRs then and in Ba/F3 cells a in is the as the EpoR. cell receptors for the of and then for to and The mutations that constitutively activate the EpoR are in activating mutations in the EpoR transmembrane domain identified by a random mutagenesis activating mutations in the EpoR transmembrane domain identified by a random mutagenesis EpoR transmembrane domain and juxtamembrane that are identified to EpoR are by a residues are the a EpoR transmembrane domain and juxtamembrane domain. that are identified to EpoR are by a residues are the mutations in the or residues of the transmembrane domain, are to residues or These mutations Ba/F3 cell proliferation in the absence of Epo, as in with Ba/F3 cells the EpoR, in the absence of Epo, cells that receptors to and at or residues the of the transmembrane domain receptor activation or cell proliferation, that cell proliferation in mutants is by the of the of the EpoR transmembrane domain that cells one of at a in the of of Epo In cells the constitutively active mutants and showed the of Epo that Ba/F3 cells the constitutively active EpoR mutants and activation of JAK2 when of for In and as activation of JAK2 when Ba/F3 cells the EpoR of Epo and for The of JAK2 activation by EpoRs is with that in Ba/F3 cells the EpoR but in a of Epo, This of JAK2 activation as that in Ba/F3 cells the EpoR of Epo for and then with a of Epo for In cells the EpoR in the of Epo a of proteins are or the signaling the that in Ba/F3 cells of the constitutively active an of JAK2 in the absence of Epo receptor mutants an active conformation in the absence of Epo that is for JAK2 of either of the two residues of the EpoR transmembrane domain, or to and JAK2 in Ba/F3 cells and the of we that the activation of EpoRs a of that the EpoR dimers in an activating mutations in the EpoR extracellular domain, R129C, and disulfide-linked constitutively active EpoR that receptors are in an active dimeric conformation (4Watowich S.S. Yoshimura A. Longmore G.D. Hilton D.J. Yoshimura Y. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2140-2144Crossref PubMed Scopus (273) Google Scholar, A. Longmore Lodish H.F. PubMed Scopus Google Scholar, S.S. Hilton D.J. Lodish H.F. Mol. PubMed Scopus Google Scholar). in mutants lead to the in the extracellular domain, the structure of EpoR transmembrane domains the active orientation of the EpoR transmembrane domain and intracellular domains. of EpoR and the of in the EpoR transmembrane and juxtamembrane domains, we of the transmembrane residues to cysteine. residues in the and cytosolic juxtamembrane domains to and and to the juxtamembrane domain in our These EpoRs the and in of Ba/F3 cells through cells by on the of X. Constantinescu S.N. Y. Lodish H.F. PubMed Scopus Google Scholar). These cells then in the with and cells in the of Epo used as only mutants in the domain and only and in the transmembrane domain supported of Ba/F3 cells and supported a proliferation of Ba/F3 in a of the cells and the of the cells the EpoR a of receptor that Ba/F3 cells In of residues with receptors with Ba/F3 cells receptors to Epo and proliferation and data This that our mutagenesis and activating in the EpoR that Ba/F3 cells EpoRs with at in the of Epo, as by the and showed an to Epo, mutants to Epo or to Epo of but for that at and constitutively activate the EpoR, we that of at positions receptor To when in Ba/F3 performed In to EpoR, EpoR mutants with cysteines at positions formed disulfide-linked of EpoR in mutants with at or to and data This the of residues to the endoplasmic that These mutants activation in Ba/F3 that in EpoR mutants with of an is for the data in that of an is for the of the disulfide-linked dimer is with the of an as by the of dimeric to EpoR the of with of EpoR in a disulfide-linked This of proliferation the of disulfide-linked dimers, proliferation in Ba/F3 The of of disulfide-linked dimers is that of either or supported the cell proliferation this and of disulfide-linked dimers formed by the and of only and supported cell These suggest that receptor activation is by two receptors as by 1999; PubMed Scopus Google Scholar). EpoR and our studies and EpoRs are in Ba/F3 cells a in of GFP, by the the as the EpoR X. Constantinescu S.N. Y. Lodish H.F. PubMed Scopus Google Scholar). cells for the of expression, and we that the of EpoR, or mutant, in cell that this is the with the that the of and EpoR is a of EpoRs the cell in Ba/F3 cells in the as an Endo and are A. A.D. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, D.J. S.S. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). that cells mutants and the of the Endo EpoR and endoplasmic Endo EpoR as cells the EpoR. This that Epo receptors to the and receptor This is by the data in and that that cells EpoR mutants and the of cell surface Epo Epo bound to cells the EpoR. EpoR mutants and the constitutively active in receptor in Ba/F3 cells EpoR and endoplasmic Endo EpoR. This indicates that a of EpoRs and are in the endoplasmic a to These mutants at a as is the for the EpoR A. Longmore Lodish H.F. PubMed Scopus Google Scholar). In the disulfide-linked Endo EpoR cells EpoR. This is by the data in and that that cells EpoR cell surface Epo Epo bound cells the EpoR. by surface EpoRs data that mutants and are through the and on the cell The activated EpoR in the ER, as receptor is formed and on the cell This in of surface to when in Ba/F3 cells EpoR Activated in Ba/F3 Activated EpoR data in that signal transduction proteins are activated by in cells EpoR mutants and in the absence of are the mutants that of Ba/F3 for cells of Epo and then with Epo for the the activation of signaling is tyrosine of JAK2 and the EpoR cells EpoR mutants and of signaling proteins in the absence of Epo in the of activation of signaling proteins is to in cells by of Epo, several cell signaling such as and activated U. U. Lodish H.F. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, U. H. A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). that cells EpoR and tyrosine of the JAK2 protein, as by with and with the of JAK2 in cells EpoR, or mutants when in the absence of a cells a of JAK2 protein tyrosine of JAK2 with the of EpoRs to cell as is in in the absence of Epo, Ba/F3 cells EpoR, or mutants or showed only of In cells EpoR mutants or showed of cells the of protein of the EpoR cytosolic domain docking sites for signaling and is for activation of STAT5, phosphatidylinositol protein and such as and proteins U. U. Lodish H.F. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, U. H. A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, U. S. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). that cells EpoR mutants or and in the absence of Epo tyrosine of the EpoR in to cells EpoR or mutants or This that the JAK2 appended to the or EpoRs are activated and in the EpoR cytosolic domains. is of EpoR in cells the The of JAK2 and in cells mediated mainly by mutants that are in the In cells EpoR, the and surface of EpoR to the of surface EpoR, and in in of in certain signaling pathways that are on surface EpoR. that mutants and are active in the absence of Epo, as activation by of signaling proteins activated by Epo the EpoR and that EpoRs activate the phosphatidylinositol and kinase pathways we we performed mutagenesis of the EpoR transmembrane and juxtamembrane domains and identified constitutively activated receptor mutations by EpoRs that proliferation of Ba/F3 identified mutants with in the first two transmembrane residues (Leu226 and as as mutants with residues at the of the transmembrane domain. we cysteine-scanning mutagenesis of the transmembrane domain and on either mutants at positions the extracellular juxtamembrane and the of the transmembrane domain, formed disulfide-linked receptor only EpoR dimers linked by cysteines at positions 223, 226, or 227 activated the signal transduction proteins STAT5, and the EpoR and supported proliferation of Ba/F3 cells in the absence of cytokines. data that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane domains in the inactive receptor dimer and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR, constitutively activating downstream signaling. EpoR activation only when two receptors are in a dimeric and certain in or the EpoR transmembrane domain the EpoR dimer in a conformation that is for signaling. in of Kubatzky K.F. Liu C. Smith S.O. Constantinescu S.N. J. Full Text Full Text PDF PubMed Scopus Google performed cysteine-scanning mutagenesis to the dimerization of the EpoR juxtamembrane and transmembrane domains, and identified the constitutively active mutants and of Ba/F3 cells that mutants at the in the absence of Epo K.F. Liu C. Smith S.O. Constantinescu S.N. J. Full Text Full Text PDF PubMed Scopus Google Scholar). studies of cells to in and we in the of activating mutants to the of cell surface These by in the of activation of JAK2 and and of the EpoR cytosolic domain. in the of the EpoRs to disulfide-linked we showed that the of dimerization with the of the that the orientation of the disulfide-linked EpoR dimers is the for we identified mutations in the EpoR transmembrane domain, such as that are constitutively activated and These in the of the EpoR in K.F. Liu C. Smith S.O. Constantinescu S.N. J. Full Text Full Text PDF PubMed Scopus Google Scholar, suggest mutants activated of EpoR transmembrane domains mutations to the receptor conformation for and for constitutively activating receptors libraries Ba/F3 cells with the receptor or an used to libraries the M. Y. S. A. T. PubMed Google Scholar). of the mutants single point mutations that only one in a and in that the changes for to point in the transmembrane domain of identified for to ligand studies on this mutant, and mutations to residues studies mainly on the of the EpoR transmembrane domain and intracellular juxtamembrane the we we to a in creating our random we to mutants with at To mutants with on the structure of EpoR transmembrane domain and juxtamembrane domain point we two or and of with random This to mutations that the receptor this we several mutants that are constitutively active and that two or mutations In we constitutively active of one or residues the extracellular of the transmembrane domain. the of point mutations in the constitutively active mutants and that only the EpoR and that the single mutants and are to of Ba/F3 cells that point mutations are to a to activate EpoR signaling. of mutants with that of EpoR on the activation of the receptor. and EpoR our residues the transmembrane domain, to with two dimerized EpoR in Ba/F3 cells by only EpoRs with cysteines to formed the of dimers, in by and and a of in a dimeric with cysteines at positions are either in the membrane or at the cytosolic disulfide-linked dimers, residues are to proteins such as that in the in mutagenesis in that only the first or residues in the transmembrane domain are to J. Scopus Google Scholar). the of of with the of EpoR showed the of dimerization but supported of the for is by the that a of EpoR is in the and a the cell the of of disulfide-linked dimers that of supported cell proliferation, and of disulfide-linked dimers formed by the and of only and supported cell In and activation of EpoR, and in cells mutants with to in the absence of cytokines. our that receptor activation is by two receptors through in the transmembrane or extracellular domains. are with the model by 1999; PubMed Scopus Google that that Epo of the EpoR extracellular domains that the EpoR transmembrane and intracellular domains S.W. 1999; PubMed Scopus Google Scholar). dimerized EpoR mutants and to in an activated the EpoR in the absence of Epo and dimerized and EpoRs are the orientation of the two transmembrane domains of an EpoR the of receptor one active for dimerized EpoR, as by the of and (7Syed R.S. Reid S.W. Li C. Cheetham J.C. Aoki K.H. Liu B. Zhan H. Osslund T.D. Chirino A.J. J. J. S. D.J. J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). to of the structure of the EpoR extracellular domain a defined structure only to The to an structure with and to one of the molecule and to the (7Syed R.S. Reid S.W. Li C. Cheetham J.C. Aoki K.H. Liu B. Zhan H. Osslund T.D. Chirino A.J. J. J. S. D.J. J. PubMed Scopus Google Scholar). the dimer to the that and are that cysteines at two positions at is at the of the domain and by the domain. at of a of the two receptors in a orientation with The dimerized transmembrane domains to or to such and this receptor is structure suggest that residues are likely to the residues and is with to in an This to is for receptor showed this is and mammalian residues at certain positions in this activate the EpoR, this is for EpoR an to with on EpoR activation by Kubatzky K.F. Liu C. Smith S.O. Constantinescu S.N. J. Full Text Full Text PDF PubMed Scopus Google used mutagenesis to the constitutively active and studies as that the segment through is and and studies that this an the structure of the to is as is in the active and inactive conformations of EpoR. is an for of the EpoR for JAK2 the of the JAK2 kinase with the segment of the EpoR intracellular domain. JAK2 is comprised of domains, to In the domain is a active tyrosine The domain is a domain that the kinase of JAK2 the domain of kinase leading to a constitutively active JAK2 Mol. PubMed Scopus Google Scholar). The domain with and the domain with domain, leading to activation J. Full Text Full Text PDF PubMed Scopus Google Scholar). JAK2 activation a in the two EpoR intracellular domains of a surface the two appended JAK2 in such an orientation that activating of the this is the that one conformation of two EpoRs that activate the crystal of and Epo, the ligand for EpoR, is an positions the two EpoR extracellular domains with a 120° angle the two D1 domains (7Syed R.S. Reid S.W. Li C. Cheetham J.C. Aoki K.H. Liu B. Zhan H. Osslund T.D. Chirino A.J. J. J. S. D.J. J. PubMed Scopus Google Scholar). The dimer positions the EpoR extracellular domains with a angle PubMed Scopus Google Scholar). These two conformations likely of the intracellular domains. Epo, activate EpoR signaling. the EpoR transmembrane domain and intracellular domain by in the constitutively activated receptors N. Royer Y. Staerk J. Kubatzky K.F. Moucadel V. Krishnakumar S. Smith S.O. Constantinescu S.N. Mol. Cell. 2003; 12: 1239-1250Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). of EpoR activation is in EpoRs as dimers on the cell The in the transmembrane domain represent the surface in the inactive EpoR the of residues in the two a in the of the EpoR extracellular domain and transmembrane domain is to an the is formed at or the dimer with and the receptor inactive the of the dimer the transmembrane domains change The a the activated conformation of the N. Royer Y. Staerk J. Kubatzky K.F. Moucadel V. Krishnakumar S. Smith S.O. Constantinescu S.N. Mol. Cell. 2003; 12: 1239-1250Abstract Full Text Full Text PDF PubMed Scopus (167) Google through that the appended are likely to an to that of the EpoR dimer (7Syed R.S. Reid S.W. Li C. Cheetham J.C. Aoki K.H. Liu B. Zhan H. Osslund T.D. Chirino A.J. J. J. S. D.J. J. PubMed Scopus Google Scholar). In this two of residues EpoR at the dimer of the transmembrane domain as a of an conformation the receptor likely on the in the EpoR transmembrane dimer R. R. B. J. 1999; Full Text Full Text PDF PubMed Scopus Google we the model in is a likely for the disulfide-linked only this conformation achieved by a of the two dimerized EpoR. Epo is an molecule that binding positions the two EpoR extracellular D1 domains in a 120° the EpoR dimer a conformation on and to EpoR with a and for and for with and