Biochemical Studies Supporting Preformed Dimers

Remy et al. [11] used an in vivo complementation assay to demonstrate that a ligand-induced conformational change of the EPOR dimer is required for activation. In these studies, chimeras that contained the extracellular and transmembrane domains of EPOR were fused to two complementary fragments of murine dihydrofolate reductase (DHFR) through flexible linkers of different lengths. Cells transfected with these chimeras express the receptors at the cell surface. In this experiment, DHFR activity is restored if the two complementary fragments are brought into close proximity. Chimeras containing a short 5-residue linker could not restore DHFR activity in the absence of EPO. In contrast, chimeras that contained the 30-residue linker allowed complementation both in the presence and absence of EPO. This linker-length correlation agrees well with the crystallo-graphic distances between the C termini of the two EBPs in their free and bound states. Thus, taken with the unliganded EPOR structure, these observations suggest a ligand-induced reorganization of the dimer that results in activation of the signal cascade.

Recent biochemical studies of the transmembrane and juxtamembrane domains of the EPO receptor further support preformed EPOR dimers and the importance of receptor orientation for productive signaling. Constantinescu et al. [25] have shown that the transmembrane domains of the EPO receptor interact in vivo using antibody-mediated immuno-flouresence copatching assays. These oligomerized receptors are not constitutively active, but rather require EPO binding to induce signaling. Furthermore, experiments that swapped the transmembrane domains of the EPO receptor for the strongly dimerizing transmembrane domain of glycophorin A still showed a dependence on EPO for JAK2 activation.

Further studies demonstrate the importance of the relative orientation of a region in the cytosolic juxtamembrane domains of the EPOR in signaling [26]. Alanine scanning has indicated that three residues, Leu253, Ile257, and

EMP1-EBP complex. (a) Weak agonist EMP1-EBP complex (EBP, cyan; EMP1, red; PDB code 1ebp) [15]. (b) Antagonist EMP33-EBP complex (EBP, salmon red; EMP33, red; PDB code 1eba) [10]. (c) Strong agonist EPO-EBP complex (receptor site 1, green; receptor site 2, purple; EPO, red; PDB code 1cn4) [16]. (d) Self-dimer EBP-EBP native complex (EBP, orange [33]; PDB code 1ern) [17]. Molecules were made using MOLSCRIPT [34] and rendered in Raster3D [33]. For color figures, see CD-ROM version of Handbook of Cell Signaling. (Adapted from Wilson, I. A. and Jolliffe, L. K., Curr. Opin. Struct. Biol. 9, 696-704, 1999.)

Trp258 (LIW), in this region were necessary for EPO-induced phosphorylation, but not for binding of JAK2. Accordingly, these residues are likely to be involved in a switch mechanism propagated from EPO binding to the EC that positions JAK2 correctly for appropriate activation (Fig. 3a). These three residues are likely to occur on the same face of the protein surface, as the secondary structure analysis predicts an a-helix continuing from the TM through this region. Experiments in which additional alanine residues were inserted into this juxtamembrane region were performed in order to assess the affect of changing the relative "register" of these regions and, hence the effect transmitted to the intermolecular domains (Figs. 3b-d). Each alanine insertion rotates the register of the predicted a-helix by 109°. A single insertion greatly diminishes signaling by EPO, whereas a three-residue alanine insertion restores signaling

Figure 3 A model of EPO-induced dimerization and activation of the EPO receptor, JAK2 transphosphoryla-tion, and tyrosine phosphorylation of the EPO cytosolic (CT) domain. (a) The schematic depicts contrasting views of ligand-induced signal activation. Top panel depicts a preformed dimer whereby the binding of cytokine (e.g., EPO) induces a structural reorganization, leading to active signaling. Bottom panel (b, c, d) depicts receptor monomers on the cell surface in which the binding of cytokine (e.g., GH) leads to dimerization, resulting in active signaling. EPO receptors are dimerized by EPO. The transmembrane (TM) domain is shown as a yellow cylinder and continues as a rigid helix into the CT domain through residue W258. L253, I257, and W258 comprise a hydrophobic patch expected to be at one face of this a-helix. L253 is shown in this helix. Upon activation (b), JAK2 transphosphorylates its partner JAK2 molecule, which in turn phosphorylates the partner EPOR CT domain tyrosine residues. Phosphorylation sites are marked as red circles. Insertion of one alanine residue prior to L253 (c) rotates the putative transmembrane a-helix by 109°, causing a change in the "register" of the hydrophobic patch. In this case, EPO is capable of inducing JAK2 phosphorylation; however, tyrosine phosphorylation of the EPO CT domains does not occur. Insertion of three alanine residues prior to L253 (d) restores wild-type activity to the EPOR. A three-alanine insertion restores the "register" of the hydrophobic patch, as the helix is rotated by 327° [26].

Figure 3 A model of EPO-induced dimerization and activation of the EPO receptor, JAK2 transphosphoryla-tion, and tyrosine phosphorylation of the EPO cytosolic (CT) domain. (a) The schematic depicts contrasting views of ligand-induced signal activation. Top panel depicts a preformed dimer whereby the binding of cytokine (e.g., EPO) induces a structural reorganization, leading to active signaling. Bottom panel (b, c, d) depicts receptor monomers on the cell surface in which the binding of cytokine (e.g., GH) leads to dimerization, resulting in active signaling. EPO receptors are dimerized by EPO. The transmembrane (TM) domain is shown as a yellow cylinder and continues as a rigid helix into the CT domain through residue W258. L253, I257, and W258 comprise a hydrophobic patch expected to be at one face of this a-helix. L253 is shown in this helix. Upon activation (b), JAK2 transphosphorylates its partner JAK2 molecule, which in turn phosphorylates the partner EPOR CT domain tyrosine residues. Phosphorylation sites are marked as red circles. Insertion of one alanine residue prior to L253 (c) rotates the putative transmembrane a-helix by 109°, causing a change in the "register" of the hydrophobic patch. In this case, EPO is capable of inducing JAK2 phosphorylation; however, tyrosine phosphorylation of the EPO CT domains does not occur. Insertion of three alanine residues prior to L253 (d) restores wild-type activity to the EPOR. A three-alanine insertion restores the "register" of the hydrophobic patch, as the helix is rotated by 327° [26].

to the same level as the wild-type protein. Furthermore, it is not the increase in distance («5 Â) from the transmembrane domain (corresponding to one turn of helix) that alters JAK2 activity; instead, variation in the relative orientation of the LIW patch is crucial for proper signal response to EPO. Further insertion of two alanine residues in the TM region confirmed the predicted secondary structure of a continuous rigid helix from the TM through the Trp258, as a two amino-acid insertion restores the register of the LIW hydrophobic patch (Fig. 3b). Sequence alignments reveal that the hydropho-bic nature of these three residues is conserved throughout nine other members of the cytokine receptor superfamily. Furthermore, the spacing of these residues relative to each other and their distance from Box 1 are strictly conserved, even though their distance from the membrane varies [26].

Two alternative models can explain the possible mechanisms of activation by cytokines and growth factors (Fig. 3a). For EPOR, the evidence is consistent with the unliganded EPOR existing as a preformed dimer on the cell surface in an "inactive" state and that binding of EPO in vivo causes a con-formational change that is propagated through the membrane to the cytosolic domain. In addition, the relative orientation of the receptor is critical for signal generation, again consistent with the distinctly different agonist and antagonist structures. This ligand-dependent structural reorganization allows for interaction of JAK2, thereby eliciting a signaling cascade.

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